Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the ...development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.
•Aging-associated inflammation by TNF-α plays an important role in the development of platelet hyperreactivity during aging.•Aging-associated platelet hyperreactivity is associated with megakaryocytic inflammatory, metabolic, and mitochondrial reprogramming.
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Two commonly used methods for cyanotoxin analysis are enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each method has its advantages and ...disadvantages, and discrepancies are commonly observed between the two methods due to various factors including the ELISA antibody cross-reacting to different cyanotoxin congeners. However, reliable cyanotoxin monitoring methods and accurate interpretation of results are needed for water utilities to guide recreational water planning and drinking water treatment operations. In this study, we explored an innovative “effective concentration-equivalent concentration” (EC-EQ) approach to improve the interpretation of ELISA results and the comparison to LC-MS/MS results. The precision of ELISA results was first improved by reporting the sample ECs and EQs derived from their ELISA dose curves. Concentrations of each cyanotoxin as measured by LC-MS/MS were then combined with their respective ELISA cross-reactivities to calculate their theoretical ELISA responses. Finally, instead of comparing the results from the two methods directly, the equivalent concentration based on one single reference cyanotoxin was used for reporting and comparison. This integrated mass balance-based approach provides a more reliable interpretation of results by considering the reactivity differences between toxins as well as their mixture effects. This approach has been successfully applied to microcystin (one main group of cyanotoxins) standard mixtures and cyanobacterial bloom samples to interpret and compare their ELISA and LC-MS/MS detection results. The study provides guidance to utilities on how to obtain more accurate cyanotoxin monitoring results and better understand the discrepancy between the two methods.
Assessing the presence of human pathogenic Cryptosporidium oocysts in surface water remains a significant water treatment and public health challenge. Most drinking water suppliers rely on fecal ...indicators, such as the well-established Escherichia coli (E. coli), to avoid costly Cryptosporidium assays. However, the use of E. coli has significant limitations in predicting the concentration, the removal and the transport of Cryptosporidium. This study presents a meta-analysis of E. coli to Cryptosporidium concentration paired ratios to compare their complex relationships in eight municipal wastewater sources, five agricultural fecal pollution sources and at 13 drinking water intakes (DWI) to a risk threshold based on US Environmental Protection Agency (USEPA) regulations. Ratios lower than the USEPA risk threshold suggested higher concentrations of oocysts in relation to E. coli concentrations, revealing an underestimed risk for Cryptosporidium based on E. coli measurements. In raw sewage (RS), high ratios proved E. coli (or fecal coliforms) concentrations were a conservative indicator of Cryptosporidium concentrations, which was also typically true for secondary treated wastewater (TWW). Removals of fecal indicator bacteria (FIB) and parasites were quantified in WWTPs and their differences are put forward as a plausible explanation of the sporadic ratio shift. Ratios measured from agricultural runoff surface water were typically lower than the USEPA risk threshold and within the range of risk misinterpretation. Indeed, heavy precipitation events in the agricultural watershed led to high oocyst concentrations but not to E. coli or enterococci concentrations. More importantly, ratios established in variously impacted DWI from 13 Canadian drinking water plants were found to be related to dominant fecal pollution sources, namely municipal sewage. In most cases, when DWIs were mainly influenced by municipal sewage, E. coli or fecal coliforms concentrations agreed with Cryptosporidium concentrations as estimated by the meta-analysis, but when DWIs were influenced by agricultural runoff or wildlife, there was a poor relationship. Average recovery values were available for 6 out of 22 Cryptosporidium concentration data sets and concomitant analysis demonstrated no changes in trends, with and without correction. Nevertheless, recovery assays performed along with every oocyst count would have enhanced the precision of this work. Based on our findings, the use of annual averages of E. coli concentrations as a surrogate for Cryptosporidium concentrations can result in an inaccurate estimate of the Cryptosporidium risk for agriculture impacted drinking water intakes or for intakes with more distant wastewater sources. Studies of upstream fecal pollution sources are recommended for drinking water suppliers to improve their interpretation of source water quality data.
