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In this work, a rapid and simplified method for extracting SARS-CoV-2 RNA from whole wastewater using a magnetic beads-based protocol is presented. The described method involves the centrifugation of a 50-mL aliquot of raw wastewater influent for 5 min to obtain a 500-μL pellet, which is eluted with 2 mL of a Tween®20-based elution buffer; 1 mL of the elute is extracted for RNA using a direct magnetic bead-based extraction method. RNA recovery was examined in several bench-scale experiments using heat-inactivated SARS-CoV-2 (HI-SCV-2) spiked into raw wastewater to assess the effects of different solids pellet : buffer ratios, inhibition mitigation strategies, and varying levels of total suspended solids. When the method was assessed using an influent wastewater sample known to contain SARS-CoV-2, the viral signal was detected in all five biological replicates, whereas direct extraction of 1-mL aliquots of the raw wastewater resulted in a 40% viral detection rate. The experimental method limit of detection (MLOD) using HI-SCV-2 spiked into raw wastewater was 50 GU mL−1 with a 95% limit of detection. Using the described protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from wastewater treatment facilities (WWTFs) in Atlantic Canada over a period of 15 weeks during the rise and fall of a COVID-19 outbreak. This method is effective and rapid and could provide potential application for laboratories with limited resources. Of approximately 50 methods that have been developed for measuring SARS-CoV-2 in wastewater referenced in the literature, this is the first to advance a robust magnetic beads-based RNA extraction technique from whole wastewater without extensive sample pre-treatment. The novel application of this method in the rapid extraction of SARS-CoV-2 RNA from municipal wastewater is an indispensable tool to potentially understand COVID-19 infection occurrence within communities.

Wastewater-based surveillance methods have been implemented in several countries as a tool for monitoring SARS-CoV-2 at a community scale. A variety of methods have been used for concentrating, extracting, and detecting the virus, with no clear consensus on the most effective approach. In this note, we report preliminary findings from a study that is tracking SARS-CoV-2 in wastewater in Halifax, Nova Scotia, with a specific focus on the use of four reverse transcriptase quantitative PCR (RT-qPCR) assays for detecting the virus in wastewater. We were able to detect the virus in wastewater samples during the initial rise of cases in the Halifax region in early November 2020. Levels of the targeted SARS-CoV-2 gene fragments increased and fell in response to reported cases of COVID-19. The CDC N1 and E RT-qPCR assays demonstrated greater relative sensitivity than the CDC N2 and N3 assays for detection of SARS-CoV-2 in raw sewage samples.

Objective Agglomeration is a common problem facing the preparation and application of nanomaterials, and whether nano-hydroxyapatite (nano HA) can modulate oral microecology left to be unclear. In this study, nano HA was disaggregated by sodium hexametaphosphate (SHMP) and ultrasonic cavitation to observe whether agglomeration would affect its effect on oral bacterial biofilm. Methods Dynamic light scattering (DLS) and scanning electronic microscope (SEM) were used to observe the treatment solutions. Single-species biofilms and multi-species biofilms were treated with 10% nano HA, 10% disaggregated nano HA, 10% micro hydroxyapatite (micro HA) and deionized water (DDW) for 30 min and analyzed via MTT assay, lactic acid measurement, SEM and confocal laser scanning microscope (CLSM). Real-time polymerase chain reaction was performed to analyze the biofilm composition. Results Ultrasonic cavitation combined with SHMP could significantly reduce the degree of agglomeration of nano HA. Disaggregated nano HA could inhibit bacterial growth and reduce the ability of bacterial biofilm to produce lactic acid and extracellular polysaccharides. There was no significant difference on composition of multi-species biofilms between nano HA and disaggregated nano HA. Significance The disaggregated nano-hydroxyapatite could inhibit the metabolism and acid production of oral bacterial biofilm, but did not significantly affect the composition of multi-species biofilms.

