Publications by authors named "manage"

47 Publications

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg and ionic strength.

ACS Bio Med Chem Au 2021 Dec 29;1(1):44-56. Epub 2021 Oct 29.

Department of Chemistry, University of Utah, 315 S. 1400 E., Salt Lake City, UT 84112-0850, United States.

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1's binding and catalytic activity with the vascular endothelial growth factor () promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K and Mg and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg found that dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg]. Studies with 50 versus physiological 140 mM K ions showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis is nearly abolished without the N-terminus. With this knowledge, we found APE1 could remodel the F-containing promoter dsDNA→G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.
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http://dx.doi.org/10.1021/acsbiomedchemau.1c00031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8740889PMC
December 2021

Diversity of microbial communities in hot springs of Sri Lanka as revealed by 16S rRNA gene high-throughput sequencing analysis.

Gene 2022 Feb 9;812:146103. Epub 2021 Dec 9.

Centre for Water Quality and Algae Research, Department of Zoology, University of Sri Jayewardenepura, Gangodawila, Nugegoda 10250, Sri Lanka; Faculty of Graduate Studies, University of Sri Jayewardenepura, Nugegoda 10250, Sri Lanka. Electronic address:

Characterization of hot spring microbiota is useful as an initial platform for exploring industrially important microbes. The present study focused on characterization of microbiota in four hot springs in Sri Lanka: Maha Oya; Wahava; Madunagala; and Kivlegama using high throughput 16S amplicon sequencing. Temperatures of the selected springs were ranged from 33.7 °C to 52.4 °C, whereas pH ranged from 7.2 to 8.2. Bacteria were found to be the dominant microbial group (>99%) compared to Archaea which represented less than 1% of microbiota. Four hot springs comprised of unique microbial community structures. Proteobacteria, Firmicutes, Bacteroidetes, Cloroflexi, Deinococcus and Actenobacteria were the major bacterial phyla. Moderately thermophilic genera such as Thermodesulfobacteria and Deinococcus-Thermus were detected as major genera that could be used in industrial applications operating at temperatures around 50 °C and alkaline reaction conditions.
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http://dx.doi.org/10.1016/j.gene.2021.146103DOI Listing
February 2022

Prevalence and Quantitative Analysis of Antibiotic Resistance Genes (ARGs) in Surface and Groundwater in Meandering Part of the Kelani River Basin in Sri Lanka.

Water Air Soil Pollut 2021 22;232(9):351. Epub 2021 Aug 22.

Center for Water Quality and Algae Research, Department of Zoology, University of Sri Jayewardenepura, Nugegoda, Sri Lanka.

Nearly 80% of the population in the Colombo district fulfill their major requirement from the Kelani river. Recent studies are interoperating: most groundwater and surface water in Sri Lanka are contaminated with waterborne pathogens and antibiotics. In the present study, nine antibiotic resistance genes (ARGs) were screened which were belonging to two common groups of antibiotic: penicillin - , , , , and - and tetracycline - , , , and . The results of the study reveled that the surface and groundwater of the entire lower part of the Kelani river basin were contaminated with TC and FC (98%). None of the penicillin and tetracycline group antibiotics were detected either surface or groundwater samples except the Kelani river mouth (amoxicillin (AMX) at 0.003 ± 0.001 µg/ml). The results showed that 5 to 15% of surface water samples were positive for penicillin resistance genes ( , , , , ) where ~ 10% of groundwater samples were positive against tetracycline resistance genes (, , , ). Among the penicillin resistance genes, the (700.576 × 10 copy/ml) was recorded as the highest concentration where the highest gene (439.875 × 10 copy/ml) was detected among the tetracycline resistance genes. Therefore, water quality management and regular monitoring are essential to maintain the quality of drinking water in the meandering part of the Kelani river basin to safeguard river water consumers.
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http://dx.doi.org/10.1007/s11270-021-05300-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380415PMC
August 2021

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting in Beef Carcass Swabs.

Micromachines (Basel) 2021 Aug 13;12(8). Epub 2021 Aug 13.

Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB T6G 2P5, Canada.

Detection sensitivity of cassette PCR was compared with a commercial BAX PCR system for detection of and genes in from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored + . For BAX PCR reactions, 84% of + swabs, 79% of + swabs, and 86% of + + swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were + with only 117 scored as + using BAX PCR for 26% positive concordance. For primers, cassette PCR scored 480 samples as + but only 215 samples were + by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring + + , but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in found in carcass swabs.
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http://dx.doi.org/10.3390/mi12080959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8398369PMC
August 2021

Modified, optimized method of determination of Tributyltin (TBT) contamination in coastal water, sediment and biota in Sri Lanka.

