Proliferation and anatoxin production of benthic cyanobacteria associated with canine mortalities along a stream-lake continuum.

Proliferations of benthic cyanobacteria are increasingly in the public eye, with rising animal deaths associated with benthic rather than planktonic blooms. In early June 2021, two dogs died after consuming material on the shore of Shubenacadie Grand Lake, Nova Scotia. Preliminary investigations indicated anatoxins produced by benthic cyanobacterial mats were responsible for the deaths. In this study, we monitored the growth of a toxic benthic cyanobacterial species (Microcoleus sp.) along a stream-lake continuum where the canine poisonings occurred. We found that the species was able to proliferate in both lentic and lotic environments, but temporal growth dynamics and the predominant sub-species were influenced by habitat type, and differed with hydrodynamic setting, nutrient and sunlight availability. Toxin concentration was greatest in cyanobacterial mats growing in the oligotrophic lakeshore environment (maximum measured total anatoxins (ATXs) >20 mg·kg-1 wet weight). This corresponded with a shift in the profile of ATX analogues, which also indicated changing sub-species dominance along the stream-lake transition.

Disease Course, Treatments, and Outcomes of Children With Systemic Juvenile Idiopathic Arthritis-Associated Lung Disease.

OBJECTIVE: Systemic juvenile idiopathic arthritis-associated lung disease (SJIA-LD) is a life-threatening disease complication. Key questions remain regarding clinical course and optimal treatment approaches. The objectives of the study were to detail management strategies after SJIA-LD detection, characterize overall disease courses, and measure long-term outcomes. METHODS: This was a prospective cohort study. Clinical data were abstracted from the electronic medical record, including current clinical status and changes since diagnosis. Serum biomarkers were determined and correlated with presence of LD. RESULTS: We enrolled 41 patients with SJIA-LD, 85% with at least one episode of macrophage activation syndrome and 41% with adverse reactions to a biologic. Although 93% of patients were alive at last follow-up (median 2.9 years), 37% progressed to requiring chronic oxygen or other ventilator support, and 65% of patients had abnormal overnight oximetry studies, which changed over time. Eighty-four percent of patients carried the HLA-DRB1*15 haplotype, significantly more than patients without LD. Patients with SJIA-LD also showed markedly elevated serum interleukin-18 (IL-18), variable C-X-C motif chemokine ligand 9 (CXCL9), and significantly elevated matrix metalloproteinase 7. Treatment strategies showed variable use of anti-IL-1/6 biologics and addition of other immunomodulatory treatments and lung-directed therapies. We found a broad range of current clinical status independent of time from diagnosis or continued biologic treatment. Multidomain measures of change showed imaging features were the least likely to improve with time. CONCLUSION: Patients with SJIA-LD had highly varied courses, with lower mortality than previously reported but frequent hypoxia and requirement for respiratory support. Treatment strategies were highly varied, highlighting an urgent need for focused clinical trials.

Effects of Bio-Aging on Mechanical Properties and Microbial Behavior of Different Resin Composites.

Under challenging oral environments, the overall performance of resin composites is affected by bio-aging. This study investigated the effects of saliva biofilm-induced bio-aging on the mechanical properties and microbial behavior of composites with different filler types. Microhybrid, nanohybrid, nano-filled and nano-filled flowable composites were bio-aged with saliva biofilm for 30 days. Surface morphology, roughness, mechanical and aesthetic properties were determined. A 48 h saliva biofilm model was used to evaluate the microbial behavior of different composites in vitro. Biofilm metabolic activity, lactic acid production and live/dead bacterial staining were tested. Six volunteers were selected to wear intra-oral appliances with composite slabs for 24 h and biofilms were collected and analyzed using 16S rRNA sequencing to assess the biofilm formation over those materials in situ. Although there were increasing trends, surface roughness, water resorption and material solubility had no significant changes for all groups after bio-aging (p > 0.05). There were no significant changes in elastic modulus for all groups after aging (p > 0.05). However, a decrease in flexural strength in all groups was observed (p < 0.05), except for the nanoflow composite group (p > 0.05). The Vickers hardness remained stable in all groups after aging (p > 0.05), except for the nano-filled group (p < 0.05). The nanoflow composite showed distinct color changes compared to the micro-hybrid group after aging (p < 0.05). Biofilm metabolic activity and lactic acid production in vitro increased slightly after bio-aging in all groups, but with no statistical significance (p > 0.05). The Shannon index diversity of biofilms in situ decreased after aging (p < 0.05), while no significant difference was shown in species composition at the genus level in all groups (p > 0.05). Resin composites with different sized fillers displayed a relatively stable mechanical performance and uncompromised microbial behavior both in vitro and in situ after 30 days of bio-aging. Based on the results, composites with different filler types can be selected flexibly according to clinical needs. However, a longer time for bio-aging is still needed to confirm the mechanical properties and microbial behaviors of composites in the long run.

Biodegradation of 1,4-dioxane by a native digestate microbial community under different electron accepting conditions.