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

Novel approaches for monitoring coastal water quality changes and identifying associated contaminant source(s) are of growing importance as climate change and population redistribution to coastal zones continue to impact coastal systems. CrAssphage, a virus found in the human gut and shed with fecal matter, is currently gaining popularity as an indicator of human fecal contamination in surface water and groundwater. Here we demonstrate that DNA assays targeting crAssphage genetic fragments can be used to detect pollution from nearshore onsite wastewater treatment systems discharging to the ocean via submarine groundwater discharge. We integrated this novel viral monitoring tool into a field study that characterized the physical hydrogeology (hydraulic gradients, hydraulic conductivity, and seepage fluxes) and surface water and groundwater quality at a study site on the north shore of Nova Scotia, Canada. Increased use of onsite wastewater treatment systems during the summer cottage season coincided with widespread detections of crAssphage in submarine groundwater discharge (4/4 samples) and coastal surface waters (3/8 samples). Conversely, classical fecal pollution indicators based on bacterial targets (Escherichia coli and human-specific Bacteroidales genetic marker (HF183)) were sparsely detected in the samples in the coastal environment (2/12 E. coli samples, 0/12 HF183 samples), likely due to greater attenuation of bacterial contaminants within the subsurface environments. Results from this first application of crAssphage in coastal groundwater contribute to a growing body of research reporting the application of this emerging tracer in various environments impacted by sewage pollution sources.

The use of magnetic beads in the extraction of nucleic acids from wastewater is presented as an approach to simplify extraction techniques for the detection of SARS-CoV-2 viral fragments in wastewater. In particular, this paper describes the development and optimization of a direct method for extracting SARS-CoV-2 RNA from municipal wastewater using magnetic beads. The recovery efficiency of the method using Accuplex SARS-CoV-2 Positive Reference Material (ASCV-2) was examined. Method factors assessed were sample volume, concentration of magnetic bead mix, elution temperature, and water matrix (deionized (DI) water and wastewater). The combination of optimized method parameters that resulted in the highest RNA recovery in both DI water (26.0 ± 0.8%) and wastewater (11.8 ± 1.4%) was a sample volume of 1.0 mL, a magnetic beads concentration of 100 µL mL-1 sample, and an elution temperature of 60 °C. The performance of this optimized method was further assessed in recovery experiments using wastewater samples spiked at 1.8×106 and 1.8×104 gene copies L-1 (GU L-1) with Gamma Inactivated SARS-COV-2 (GI-SCV-2) and 1.0×106 and 1.0×104 infectious units L-1 of Human Coronavirus 229E (HCV 229E) as viral surrogates. Recoveries of 86.1 and 4.6% were achieved for wastewater samples spiked with GI-SCV-2 at low and high concentrations, respectively. In assessing the effects of wastewater pre-filtration and addition of DL-Dithiothreitol (DTT, used to inactivate RNases that may degrade RNA) on recovery efficiency of ASCV-2, the magnetic bead-based extraction protocol performed optimally with unfiltered wastewater without DTT (recovery = 17.4 ± 0.4%). The method limit of detection (MLOD) for ASCV-2 recovered from pre-filtered wastewater was determined to be 4.6×104 GU L-1 (95% degree of confidence). Using this optimized magnetic bead-based extraction protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from sewershed locations in Atlantic Canada. This emerging RNA extraction method is direct, rapid, and does not require the use of specialized equipment, thus offering advantageous application for laboratories with limited resources. As such, this method is an indispensable tool in the monitoring of wastewater for SARS-CoV-2 to potentially understand COVID-19 infection occurrence within communities and inform public health leaders.Graphical Abstract

Quaternary ammonium methacrylates (QAMs) are useful antimicrobial compounds against oral bacteria. Here, we investigated the effects of two QAMs, dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM), on biofilm formation, survival and development of tolerance by biofilm, and survival and development of tolerance against QAMs after prolonged starvation. Enterococcus faecalis (E. faecalis), Streptococcus gordonii (S. gordonii), Lactobacillus acidophilus (L. acidophilus), and Actinomyces naeslundii (A. naeslundii) were used. Minimum inhibitory concentration (MIC) of QAMs against multispecies biofilm was determined. Biofilm formed under sub-MIC was observed by crystal violet staining and confocal laser scanning microscopy (CLSM). Metabolic activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactic acid production measurement. Development of tolerance was determined by MIC values before and after exposure to QAMs or after prolonged starvation. It was found that E. faecalis and S. gordonii could survive and form biofilm under sub-MIC of QAMs. Lactic acid production from biofilms formed under sub-MIC was significantly higher than control specimens ( p < 0.05 ). The exposure to sub-MIC of QAMs promoted biofilm formation, and prolonged starvation or prolonged contact with sub-MIC helped bacteria develop tolerance against killing by QAMs.

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.

