Hot water systems are the most frequent environment associated with the prevalence and growth of opportunistic premise plumbing pathogens (OPPPs). Previous studies identified water heaters as a source of waterborne diseases and concluded that design variables may contribute to their prevalence. A multifaceted approach was used to investigate the vertical stratification of the microbiome and selected OPPPs in an electric water heater tank connected to a simulated home plumbing system. Results show that the microbiome is highly diverse with evidence of temperature stratification and temporal structuring influenced by the partial drainage of the water tank. Representatives of the Mycobacterium spp. were the most prevalent taxa, followed by Legionella spp., and a relatively low prevalence of free-living amoeba Vermamoeba vermiformis. Higher concentrations of Legionella pneumophila at the bottom of the tank indicated the potential growth and protection of this opportunistic pathogen at this location. Overall, partial drainage of the water tank (60% of the tank capacity) did not significantly mitigate the microbiome and selected OPPPs. The outcome of this study sheds light on the role of vertical stratification on water quality, and demonstrates the resilience of the microbial community residing in an electric water heater tank and the implications for public health.

The biological treatment process consisting of an aerated contactor and filter is effective for groundwaters containing elevated ammonia and other reduced contaminants, including iron, manganese, arsenic, and methane. Depth profiles characterizing microbial activity across aerated contactors are lacking. A 1-year pilot study comparing gravel-and ceramic-packed contactors was conducted, and media depth profile samples were collected at the conclusion of the study. Media and water samples also were collected from pilot-scale aerated contactors at 4 other water systems. Water quality, media surface metals concentrations, and a suite of biofilm parameters were analyzed. Media surface metals concentrations were greatest at the influent end. ATP concentrations, extracellular polymeric substances, and extracellular enzyme activities tended to be similar across depth. Bacteria and functional genes involved in contaminant oxidation co-occurred and tended to decrease across depth, but were not correlated to the media metals concentration. Microbial community composition changed with depth, and the diversity either decreased or remained similar. The microbial activity profiles through aerated contactors differed from what is typically reported for groundwater biofilters, suggesting that the different reactor flow and dissolved oxygen profiles impacted the microbial community.

Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFβ/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.

Aerobic biotreatment systems can treat multiple reduced inorganic contaminants in groundwater, including ammonia (NH3), arsenic (As), iron (Fe), and manganese (Mn). While individual systems treating multiple contaminants simultaneously have been characterized and several systems treating one contaminant have been compared, a comparison of systems treating co-occurring contaminants is lacking. This study assessed the treatment performance and microbial communities within 7 pilot- and full-scale groundwater biotreatment systems in the United States that treated waters with pH 5.6-7.8, 0.1-2.0 mg/L dissolved oxygen, 75-376 mg CaCO3/L alkalinity, < 0.03-3.79 mg NH3-N/L, < 4-31 mu g As/L, < 0.01-9.37 mg Fe/L, 2-1220 mu g Mn/L, and 0.1-5.6 mg/L total organic carbon (TOC). Different reactor configurations and media types were represented, allowing for a broad assessment of linkages between water quality and microbial communities via microscopy, biofilm quantification, and molecular methods. Influent NH3, TOC, and pH contributed to differences in the microbial communities. Mn oxidase gene copy numbers were slightly negatively correlated with the influent Mn concentration, but no significant relationships between gene copy number and influent concentration were observed for the other contaminants. Extracellular enzyme activities, community composition, and carbon transformation pathways suggested heterotrophic bacteria may be important in nitrifying biofilters. Aerobic groundwater biofilters are complex, and improved understanding could lead to engineering enhancements.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings' plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water.

The United States (US) Environmental Protection Agency (EPA) included strontium (Sr) in the Contaminant Candidate List 3 as a chemical for potential future regulatory consideration.

Existing heterotrophic denitrification reactors rely on microorganisms to consume dissolved oxygen (DO) and create conditions suitable for denitrification, but this practice leads to excessive microbial growth and increased organic carbon doses. An innovative reactor that uses nitrogen gas sparging through a contactor to strip DO was developed and tested in the lab. It reduced influent nitrate from 15 to <1 mg/L as N with nitrite accumulation <1 mg/L as N. It maintained a consistent flow rate and developed minimal headloss, making it easier to operate than the denitrifying dual-media filter that was operated in parallel. Gravel, polyvinyl chloride pieces, and no packing media were assessed as options for the nitrogen-sparged contactor, and gravel was found to support denitrification at the highest loading rate and was resilient to nitrogen-sparging shutoffs and intermittent operation. This innovative reactor appears promising for small drinking water systems.