Mar Pollut Bull 2021 May 4;166:112202. Epub 2021 Mar 4.

Centre for Water Quality and Algae Research, Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda 10250, Sri Lanka; Faculty of Graduate Studies, University of Sri Jayewardenepura, Gangodawila, Nugegoda 10250, Sri Lanka. Electronic address:

Tributyltin (TBT) is a toxic organotin compound that belongs to the group of Persistent Organic Pollutants (POPs) and it is documented to cause severe sexual disorders development in aquatic fauna. According to the present study, The TBT concentration in coastal water ranged from 303 ± 7.4 ngL to 25 ± 4.2 ngL wherein sediment was from 107 ± 4.1 ngKg to 17 ± 1.4 ngKg. TBT in Perna viridis was found to range from 4 ± 1.2 ngKg to 42 ± 2.2 ngKg wet weight and in ascending order of the body weight. The highest TBT level in water and sediment was found in the Colombo port where the highest level of TBT in P. viridis (42 ± 2.2 ngKg) was recorded from the Dikkowita fishery harbor. A positive correlation between the number of male P. viridis and TBT level (p < 0.05) suggests possible reproductive impairment in aquatic animals exposed continuously to a high concentration of TBT.
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http://dx.doi.org/10.1016/j.marpolbul.2021.112202DOI Listing
May 2021

SIMR foci are found in the progenitor germ cells of embryos.

MicroPubl Biol 2021 Feb 22;2021. Epub 2021 Feb 22.

Department of Biological Sciences, University of Southern California, Los Angeles, California, USA.

RNA interference is a widely conserved mechanism of gene regulation and silencing across eukaryotes. In , RNA silencing is coordinated through perinuclear nuage containing at least four granules: P granules, Z granules, foci, and SIMR foci. Embryonic localization of these granules is known for all except SIMR foci. Here we establish that SIMR foci first appear at the nuclear periphery in the P germline blastomere and become numerous and bright in the Z2 and Z3 progenitor germ cells. This timing coincides with the appearance or de-mixing of other germline granules, providing further evidence for coordinated germ granule reorganization.
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http://dx.doi.org/10.17912/micropub.biology.000374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900827PMC
February 2021

Phytoremediation of synthetic textile dyes: biosorption and enzymatic degradation involved in efficient dye decolorization by Eichhornia crassipes (Mart.) Solms and Pistia stratiotes L.

Environ Sci Pollut Res Int 2021 Apr 6;28(16):20476-20486. Epub 2021 Jan 6.

Centre for Water Quality and Algae Research, Department of Zoology, University of Sri Jayewardenepura, Nugegoda, 10250, Sri Lanka.

The effectiveness of four aquatic floating plants: Eichhornia crassipes, Pistia stratiotes, Lemna minor, Salvinia sp., and a submerged plant Hydrilla sp. on decolorization and detoxification of five structurally different textile dyes: CI Direct Blue 201 (DB 201), Cibacron Blue FR, Cibanone Gold Yellow RK, Vat Green FFB, and Moxilon Blue GRL were studied. The E. crassipes and P. stratiotes showed complete decolorization of all the dyes tested, while Salvinia sp. (79-86%), L. minor (16-24%), and Hydrilla sp. (6-13%) were recorded as the least tolerance for all the dyes even after 14 days of incubation. Therefore, E. crassipes and P. stratiotes were selected for further studies using DB 201 as the model dye. E. crassipes and P. stratiotes showed complete decolorization of DB 201 at 48 and 84 h of incubation, respectively, and decolorization was well effective in the pH range 6-9. The crude extract of intracellular enzymes obtained from the roots of E. crassipes (46%) and P. stratiotes (20%) showed significant involvement on decolorization of DB 201, compared with the activity of crude extracellular extract and isolated endophytic bacteria and fungi (p ≤ 0.05). Further, 18 and 22% of biosorption of DB 201 dye were recorded by E. crassipes and P. stratiotes, respectively, suggesting that decolorization mechanisms of DB 201 dye by E. crassipes and P. stratiotes were based on biosorption and intracellular enzyme activities. The FTIR spectra and seed germination assay confirmed biodegradation and detoxification of DB 201 dye by E. crassipes and P. stratiotes plants along with complete color removal. Thus, present study confers the potential applicability of E. crassipes and P. stratiotes plants for textile dye removal and release to the environment without further treatment.
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http://dx.doi.org/10.1007/s11356-020-11699-8DOI Listing
April 2021

Kidney developmental effects of metal-herbicide mixtures: Implications for chronic kidney disease of unknown etiology.