The ability of lateral flow sand filters, used as on-site wastewater treatment systems (OWTS), to remove antibiotic resistance genes (ARGs), antibiotic resistant bacteria (ARB), and other relevant genetic markers (HF183, 16S rRNA, and int1) was assessed. Municipal wastewater was settled in a septic tank prior to loading into six pilot-scale lateral flow sand filters comprised of three different sand media types, at 5 and 30% slopes. The sand filters were sampled bi-weekly for: 9 ARGs and 3 other complimentary gene markers (sul1, sul2, qnrS, tetO, ermB, blaTEM, blaCTX-M, mecA, vanA, int1, HF183, 16S rRNA), and conventional microbial and water quality indicators, from July to November in 2017, and four times in the summer of 2018. The sand filters were observed to attenuate 7 of the ARGs to mostly below 2 log gene copies per mL. Log reductions ranging from 2.9 to 5.4 log were observed for the removal of absolute abundances of ARGs from septic tank effluent in 5 of the 6 sand filters. The fine-grained filter on the 5% slope did not perform as well for ARG attenuation due to hydraulic failure. The apportionment of cell-associated versus cell-free DNA was determined for the gene markers and this indicated that the genes were primarily carried intracellularly. Average log reductions of ARB with resistance to either sulfamethoxazole, erythromycin, or tetracycline were approximately 2.3 log CFU per mL within the filters compared to the septic tank effluent. This field study provides in-depth insights into the attenuation of ARB, ARGs, and their genetic compartmentalization in variably saturated sand OWTS. Overall, this type of OWTS was found to pose little risk of antimicrobial resistance contamination spread into surrounding environments when proper hydraulic function was maintained.

This paper first describes the characteristics of VR technology and its realization in art design. Secondly, this paper analyzes the practical significance of VR technology in Environmental art design(EDA), and finally discusses the practical application of VR (hereinafter referred to as VR) technology in EAD. In a word, the application of VR technology in EAD (hereinafter referred to as EAD) is helpful to the development and improvement of EAD.

Persisting apical periodontitis is a primary reason for multiple intervention in root canal. Persisting bacteria in root canal is related with the persisting infection. Despite the advancement in treatment strategies the persisting infection is a major challenge for endodontist. Here we tested two newly developed quaternary ammonium methacrylates (QAMs) against endodontic bacteria and their biofilms. Their antibacterial and antibiofilm efficiency were compared with chlorhexidine (CHX) and sodium hypochlorite (NaOCl). We measured the MIC, MBC and MBIC of DMADDM and DMAHDM respectively. We also detected the ratio of live/dead bacteria and bacterial composition in the biofilms treated by DMADDM and DMAHDM. We found that DMADDM and DMAHDM could inhibit the growth of bacteria and biofilms formation. The result showed that novel QAMs were remarkably efficient than CHX against biofilms. In addition, we found that Streptococcus gordonii (S. gordonii) and Enterococcus faecalis (E. faecalis) were frequent isolates after treatment with antimicrobial compounds.

To investigate the antibacterial effects of two newly developed quaternary ammonium salts (QAMs)-dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM), in the presence of various root canal inhibitors. Streptococcus gordonii, Enterococcus faecalis, Lactobacillus acidophilus and Actinomyces naeslundii were used. Dentine, dentine matrix and dead bacteria were selected as inhibitors. The antimicrobial efficacy of monomers of DMADDM and DMAHDM was tested against suspensions formed by mixtures of four bacterial species in or without the presence of inhibitors. The inhibition results were compared with chlorhexidine (CHX) and sodium hypochlorite (NaOCl). One-way analyses of variance (ANOVA) followed by Tukey&rsquo, s multiple comparison test was performed to determine significant differences. The antibacterial effects of DMADDM and DMAHDM were variably inhibited dentine, dentine matrix and dead bacteria. CHX and NaOCl showed substantivity and they inhibited bacteria present in suspension. The concentration of compound decreased in the dentine block due to constant release. Bacterial colonies on the dentine surface and dentine tubules were significantly inhibited by DMADDM and DMAHDM. The antibacterial effects of DMADDM and DMAHDM could be inactivated by dentine, dentine matrix and dead bacteria, while DMADDM and DMAHDM could inhibit bacteria colonization on the dentine surface and kill bacteria present in dentinal tubules. The antibacterial effects of DMADDM and DMAHDM as free monomers in the presence of inhibitors was evaluated for the first time. They could help inhibit the residual bacteria on the dentine surface and in dentinal tubules that may cause persisting infection. Therefore the novel QAMs showed great potentials as root canal medication.