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3. One-Sentence Summary NLRP3 senses disruptions in endosome trafficking to trigger the formation of an inflammasome complex and initiate an inflammatory response.

Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. ABSTRACT Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as Aminicenantes, Atribacteria, Chloroflexi, and Actinobacteria within bacteria and Thermoprofundales within archaea. MAGs from these taxa have been obtained previously from nonpermafrost environments and have been suggested to show adaptations to long-term energy starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had greater proportions in the Clusters of Orthologous Groups (COGs) categories of energy production and conversion and carbohydrate transport and metabolism than did their nonpermafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost genomes. Many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of high enough quality to assemble MAGs from microorganisms with adaptations to survive long-term freezing in this extreme environment. IMPORTANCE Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. The fate of permafrost microorganisms is connected to these predicted rapid environmental changes. Studying ancient permafrost with culture-independent techniques can give a glimpse into how these microorganisms function under these extreme low-temperature and low-energy conditions. This will facilitate understanding how they will change with the environment. This study presents genomic data from this unique environment ∼20,000 to 1,000,000 years of age.

Orthophosphate (PO4) addition is a common corrosion control treatment used to lower lead (Pb) concentrations at the consumer's tap by forming relatively insoluble Pb-phosphate (Pb-PO4) minerals. However, some Pb-PO4 particles that can form in drinking water are mobile nanoparticles (i.e., 0.001-0.1 µm) that have the potential to reach the tap. Point-of-use (POU) or pitcher filters are often used to manage risks during distribution system upsets, when corrosion control treatment is not optimized, or following Pb service line replacements. To abide by industry convention, POU and pitcher filters must be NSF/ANSI-certified for Pb reduction (NSF/ANSI-53) using a test water containing dissolved Pb and large Pb particles. Certification for particulates reduction (NSF/ANSI-42) is done using a test water that contains particles, but not leaded particles. To address the lack of testing for Pb nanoparticles, this study challenged six certified commercially available faucet-mounted POU (3) and pitcher (3) filters with aqueous suspensions of Pb-PO4 nanoparticle. For the water quality investigated, the Pb particles formed ranged between 0.016 and 0.098 µm, based on scanning electron microscopy, transmission electron microscopy, and dynamic light scattering analysis. These particles represented 98.5% of total Pb in suspension. The total Pb removals were between 44.6 and 65.1% for the POU filters, and between 10.9 and 92.9% for the pitcher filters. The electron microscopy results confirm that Pb-PO4 nanoparticles passed through the filters. The findings can inform future efforts to re-examine the test waters used in the certification challenge tests.

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1β release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K+ efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors.

Following a pH reduction in their drinking water over a span of more than 20 years, the City of Newark, New Jersey, has struggled with elevated lead (Pb) release from Pb service lines and domestic plumbing in the zone fed by the Pequannock Water Treatment Plant. In response, Newark initiated orthophosphate addition and provided faucet-mounted point-of-use (POU) filters and pitcher filters certified for Pb and particulate reduction under NSF/ANSI Standards 53 and 42 to residential homes in that zone. Water chemistry analysis and size fractionation sampling were performed at four of these houses. Analysis of the particulate material retained by the fractionation filters revealed that Pb was dominantly present in the water as fine Pb(II) orthophosphate particles. A considerable amount of the particulates occurred as a nanoscale fraction that sometimes passed through the POU faucet or pitcher filtration units. Scanning electron microscopy, transmission electron microscopy, and energy-dispersive spectroscopy analyses showed that the nanoparticles (<100 nm) and their aggregates were composed of Pb, phosphorus, and chlorine, which are consistent with pyromorphite, Pb5(PO4)3Cl. Electron diffraction and X-ray analyses supported the presence of hydroxypyromorphite and chloropyromorphite nanoparticles and the size range estimates from the imaging. This research confirmed that nonadherent Pb(II)-orthophosphate nanoparticles were an important form of Pb in drinking water in the Pequannock water quality zone of Newark.