Environ Int 2020 11 17;144:106019. Epub 2020 Aug 17.

Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469, USA; Nicholas School of the Environment, Duke University, Durham, NC 27708, USA; School of Marine Sciences, University of Maine, Orono, ME 04469, USA.

Chronic kidney disease of unknown etiology (CKDu) is an emerging global concern affecting several agricultural communities in the Americas and South Asia. Environmental contaminants such as heavy metals (e.g., Cd, As, Pb, and V) and organic pesticides (e.g., glyphosate) in the drinking water have been hypothesized to play a role in childhood onset and progression of this disease. However, a comprehensive analysis of chemical contaminants in the drinking water and effects of these compounds and their mixtures on kidney development and function remains unknown. Here, we conducted targeted and non-targeted chemical analyses of sediment and drinking water in CKDu affected regions in Sri Lanka, one of the most affected countries. Using zebrafish Danio rerio, a toxicology and kidney disease model, we then examined kidney developmental effects of exposure to (i) environmentally derived samples from CKDu endemic and non-endemic regions and (ii) Cd, As, V, Pb, and glyphosate as individual compounds and in mixtures. We found that drinking water is contaminated with various organic chemicals including nephrotoxic compounds as well as heavy metals, but at levels considered safe for drinking. Histological studies and gene expression analyses examining markers of kidney development (pax2a) and kidney injury (kim1) showed novel metal and glyphosate-metal mixture specific effects on kidney development. Mitochondrial dysfunction is directly linked to kidney failure, and examination of mixture specific mitochondrial toxicity showed altered mitochondrial function following treatment with environmental samples from endemic regions. Collectively, we show that metals in drinking water, even at safe levels, can impede kidney development at an early age, potentiating increased susceptibility to other agrochemicals such as glyphosate. Drinking water contaminant effects on mitochondria can further contribute to progression of kidney dysfunction and our mitochondrial assay may help identify regions at risk of CKDu.
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http://dx.doi.org/10.1016/j.envint.2020.106019DOI Listing
November 2020

A tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in .

Elife 2020 04 27;9. Epub 2020 Apr 27.

Department of Biological Sciences, University of Southern California, Los Angeles, United States.

piRNAs play a critical role in the regulation of transposons and other germline genes. In , regulation of piRNA target genes is mediated by the complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the complex.
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http://dx.doi.org/10.7554/eLife.56731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255803PMC
April 2020

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR.

BMC Microbiol 2019 07 30;19(1):175. Epub 2019 Jul 30.

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada.

Background: Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed "cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145).

Results: Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison.

Conclusions: Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.
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http://dx.doi.org/10.1186/s12866-019-1541-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668150PMC
July 2019

Accumulation of Microcystin-LR in Grains of Two Rice Varieties () and a Leafy Vegetable, .

Toxins (Basel) 2019 07 24;11(8). Epub 2019 Jul 24.

Center for Water Quality and Algae Research, Department of Zoology, University of Sri Jayewardenepura, Gangodawila, Nugegoda 10250, Sri Lanka.

The potential transfer of microcystin-LR (MC-LR) to humans via crop plants irrigated with MC-contaminated water is causing serious concern. In this study, two variants, a hybrid (BG358), a traditional (Suwandel) variety, and a leafy green vegetable crop, , were exposed under laboratory conditions to natural blooms of sampled from a hypereutrophic lake contaminated with MC-LR (3,197.37 ± 1.04 µg/L). Field samples of and were collected from farmlands that had been irrigated from a reservoir, containing MC-LR (180 µg/L). MC-LR was quantified by high performance liquid chromatography followed by photodiode-array detection (HPLC-PDA). From the laboratory study, we calculated the potential human health exposure from BG358, Suwandel and as 2.84 ± 0.01, 0.22 ± 0.01, and 0.06 ± 0.01 µg/kg of body weight/day, respectively, whereas the potential health exposures from BG358, Suwandel and collected from the field were 0.10 ± 0.01, 0.009 ± 0.005, and 0.03 ± 0.01 µg/kg of body weight/day, respectively. In certain instances, the results exceeded the World Health Organization's (WHO) tolerable daily intake of MC-LR, posing a potential health risk to humans. Thus, our results emphasize the importance of continuous screening programs for cyanotoxins in edible plants in the future to prevent the consumption of contaminated crops.
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http://dx.doi.org/10.3390/toxins11080432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722703PMC
July 2019

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli.