The potential of a native digestate microbial community for 1,4-dioxane (DX) biodegradation was evaluated under low dissolved oxygen (DO) concentrations (1-3 mg/L) under different conditions in terms of electron acceptors, co-substrates, co-contaminants and temperature. Complete DX biodegradation (detection limit of 0.01 mg/L) of initial 25 mg/L was achieved in 119 days under low DO concentrations, while complete biodegradation happened faster at 91 and 77 days, respectively in nitrate-amended and aerated conditions. In addition, conducting biodegradation at 30 ˚C showed that the time required for complete DX biodegradation in unamended flasks reduced from 119 days in ambient condition (20-25 °C) to 84 days. Oxalic acid, which is a common metabolite of DX biodegradation was identified in the flasks under different treatments including unamended, nitrate-amended and aerated conditions. Furthermore, transition of the microbial community was monitored during the DX biodegradation period. While the overall richness and diversity of the microbial community decreased, several families of known DX-degrading bacteria such as Pseudonocardiaceae, Xanthobacteraceae and Chitinophagaceae were able to maintain and grow in different electron-accepting conditions. The results suggested that DX biodegradation under low DO concentrations, where no external aeration was provided, is possible by the digestate microbial community, which can be helpful to the ongoing research for DX bioremediation and natural attenuation.

Fate and distribution of determinants of antimicrobial resistance in lateral flow sand filters used for treatment of domestic wastewater.

Residuals of antimicrobial products from anthropogenic uses can create a selective environment in domestic wastewater treatment systems and receiving environments and contribute to the spread of antimicrobial resistance (AMR). On-site wastewater treatment systems are widely used for domestic wastewater management in rural and remote regions, but the fate of determinants of AMR in these types of environments has received little attention. In this study, the mechanisms responsible for the attenuation of determinants of AMR in lateral flow sand filters were explored using a combination of lab, field and modeling investigations. The degradation kinetics and adsorption potential in the sand filter medium of three antibiotic resistance genes (ARGs; sul1, tetO, and ermB) and culturable bacteria resistant to sulfamethoxazole, tetracycline, and erythromycin were measured using lab experiments. The spatial distribution of ARGs and antibiotic resistant bacteria were also assessed in field scale sand filters, and mechanistic modeling was conducted to characterize filtration processes. The results indicated that the primary mechanisms responsible for AMR attenuation within the sand filters were degradation and filtration. The spatial distribution of AMR determinants illustrated that attenuation was occurring along the entire length of each filter. This study provides new insights on primary mechanisms of AMR attenuation in on-site wastewater treatment systems and supports the use of conservative design guidelines and separation distances for reducing AMR transmission.

Starvation Survival and Biofilm Formation under Subminimum Inhibitory Concentration of QAMs.

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.

Research progress on nano-calcium phosphate modified dental materials for the prevention and treatment of dental pulp diseases / 口腔疾病防治

@#Nanomaterials usually refer to tiny particles with a diameter of 1-100 nm, which often have unique physicochemical properties and are one of the main areas of research interest for development of dental biomaterials. Nano-calcium phosphate modified dental materials have been widely used in pit and fissure sealing, dental resin restoration, tooth adhesion, and root canal sealing. The current research shows that the dental material modified by nano-calcium phosphate has stronger mechanical properties and shows long-term calcium and phosphorus ion release and excellent ion recharging ability, which can promote the remineralization of tooth hard tissue and has good prospects for application. However, it is difficult to accurately simulate the complex environment of the oral cavity. Therefore, the biocompatibility, cytotoxicity and effect of clinical application of nano-calcium phosphate modified dental materials still needs further study. This review summarizes and discusses the recent research progress regarding nano-calcium phosphate modified dental materials in the prevention and treatment of dental pulp diseases.

The effect of disaggregated nano-hydroxyapatite on oral biofilm in vitro.

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 30min 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.

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae.

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 ß-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a ß-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 inhibitory effect of quaternary ammonium salt on bacteria in root canal.

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.

Lateral flow sand filters are effective for removal of antibiotic resistance genes from domestic wastewater.

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.

The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry.

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.

Chemical and microbial characteristics of municipal drinking water supply systems in the Canadian Arctic.

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 of <2000) 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 of <1 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.

Disinfection and removal of human pathogenic bacteria in arctic waste stabilization ponds.

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 < 0.05) affected the disinfection efficiency. Improved disinfection and pathogen removal was observed for the two-cell system in Clyde River as compared to the one-cell system in Pond Inlet. A quantitative microbial risk assessment should be performed to determine if the release of low levels of human pathogens into the arctic environment poses a human health risk.

Role of sigB and osmolytes in desiccation survival of Listeria monocytogenes in simulated food soils on the surface of food grade stainless steel.

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.

Dominant negative protein kinase Cbeta improves 1alpha, 25-dihydroxy vitamin D3-induced insulin resistance.

1alpha,25-Dihydroxy vitamin D3 (1,25D3) activates conventional PKC and may subsequently lead to insulin resistance. Previous studies from our laboratory have shown that pretreatment with 10 nM-10 microM 1,25D3 dose-responsively suppressed insulin-induced glucose. To assess PKC(beta)-mediated inhibition of insulin-induced glucose uptake in rat adipocytes, we preincubated with Go6976 and LY379196, conventional PKC inhibitors, and found they abolished the 1,25D3-mediated inhibitory effect on insulin-induced 2-deoxyglucose (DOG) uptake. Moreover, the inhibitory effect of 1,25D3 on insulin-induced DOG uptake was abrogated in adipocytes overexpressed with dominant negative PKC(beta), but not in those overexpressed with wild type PKC(beta). These results suggest that 1,25D3 reduces insulin-induced glucose uptake via activation of PKC(beta) in rat adipocytes.

Effect of 1 alpha,25-dihydroxy vitamin D3 and vitamin E on insulin-induced glucose uptake in rat adipocytes.

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.