To realize the noncontact gap measurement based on eddy current effect when the object is not conductive, we fabricate a film made of conductive polymer composite and cover it on the surface of the nonconductive object. The experimental results on the lift-off effect show that the impedance of the coil with alternating current changes regularly with the variations in the distance between the coil and the composite object. This phenomenon indicates that there exists an eddy current composed of the tunneling current and the conduction current in the composite object. The results indicate that conductive polymer composite has the potential to replace metal as an object of an eddy current sensor system.

Drinking water in the vast Arctic Canadian territory of Nunavut is sourced from surface water lakes or rivers and transferred to man-made or natural reservoirs. The raw water is at a minimum treated by chlorination and distributed to customers either by trucks delivering to a water storage tank inside buildings or through a piped distribution system. The objective of this study was to characterize the chemical and microbial drinking water quality from source to tap in three hamlets (Coral Harbour, Pond Inlet and Pangnirtung-each has a population of2000) on trucked service, and in Iqaluit (population ~6700), which uses a combination of trucked and piped water conveyance. Generally, the source and drinking water was of satisfactory microbial quality, containing Escherichia coli levels of1 MPN/100 mL with a few exceptions, and selected pathogenic bacteria and parasites were below detection limits using quantitative polymerase chain reaction (qPCR) methods. Tap water in households receiving trucked water contained less than the recommended 0.2 mg/L of free chlorine, while piped drinking water in Iqaluit complied with Health Canada guidelines for residual chlorine (i.e.0.2 mg/L free chlorine). Some buildings in the four communities contained manganese (Mn), copper (Cu), iron (Fe) and/or lead (Pb) concentrations above Health Canada guideline values for the aesthetic (Mn, Cu and Fe) and health (Pb) objectives. Corrosion of components of the drinking water distribution system (household storage tanks, premise plumbing) could be contributing to Pb, Cu and Fe levels, as the source water in three of the four communities had low alkalinity. The results point to the need for robust disinfection, which may include secondary disinfection or point-of-use disinfection, to prevent microbial risks in drinking water tanks in buildings and ultimately at the tap.

Wastewater stabilization ponds (WSPs) are commonly used to treat municipal wastewater in Arctic Canada. The biological treatment in the WSPs is strongly influenced by climatic conditions. Currently, there is limited information about the removal of fecal and pathogenic bacteria during the short cool summer treatment season. With relevance to public health, the objectives of this paper were to determine if treatment in arctic WSPs resulted in the disinfection (i.e., removal of fecal indicator bacteria, Escherichia coli) and removal of selected human bacterial pathogens from the treated effluent. The treatment performance, with focus on microbial removal, was assessed for the one-cell WSP in Pond Inlet (Nunavut [NU]) and two-cell WSP in Clyde River (NU) over three consecutive (2012–2014) summer treatment seasons (late June-early September). The WSPs provided a primary disinfection treatment of the wastewater with a 2–3 Log removal of generic indicator E. coli. The bacterial pathogens Salmonella spp., pathogenic E. coli, and Listeria monocytogenes, but not Campylobacter spp. and Helicobacter pylori, were detected in the untreated and treated wastewater, indicating that human pathogens were not reliably removed. Seasonal and annual variations in temperature significantly (p

This research aimed to determine whether the SigB (σ(B)) regulon and osmolytes impact the survival of the foodborne pathogen, Listeria monocytogenes, during desiccation in simulated food soils with varying salt and nutrient contents on food grade stainless steel (SS) surfaces. L. monocytogenes 568 (Lm568, serotype 1/2a), its isogenic sigB mutant (ΔsigB) and the back-complemented ΔsigB were desiccated in BHI, TSB with 1% glucose (TSB-glu), peptone physiological saline (PPS) and minimal media (MM) for 21 days at 43% relative humidity (RH) and 15 °C on SS. The effect of food related osmolytes (proline, betaine and carnitine) on desiccation survival was studied by (a) pre-culturing strains in MM with an osmolyte followed by desiccation in MM and (b) by desiccating strains in MM with an osmolyte. Desiccation survival of L. monocytogenes was positively correlated to the nutrient and osmolyte concentrations in the desiccation substrates. Initial Lm568 levels of 8 Log(CFU/cm(2)) decreased by 0.9 Log(CFU/cm(2)) in BHI and 1.1-2.9 Log(CFU/cm(2)) in TSB-glu, PPS and MM after 21 days. Comparatively, the initial survival of ΔsigB was reduced in PS and MM, while no differences were observed among the three strains in BHI and TSB-glu. Pre-culture in osmolyte containing MM enhanced (p < 0.05) desiccation survival of all strains. Desiccation in osmolyte-containing MM improved desiccation survival of all strains, albeit the protection was less than that observed after pre-culture with the osmolytes. Complementation of the ΔsigB mutant restored the wildtype phenotype. In conclusion, this work shows the protective effect of osmolytes in desiccation survival of L. monocytogenes, while the σ(B) regulon only improved the initial survival in nutrient and osmolyte poor environments.