BMC Microbiol 2019 05 14;19(1):93. Epub 2019 May 14.

Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2, Canada.

Background: Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient.

Results: In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system.

Conclusions: Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.
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http://dx.doi.org/10.1186/s12866-019-1463-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515682PMC
May 2019

Antidotal efficacies of the cyanide antidote candidate dimethyl trisulfide alone and in combination with cobinamide derivatives.

Toxicol Mech Methods 2019 Jul 6;29(6):438-444. Epub 2019 May 6.

e US Army Medical Research Institute of Chemical Defense , APG , MD , USA.

Formulation optimization and antidotal combination therapy are the two important tools to enhance the antidotal protection of the cyanide (CN) antidote dimethyl trisulfide (DMTS). The focus of this study is to demonstrate how the formulation with polysorbate 80 (Poly80), an excipient used in pharmaceutical technology, and the combinations with other CN antidotes having different mechanisms of action enhance the antidotal efficacy of the unformulated (neat) DMTS. The LD for CN was determined by the statistical Dixon up-and-down method on mice. Antidotal efficacy was expressed as antidotal potency ratio (APR). CN was injected subcutaneously one minute prior to the antidotes' injection intramuscularly. The APR values of 1.17 (dose: 25 mg/kg bodyweight) and 1.45 (dose: 50 mg/kg bodyweight) of the neat DMTS were significantly enhanced by the Poly80 formulation at both investigated doses to 2.03 and 2.33, respectively. The combination partners for the Poly80 formulated DMTS (DMTS-Poly80; 25 and 50 mg/kg bodyweight) were 4-nitrocobinamide (4NCbi) (20 mg/kg bodyweight) and aquohydroxocobinamide (AHCbi; 50, 100, and 250 mg/kg bodyweight). When DMTS-Poly80 (25 and 50 mg/kg bodyweight; APR = 2.03 and 2.33, respectively) was combined with 4NCbi (20 mg/kg bodyweight; APR = 1.35), significant increase in the APR values were noted at both DMTS doses (APR = 2.38 and 3.12, respectively). AHCbi enhanced the APR of DMTS-Poly80 (100 mg/kg bodyweight; APR = 3.29) significantly only at the dose of 250 mg/kg bodyweight (APR = 5.86). These studies provided evidence for the importance of the formulation with Poly80 and the combinations with cobinamide derivatives with different mechanisms of action for DMTS as a CN antidote candidate.
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http://dx.doi.org/10.1080/15376516.2019.1585504DOI Listing
July 2019

Distinct regions of the intrinsically disordered protein MUT-16 mediate assembly of a small RNA amplification complex and promote phase separation of Mutator foci.

PLoS Genet 2018 07 23;14(7):e1007542. Epub 2018 Jul 23.

Department of Biological Sciences, University of Southern California, Los Angeles, California, United States of America.

In C. elegans, efficient RNA silencing requires small RNA amplification mediated by RNA-dependent RNA polymerases (RdRPs). RRF-1, an RdRP, and other Mutator complex proteins localize to Mutator foci, which are perinuclear germline foci that associate with nuclear pores and P granules to facilitate small RNA amplification. The Mutator complex protein MUT-16 is critical for Mutator foci assembly. By analyzing small deletions of MUT-16, we identify specific regions of the protein that recruit other Mutator complex components and demonstrate that it acts as a scaffolding protein. We further determine that the C-terminal region of MUT-16, a portion of which contains predicted intrinsic disorder, is necessary and sufficient to promote Mutator foci formation. Finally, we establish that MUT-16 foci have many properties consistent with a phase-separated condensate and propose that Mutator foci form through liquid-liquid phase separation of MUT-16. P granules, which contain additional RNA silencing proteins, have previously been shown to have liquid-like properties. Thus, RNA silencing in C. elegans germ cells may rely on multiple phase-separated compartments through which sorting, processing, and silencing of mRNAs occurs.
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http://dx.doi.org/10.1371/journal.pgen.1007542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072111PMC
July 2018

Monitoring food pathogens: Novel instrumentation for cassette PCR testing.