Although not regulated in United States drinking water, ammonia has the potential to increase chlorine consumption and cause nitrification problems in the distribution system. Many groundwaters with elevated ammonia are also contaminated with other inorganic analytes such as arsenic, iron, and manganese, all of which have primary or secondary maximum contaminant levels (MCLs). The objective of this work was to demonstrate the effectiveness of an innovative biological treatment process to simultaneously remove ammonia (2.9 mg N per L), arsenic (23 μg L-1), iron (2.9 mg L-1) and manganese (80 μg L-1) from a groundwater source in Iowa. The biological treatment system consisted of an "aeration contactor" followed by a conventional granular media filter. Orthophosphate was also added, as a biological nutrient, at 0.3 mg PO4 per L. Ammonia, manganese, and iron were consistently reduced through the pilot system by 98 to 99%. Complete oxidation of ammonia to nitrate was observed (i.e., no nitrite was released) and arsenic was consistently removed to below the 10 μg L-1 MCL. Ammonia was oxidized by ammonia and nitrite oxidizing bacteria and arsenic by bacteria which converted As(III) in the source water to more readily removable As(V). Iron was presumably oxidized by oxygen during aeration although some biologically assisted oxidation could not be ruled out. As(V) bound iron particles were removed in the filter resulting in effective arsenic (and iron) reduction. A surprising treatment benefit was the effective manganese reduction, the mechanism of which was not so clear, but was attributed to biologically assisted oxidation of Mn(II). While some system acclimation time was necessary to achieve desired ammonia and manganese reductions, acceptable arsenic and iron reductions were observed shortly after start-up.

There is great interest in producing nanoparticles for various applications. The objective of this work was to develop a procedure for reproducibly creating stable lead (Pb) phosphate nanoparticle aqueous suspensions. A stable 5 mg/L Pb-phosphate nanoparticle "stock" suspension was synthesized via chemical precipitation of Pb and orthophosphate in water at pH 7.5 with 4.4 mg soluble PO4/L and 7 mg C/L dissolved inorganic carbon. The stock suspension was subsequently diluted to produce stable 0.10 mg/L Pb "challenge" water suspensions without compromising the nanoparticle size, structure, mineralogy and solubility. Specifically, the hexagonal hydroxypyromorphite nanoparticles had an average diameter of 38 nm based on transmission electron microscopy analysis and an associated Pb solubility of 0.001 mg/L. The properties of the stock suspensions were not impacted by further dilutions, and the challenge water suspensions remained stable for 24 hours. In the context of drinking water, a protocol to produce such a stable Pb nanoparticle challenge water suspension would be very useful in evaluating Pb bioavailability, identifying Pb remediation strategies, and testing filter effectiveness to remove Pb from water.

The US Environmental Protection Agency's Office of Research and Development designed, fabricated, and installed four pipe rigs in Flint, Mich., to help the city optimize corrosion control. The lead service line (LSL) pipe loops were constructed of polyvinyl chloride pipe and fittings and welded steel channel frames. Each pipe rig consisted of four approximately 4 ft‐long sections of 0.75 in. inside diameter lead pipe that were excavated from homes fed by the Flint distribution system. The rigs were operated on a set daily on/off schedule such that a specified amount of water passed through each pipe using a solenoid valve‐operated timer system. Solenoid problems resulted in sporadic and enhanced daily flow volume (but no flow rate changes) that prevented restabilization of pipe scales. Lead levels were relatively sporadic and statistically different during this period. After the solenoids were replaced, the lead pipe rigs were successfully conditioned, and lead release with consistent influent water chemistry became relatively stable. Average lead levels across all 16 loops ranged between approximately 2 and 5 μg/L after the solenoid replacement. The lead results were consistent with levels measured from LSLs from homes in the city during sequential sampling efforts.

ABSTRACT The benefits of using transgenic switchgrass with decreased levels of caffeic acid 3- O -methyltransferase (COMT) as biomass feedstock have been clearly demonstrated. However, its effect on the soil microbial community has not been assessed. Here we report metagenomic and metatranscriptomic analyses of root-associated soil from COMT switchgrass compared with nontransgenic counterparts.

ABSTRACT Microbial release of greenhouse gases from thawing permafrost is a global concern. Seventy-six metagenomes were generated from low-soil-organic-carbon mineral cryosols from Axel Heiberg Island, Nunavut, Canada, during a controlled thawing experiment. Permafrost thawing resulted in an increase in anaerobic fermenters and sulfate-reducing bacteria but not methanogens.

The objective of this work was to demonstrate the effectiveness of an innovative and simple biological water treatment approach for removing 3.3 mg nitrogen (N) per litre ammonia and iron from water using a pilot study conducted at a utility in Iowa. Biological water treatment can be an effective approach to addressing low ammonia levels; however, providing adequate oxygen to address source water ammonia levels greater than 1.5 mg/L N is challenging. The treatment consisted of a biological “contactor” combined with aeration, followed in series with a dual granular media filter. Orthophosphate was necessary to satisfy biological nutrient requirements, and maintaining near‐saturated dissolved oxygen levels throughout the contactor was important. The pilot project demonstrated that a contactor and filter operated at loading rates of 2.2 gpm/sq ft and 2.0 gpm/sq ft, respectively, met desired finished water quality objectives. The contactor alone was able to completely biologically oxidize all of the ammonia to nitrate. Contactor maintenance was minimal.