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•High Escherichia coli/Cryptosporidium ratios for sewage show E. coli was a suitable indicator.•Low E. coli/Cryptosporidium ratios for rural runoff show E. coli was a poor indicator.•E. coli explains Cryptosporidium concentrations at DWI impacted by municipal sewage.•E. coli was not a good indicator of Cryptosporidium for agriculture impacted water.
Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell ...culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.
Solar irradiation of aqueous solutions containing free available chlorine (FAC) dramatically enhances inactivation of Cryptosporidium parvum oocysts compared to FAC or sunlight alone. In pH 8, 10 mM ...phosphate buffer at 25 °C, exposure to FAC alone yields no oocyst inactivation at CT FAC ≤ 832 (mg min) L–1, while exposure to simulated sunlight alone for 60 min yields <0.5 log inactivation. In contrast, exposure to simulated sunlight for 60 min in the presence of FAC0 = 8 mg L–1 as Cl2 results in photolytic decomposition of FAC to ∼1 mg L–1 as Cl2 yielding CT FAC ∼ 200 (mg min) L–1 accompanied by >2 log oocyst inactivation. Similar enhancement effects are observed in natural water under natural sunlight. Experiments undertaken in the presence of the reactive oxygen species (ROS) scavenger tert-butanol or in the absence of oxygen indicate that these enhancements are due to in situ ROS and ozone production via FAC photolysis.
Most library-dependent bacterial source tracking studies using
Escherichia coli (
E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library ...development. In contrast, this study evaluated the genotype variation of
E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for
E. coli, yielding a total of 631
E. coli isolates (1–12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product–moment correlation coefficient. The 555
E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2–5 isolates each. Using ERIC-PCR, the 555
E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the
E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted
E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.
Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and ...bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations
Variabilities in commercial cyanotoxin standards Jia, Ai; Prescott, Matthew D.; Guo, Yingbo C. ...
AWWA water science,
March/April 2023, 2023-03-00, 20230301, Volume:
5, Issue:
2
Journal Article
Peer reviewed
Cyanotoxin standards are commercially available from various suppliers. To investigate the potential impact of different sources on the comparability of cyanotoxin monitoring results among different ...methods and studies, this study evaluated the quality of 86 cyanotoxin standards from nine vendors via enzyme‐linked immunosorbent assay and liquid chromatography–tandem mass spectrometry. Substantial variabilities between vendors (up to 60%) and between lots (up to 98%) were observed for most of the standards. In addition, some of the microcystin standards had up to 11% of other microcystins as impurities. Compared to non‐certified analytical standards, certified standards showed much better agreement, with standards variations below 15% for all tested toxins. This study highlights the importance of developing and applying unified and certified standards for cyanotoxin analysis to improve the consistency and comparability of results. If different sources/lots of standards are used, they need to be cross‐checked to evaluate the potential impacts on results.
Two commonly used methods for cyanotoxin analysis are enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC–MS/MS). Two rounds of interlaboratory ...comparisons of ELISA and LC–MS/MS analyses were conducted with 12 participating laboratories to evaluate method performances in various matrices, including cyanobacterial bloom and drinking water samples. Fifteen cyanotoxins, including 12 microcystin variants, nodularin, anatoxin‐a, and cylindrospermopsin were evaluated. The impact of sample matrices, preservatives, and quenching reagents was assessed, and no substantial effects were observed. Overall, comparable results were obtained among laboratories performing ELISA and LC–MS/MS analyses, respectively. ELISA results for fortified samples matched more closely with those from LC–MS/MS when microcystin cross‐reactivities were considered, providing data 26% closer to theoretical values on average. This study demonstrates that understanding the effect of cross‐reactivities when comparing ELISA and LC–MS/MS results and considering potential variabilities in commercial standards is important when interpreting data from these two methods.
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