PLoS One 2018 10;13(5):e0197100. Epub 2018 May 10.

Division of Physical Medicine & Rehabilitation, Department of Medicine, University of Alberta, 5-005 Katz Group Centre for Pharmacy and Health Research, Edmonton, AB, Canada.

In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197100PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945031PMC
July 2018

Effective use of iron-aluminum rich laterite based soil mixture for treatment of landfill leachate.

Waste Manag 2018 Apr 12;74:347-361. Epub 2018 Jan 12.

University of Sri Jayewardenepura, Faculty of Applied Science, Department of Forestry and Environmental Science, Gangodawila, Nugegoda, Sri Lanka. Electronic address:

Landfill leachate poses environmental threats worldwide and causes severe issues on adjacent water bodies and soil by direct discharge. The primary objective of this study is to analyze the efficient use of compost and laterite mixtures (0, 10, 20, 30 and 40 wt% compost/laterite) on leachate treatment and to investigate the associated removal efficiencies under different sorption processes. Therefore, in the experimental design, laterite is used for providing adsorption characteristics, and compost for activating biological properties of the filter. The filtering process is continued until major physical changes occur in the filter at approximately 100 days. The raw leachate used for the experiment shows higher average values for many analyzed parameters. Parameters for the experiment are selected based on their availability in raw leachate in the Sri Lanka. During filtering, removal efficiencies of BOD (>90%), COD (>85%), phosphate (>90%) and nitrate (75-95%) show higher values for all filters. These removals are mainly associated with biodegradation, which is activated by the added compost. Perhaps the removal of nitrate steadily increases with time, which indicates in denitrification by the added excess carbon from the leachate. The removal of total suspended solids (TSS) is moderate to high, but conversely, the electric conductivity (EC) is unsteady, indicating an association between iron exchange and carbonate degradation. A very high removal efficiency is reported in Fe (90-100%), and wide ranges of efficiencies in Mn (30-90%), Cu (45-85%), Ni (30-93%), Cd (37-98%), Zn (15-98%), and Pb (35-98%) involve heterogeneous sorption processes. Furthermore, the normalization of raw leachate by the liquid filtrate has apparent improvements. The differences (p > .05) in removal efficiencies between the filters are significant. It can be concluded that the filter with laterite mixed with 20% of compost has the optimum conditions. Further, the Fourier-transforminfrared (FT-IR) models for filter media conclude multiple sorptions and reveal evidence on vacant sites. X-ray diffraction (XRD) analyses indicate secondary minerals gibbsite, hematite, goethite and kaolinite as the major minerals that involved on the sorption process.
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http://dx.doi.org/10.1016/j.wasman.2018.01.013DOI Listing
April 2018

The host effects of Gambusia affinis with an antibiotic-disrupted microbiome.

Comp Biochem Physiol C Toxicol Pharmacol 2015 Dec 18;178:163-168. Epub 2015 Oct 18.

Department of Biological Sciences, Sam Houston State University, Huntsville, TX, USA. Electronic address:

While serving as critical tools against bacterial infections, antimicrobial therapies can also result in serious side effects, such as antibiotic-associated entercolitis. Recent studies utilizing next generation sequencing to generate community 16S gene profiles have shown that antibiotics can strongly alter community composition and deplete diversity. However, how these community changes in the microbiota are related to the host side effects is still unclear. We have used the freshwater Western mosquitofish (Gambusia affinis) as a tractable vertebrate model system to study host effects following exposure to a broad spectrum antibiotic, rifampicin. After 3days of exposure, the bacterial communities of the mucosal skin and gut microbiomes lost diversity and shifted composition. Compared to unexposed controls, treated fish were more susceptible to a specific pathogen, Edwardsiella ictaluri, yet displayed no survival differences when subjected to a polymicrobial water challenge of soil or feces. Treated fish were more susceptible to osmotic stress from NaCl, but not to the toxin nitrate. Treated fish failed to gain weight as well as controls over one month when fed a matched diet. Because of small sample sizes, pathogen susceptibility and weight gain differences were not statistically significant. This study provides supporting evidence in an experimental laboratory system that an antibiotic can have significant and persistent negative host effects, and provides for future study into the mechanisms of these effects.
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http://dx.doi.org/10.1016/j.cbpc.2015.10.004DOI Listing
December 2015

Miniaturized technology for DNA typing: cassette PCR.