Insulin stimulates glucose uptake in association with phosphatidylinositol (PI) 3-kinase activation mechanisms in rat adipocytes. Insulin stimulated glucose uptake to 6.5-fold, and 12-o-tetradecanoyl phorbol 13-acetate (TPA) also stimulated glucose uptake to 4.5-fold in rat adipocytes. We examined these differences in glucose uptake, PKCzeta activation, and PI 3-kinase activation after stimulation with insulin and TPA. TPA stimulated PI 3-kinase activity and increased the p85 subunit of PI 3-kinase immunoreactivity in anti-phosphotyrosine antibody-immunoprecipitated protein. Insulin and TPA provoked increases in membrane PKCzeta immunoreactivity. The PI 3-kinase inhibitor, wortmannin, suppressed insulin-induced increases in glucose uptake, PI 3-kinase activity, and PKCzeta activation. Wortmannin also suppressed TPA-induced PI 3-kinase activity and PKCzeta activation but suppressed TPA-induced glucose uptake to only a small extent. The PKC inhibitor, Go6976, which only inhibits conventional PKCalpha and _, suppressed TPA-induced glucose uptake, but suppressed insulin-induced glucose uptake to only a small extent. On the other hand, the PKC inhibitor, RO32-0432, which inhibits conventional, novel, and atypical PKCs, markedly suppressed both insulin- and TPA-induced glucose uptake. These results suggest that insulin-induced glucose uptake is mainly mediated by PI 3-kinase-PKCzeta signaling, whereas phorbol ester-induced glucose uptake is mainly mediated by conventional PKC despite PI 3-kinase and PKCzeta activations.

Vitamin E, an antioxidant, improves insulin sensitivity through the suppression of conventional PKC in vascular smooth muscle cells. It has been reported that vitamin E reduces platelet aggregation through the suppression of PKC alpha and beta (Diabetes 47 (1998) 1494). On the other hand, 1 alpha,25-dihydroxy vitamin D3 (1,25D3) activates conventional PKC and may subsequently cause insulin resistance. Against this background, we examined the effect of vitamin E and 1,25D3 on PKC beta and PKC zeta/lambda activities in vitro and 10 nM insulin-induced glucose uptake in rat adipocytes. In vitro PKC beta activity of adipocytes was slightly decreased by the addition of 1 microM vitamin E, but not PKC zeta/lambda activity. In contrast, a 10-1000 nM 1,25D3 dose responsively activated PKC beta activity of adipocytes (ED 50%, 10 nM), but not PKC zeta/lambda activity. Pretreatment with 1 microM vitamin E for 60 min did not improve the insulin-induced glucose uptake. On the other hand, pretreatment with a 10-1000 nM 1,25D3 dose responsively suppressed insulin-induced glucose uptake. Moreover, 1,25D3 increased membrane-associated PKC beta immunoreactivity for 60 min, but no additional increase in membrane-associated PKC beta immunoreactivity during treatment with insulin was observed. These results suggest that 1,25D3 reduces insulin-induced glucose uptake via activation of PKC beta, but not vitamin E in rat adipocytes.

Conductive polymer composite, which can be used as the piezoresistive material of a flexible pressure sensor, has viscoelasticity. Therefore, the piezoresistivity is time dependent, introducing detrimental effects for sensor development. To reduce the time dependence, we designed a piezoresistive element with a novel structure called a ?nonalignment electrodes element (NAEE)?, which is different from a traditional sandwich element (TSE). The experimental data under constant pressure show that the attenuation of the output resistance of a NAEE is much lower than that of a TSE, and the tend-to-stable-time (i.e. the shortest time needed to ensure that the output resistance of the element goes to a stable level) of a NAEE is far less than that of a TSE. The results verify the feasibility to reduce the time dependence substantially by using a NAEE.