Methods Mol Biol 2015 ;1310:175-91

Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Avenue, Edmonton, AB, Canada, T6G 1Z2,

With the smaller size, low cost, and rapid testing capabilities, miniaturized lab-on-a-chip devices can change the way medical diagnostics are currently performed in the health-care system. We have demonstrated such a device that is self-contained, simple, disposable, and inexpensive. It is capable of performing DNA amplification on an inexpensive instrument suitable for near point of care settings. This technology will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices. Our device, a gel capillary cassette, termed cassette PCR, contains capillary reaction units each holding a defined primer set, with arrays of capillary reaction units for simultaneously detecting multiple targets. With the exception of the sample to be tested, each capillary reaction unit holds all the reagents needed for PCR in a desiccated form that can be stored at room temperature for up to 3 months and even longer in colder conditions. It relies on capillary forces for sample delivery of microliter volumes through capillaries, hence avoiding the need for pumps or valves. In the assembled cassette, the wax architecture supporting the capillaries melts during the PCR and acts as a vapor barrier as well as segregating capillaries with different primer sets. No other chip sealing techniques are required. Cassette PCR accepts raw samples such as urine, genital swabs, and blood. The cassette is made with off-the-shelf components and contains integrated positive and negative controls.
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http://dx.doi.org/10.1007/978-1-4939-2690-9_15DOI Listing
February 2016

Spatial and vertical distributions of sedimentary halogenated polycyclic aromatic hydrocarbons in moderately polluted areas of Asia.

Environ Pollut 2015 Jan;196:331-40

Faculty of Agriculture, Meijo University, 1-501 Shiogamaguchi, Nagoya 468-8502, Japan.

The sedimentary halogenated (chlorinated and brominated) polycyclic aromatic hydrocarbons (Cl/BrPAHs), PAHs, and elements were analyzed to investigate contamination processes and sources. Assessments were conducted in sediments from three sites: surface sediments from the Yellow Sea and sediment cores from Kandy Lake and Negombo Lagoon, Sri Lanka. Most of ClPAHs targeted were detected in all sediments. Spatial distributions of total ClPAH concentrations in the Yellow Sea showed the presence of multiple hot spots that differed from those of total PAHs. In Kandy and Negombo sediments,total ClPAH concentrations were slightly higher in surface layers than in bottom layers; the opposite trend was observed for PAHs. Principal component analysis showed that the clusters of most ClPAHs were similar to those of anthropogenically derived elements, but were far from those of PAHs. Consequently, ClPAHs in sediments appear to be persistent contaminants, which may make them appropriate as indicators of anthropogenic sources.
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http://dx.doi.org/10.1016/j.envpol.2014.10.028DOI Listing
January 2015

Genotyping single nucleotide polymorphisms in human genomic DNA with an automated and self-contained PCR cassette.

J Mol Diagn 2014 Sep 2;16(5):550-557. Epub 2014 Jul 2.

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta. Electronic address:

Point-of-care devices can lower costs through reduced reagent costs, shifting diagnostics from centralized laboratories to local clinics or hospitals, rapidly informing on the spot medical decision making, and enabling personalized treatment options. We have previously described a self-contained miniaturized device that uses an array of gel-based reaction units that can simultaneously detect multiple biomarkers and/or multiple patients in one PCR cassette and can be stored for up to 7 months. In this article, we document the ability of cassette PCR to detect single nucleotide polymorphisms (SNPs) in human genomic DNA from buccal swabs. Swab processing takes 8 minutes, and PCR is completed in just more than an hour. To demonstrate potential for genotyping, we used allele-specific PCR and melt curve analysis to detect major and minor alleles of two SNPs in the fibroblast growth factor receptor 2 gene (FGFR2) that are linked with breast cancer. After allele-specific PCR, seamless melt curve analysis and the presence or absence of melt peaks from melt curve analysis identifies the FGFR2 SNP genotypes for each patient. The near point-of-care/point-of-need genotyping methods reported here can be applied for detecting and assessing risks of diseases such as cancer and to detect SNPs that alter drug metabolism and hence response to therapy.
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http://dx.doi.org/10.1016/j.jmoldx.2014.04.004DOI Listing
September 2014

A lab-on-chip for malaria diagnosis and surveillance.

Malar J 2014 May 9;13:179. Epub 2014 May 9.

School of Public Health, University of Alberta, WMC 2B4,59, 8440 - 112th Street, Edmonton, AB, Canada.

Background: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.

Methods: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection.

Results: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/μL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%.

Conclusions: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.
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http://dx.doi.org/10.1186/1475-2875-13-179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029813PMC
May 2014

A novel method for sample delivery and testing of whole blood: gel strip PCR for point of care (POC) molecular diagnostics.

Lab Chip 2013 Oct 22;13(20):4011-4. Epub 2013 Aug 22.

Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada.

Testing of whole blood in miniaturized PCR is compromised by the opaque nature of whole blood that leads to physical masking of a fluorescent signal. We demonstrate a method to perform real-time PCR with whole blood that avoids interference from the opacity of whole blood.
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http://dx.doi.org/10.1039/c3lc50755fDOI Listing
October 2013

Storing self-contained gel capillary cassettes for POC medical diagnostics.

Lab Chip 2013 Oct 22;13(20):4087-95. Epub 2013 Aug 22.

Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada.

For effective clinical uptake of the lab on a chip/point of care technology (LOC-POC), in addition to cost advantages LOC-POC devices should offer multiple patient screening panels for related diseases as well as cold-chain transportation and storage abilities. We recently described a device that performs polymerase chain reaction (PCR) to simultaneously screen raw clinical samples from up to 16 patients for multiple infectious agents (Manage et al., Lab Chip, 2013, 13, 2576-2584). This cassette contains glass capillaries with desiccated semi-solid acrylamide gels that include all the reagents except for the sample, with integrated quality control. Here we report the development of protocols to store assembled PCR cassettes at room temperature, 4 °C or -20 °C as well as at +40 °C. We show that our cassettes are stable, with no loss of activity for at least 3 months at RT and at least 7 months at 4 °C and -20 °C. However, the activity of desiccated cassettes degrades when stored for more than 2 weeks at 40 °C, insufficient time for post-manufacture delivery and use of cassette PCR. To address this, we have evaluated two stage storage protocols. PCR cassettes can initially be stored at 4 °C and -20 °C for prolonged periods of time and removed for shorter term storage at RT, retaining activity for at least a month, which would facilitate transport to remote areas for testing. Effective use of cassette PCR in high temperature regions of the world, for experimental purposes defined here as 40 °C, appears to be feasible only after a first stage storage in the cold, followed by no more than 1 week at 40 °C. This should allow sufficient time for delivery by the manufacturer to a central area well served by power and refrigeration, for later ambient temperature transport and use in under-resourced areas that lack refrigeration.
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http://dx.doi.org/10.1039/c3lc50655jDOI Listing
October 2013

Confidence limit calculation for antidotal potency ratio derived from lethal dose 50.

World J Methodol 2013 Mar 26;3(1):7-10. Epub 2013 Mar 26.

Ananda Manage, Department of Mathematics and Statistics, Sam Houston State University, Huntsville, TX 77341, United States.

Aim: To describe confidence interval calculation for antidotal potency ratios using bootstrap method.

Methods: We can easily adapt the nonparametric bootstrap method which was invented by Efron to construct confidence intervals in such situations like this. The bootstrap method is a resampling method in which the bootstrap samples are obtained by resampling from the original sample.

Results: The described confidence interval calculation using bootstrap method does not require the sampling distribution antidotal potency ratio. This can serve as a substantial help for toxicologists, who are directed to employ the Dixon up-and-down method with the application of lower number of animals to determine lethal dose 50 values for characterizing the investigated toxic molecules and eventually for characterizing the antidotal protections by the test antidotal systems.

Conclusion: The described method can serve as a useful tool in various other applications. Simplicity of the method makes it easier to do the calculation using most of the programming software packages.
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http://dx.doi.org/10.5662/wjm.v3.i1.7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145567PMC
March 2013

An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics.

Lab Chip 2013 Jul 7;13(13):2576-84. Epub 2013 Mar 7.

Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada.

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis (MCA). To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma urealyticum (UU) and Mycoplasma homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50 min. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for some clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices.
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http://dx.doi.org/10.1039/c3lc41419aDOI Listing
July 2013

Millimeter scale separation of DNA with a replaceable polymer matrix.

Electrophoresis 2012 Nov 2;33(21):3213-21. Epub 2012 Oct 2.

Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada.

Electrophoresis is a powerful method that has seen a wide range of applications, often in automated genetic diagnostic instruments that require the use of a replaceable sieving matrix. The power and simplicity of electrophoresis as an analysis technique would be ideal for highly integrated and low-cost analysis systems if the method could be implemented in microfluidics on the scale of several mm. We demonstrate the electrophoretic analysis of DNA with separation lengths as small as 2 mm and with a resolution adequate for the analysis of PCR products, i.e. resolutions of 10-20 base pairs. Such small-scale separations enable analysis systems consisting of microfluidics and microelectronics integrated into a single inexpensive package, thereby overcoming a key challenge facing the development of the lab on chip technologies.
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http://dx.doi.org/10.1002/elps.201200188DOI Listing
November 2012

Microfluidic approach to genotyping human platelet antigens.

IET Nanobiotechnol 2012 Jun;6(2):33-9

Canadian Blood Services, Research and Development, Edmonton, Canada.

Centralised laboratories routinely determine blood types by serological and molecular methods. Current practices have limitations in terms of cost, time and accessibility. Miniaturised microfluidic platforms offer an alternative to conventional genotyping methods, since they consume fewer reagents, provide faster analysis and allow for complete integration and automation. As these 'lab-on-a-chip' devices have been used for bacterial and viral detection, the authors investigated blood group genotyping as a novel application of microfluidic technology. To demonstrate the feasibility of microfluidic chip-based genotyping, the authors compared human platelet antigen 1 (HPA-1) genotype results from conventional and chip-based analysis for 19 blood donor specimens. DNA purification was performed with ChargeSwitch™ magnetic beads, DNA amplification (PCR), restriction length polymorphism (RFLP) and capillary electrophoresis (CE) for identification of the DNA on microfluidic chips. It was found that nine donors were HPA-1a/1a and ten were HPA-1a/1b. Concordance between the conventional and on-chip methods was achieved for all but one sample. All the steps were demonstrated for complete blood group genotyping analysis of patient whole blood specimens on separate microfluidic chips. Future work will focus on integration of all the genotyping protocols on a single microfluidic chip.
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http://dx.doi.org/10.1049/iet-nbt.2011.0044DOI Listing
June 2012

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.

Lab Chip 2012 May 19;12(9):1664-71. Epub 2012 Mar 19.

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, AB, Canada.

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 μL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.
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http://dx.doi.org/10.1039/c2lc40061hDOI Listing
May 2012

How well do multiple testing methods scale up when both n and k increase?

J Biopharm Stat 2011 Jul;21(4):583-94

Department of ISQS, Texas Tech University, Lubbock, TX 794049-2101, USA.

With increasingly massive data sets in biopharmaceutical research, particularly in genomic and related applications, there is concern about how well multiple comparisons methods "scale up" with increasing number of tests (k). Familywise error rate-controlling methods do not scale up well, and false discovery rate-controlling methods do scale up well with increasing k. But neither method scales up well with increasing sample size (n) when testing point nulls. We develop a loss function approach to investigate scale-up properties of various methods; we find that while Efron's recent proposal scales up best when both sample size n and number of tests k increase, but its performance otherwise can be erratic.
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http://dx.doi.org/10.1080/10543406.2011.551326DOI Listing
July 2011

Novel bacterial strains for the removal of microcystins from drinking water.

Water Sci Technol 2011 ;63(6):1137-42

Institute for Innovation, Design, and Sustainability Research, Robert Gordon University, Aberdeen, AB25 1HG, UK.

Microcystins (MC) and nodularin (NOD) are common contaminants of drinking water around the world and due to their significant health impact it is important to explore suitable approaches for their removal. Unfortunately, these toxins are not always removed by conventional water treatments. One of the most exciting areas that hold promise for a successful and cost effective solution is bioremediation of microcystins. Recent work resulted in successful isolation and characterisation of 10 novel bacterial strains (Rhodococcus sp., Arthrobacter spp. and Brevibacterium sp.) capable of metabolizing microcystin-LR (MC-LR) in a Biolog MT2 assay. The work presented here aims to further investigate and evaluate the metabolism and the degradation of multiple microcystins (MC-LR, MC-LF, MC-LY, MC-LW and MC-RR) and nodularin by the bacterial isolates. A total of five bacterial isolates representing the three genera were evaluated using Biolog MT2 assay with a range of MCs where they all demonstrated an overall metabolism on all MCs and NOD. Subsequently, the results were confirmed by observing the degradation of the range of toxins in a separate batch experiment.
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http://dx.doi.org/10.2166/wst.2011.352DOI Listing
June 2011
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