Impacts of caffeine on fathead minnow behaviour and physiology.

Pollution from regularly used substances such as pharmaceuticals, cleaning agents, and even food and beverages is an increasing problem in the environment. Caffeine, a commonly ingested stimulant, is one such contaminant that has been detected in aquatic environments worldwide. Yet, little is known about how ecologically relevant concentrations of caffeine influence the morphology, behaviour, and physiology of exposed organisms. To address this knowledge gap, we exposed fathead minnow (Pimephales promelas) to three caffeine treatments: a freshwater control (nominal: 0 ng/L), a low (nominal: 1,000 ng/L) and high environmentally relevant dose (nominal: 10,000 ng/L), for 35 days. We tested the learning abilities, anxiety, metabolic rates, and morphological features of exposed vs. control fish. Caffeine exposure did not affect the ability of fish to learn but did influence anxiety levels. Over the course of repeated anxiety testing, unexposed control fish visited a black square more often while fish exposed to low levels of caffeine did not, potentially indicating that these fish remained in a more anxious state. While caffeine did not impact metabolism, fish growth, or body size, it was associated with lower liver investment-although this response was only observed in our low caffeine treatment. Overall, our results suggest that even relatively low concentrations of caffeine may impact the liver size and anxiety of exposed fish, but further research is needed to assess how extended exposure to caffeine impacts fitness. Given the increase in anthropogenic contaminants in aquatic environments, it is important that we continue to investigate their effects on the organisms exposed to them.

Venlafaxine exposure alters mitochondrial respiration and mitomiR abundance in zebrafish brains.

Wastewater treatment plant (WWTP) effluent often releases pharmaceuticals like venlafaxine (a serotonin-norephinephrine reuptake inhibitor antidepressant) to freshwater ecosystems at levels causing adverse metabolic effects on fish. Changes to fish metabolism can be regulated by epigenetic mechanisms like microRNA (small RNA molecules that regulate mRNA translation), including regulating mitochondrial mRNAs. Nuclear-encoded microRNAs regulate mitochondrial gene expression in mammals, and have predicted effects in fish. We aimed to identify whether venlafaxine exposure changed mitochondrial respiration and resulted in differentially abundant mitochondrial microRNA (mitomiRs) in zebrafish brains. In vitro exposure of brain homogenate to below environmentally relevant concentrations of venlafaxine (<1 µg/L) caused a decrease in mitochondrial respiration, although this was not driven by changes to mitochondrial Complex I or II function. To identify whether these effects occur in vivo, zebrafish were exposed to 1 µg/L venlafaxine for 0, 1, 6, 12, 24, and 96 h. In vivo, venlafaxine exposure had no significant effects on brain mitochondrial respiration; however, select mitomiRs (dre-miR-301a-5p, dre-miR-301b-3p, and dre-miR-301c-3p) were also measured, because they were bioinformatically predicted to regulate mitochondrial cytochrome c oxidase subunit I (COI) abundance. These mitomiRs were differentially regulated based on venlafaxine exposure (with miR-301c-3p abundance differing during the day and miR-301b-3p being lower in exposed fish at night), and with respect to sex and time sampled. Overall, the results demonstrated that in vitro venlafaxine exposure to zebrafish brain caused a decrease in mitochondrial respiration, but these effects were not seen after acute in vivo exposure. Results may have differed because in vivo exposure allows for fish to mitigate effects through mechanisms that could include mitomiR regulation, and because fish were only acutely exposed. Environ Toxicol Chem 2024;00:1-14. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Microparticles in Wild and Caged Biota, Sediments, and Water Relative to Large Municipal Wastewater Treatment Plant Discharges.

Anthropogenically modified microparticles including microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether biotic exposures are elevated downstream of these outfalls. In the fall of 2019, the present study examined whether microparticle levels in resident fish, environmental samples, and caged organisms were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River, Ontario, Canada. Wild rainbow darters (Etheostoma caeruleum) were collected from a total of 10 sites upstream and downstream of both WWTPs, along with surface water and sediment samples to assess spatial patterns over an approximately 70-km river stretch. Amphipods (Hyalella azteca), fluted-shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were also caged upstream and downstream of one WWTP for 14 or 28 days. Whole amphipods, fish digestive tracts, and mussel tissues (hemolymph, digestive glands, gills) were digested with potassium hydroxide, whereas environmental samples were processed using filtration and density separation. Visual identification, measurement, and chemical confirmation (subset only) of microparticles were completed. Elevated abiotic microparticles were found at several upstream reference sites as well as at one or both wastewater-impacted sites. Microparticles in amphipods, all mussel tissues, and wild fish did not show patterns indicative of increased exposures downstream of effluent discharges. In contrast, elevated microparticle counts were found in trout caged directly downstream of the outfall. Across all samples, cellulose fibers (mainly blue and clear colors) were the most common. Overall, results suggest little influence of WWTP effluents on microparticles in biota but rather a ubiquitous presence across most sites that indicates the importance of other point and nonpoint sources to this system. Environ Toxicol Chem 2024;43:1047-1061. © 2024 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant.

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid-liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2. •Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect.

Identifying transcriptomic indicators of tertiary treated municipal effluent in longnose dace (Rhinichthys cataractae) caged under semi-controlled conditions in experimental raceways.

To evaluate effects of tertiary treated wastewater treatment plant effluent (MWWE) on transcriptomic responses in longnose dace (Rhinichthys cataractae; LND) we conducted a semi-controlled study in experimental raceways (Advancing Canadian Water Assets facility) imbedded in the Pine Creek treatment plant (Calgary, AB). LND collected from a reference site in the Bow River (REF) were caged in raceways containing either 5 % Pine Creek effluent (PC) or Bow River water (BR; control) over 28 d. Liver transcriptomes were analyzed in males and females sampled on days 7, 14 and 28 from BR and PC, and compared to REF fish on day 0. Concurrent with the caging, selected environmental substances of concern were analyzed in the BR and PC. Significantly different unigenes (SDUs) in females (vs males) within both BR and PC raceways increased over time and compared to REF fish. Moreover, SDUs in females and males within the same treatment (i.e., BR, PC) showed a temporal increase as well as compared to REF fish. Time was the dominant factor affecting SDUs, whereas sex and treatment had less of an impact on the transcriptome profiling. Gene Set Enrichment Analysis of BR vs PC over time revealed effects on genes involved in growth, metabolism of carbohydrates and lipids, and immune system on day 7; however, by day 28, 80-100 % of the transcripts localized to enriched biomarkers were associated with tissue immune responses in both sexes. Exposure to 5 % effluent had significant effects on female liver somatic index but no effects were observed on other phenotypic health indices in either sex. BR was used as the source of reference water, but analyses showed trace amounts of ESOCs. Analyses did not point towards definitive response patterns that could be used in field-based ecotoxicogenomic studies on the impacts of well-treated MWWE but suggested compromised adaptive immune responses.

Accelerated cascade melanoma therapy using enzyme-nanozyme-integrated dissolvable polymeric microneedles.

Dissolvable polymeric microneedles (DPMNs) have emerged as a powerful technology for the localized treatment of diseases, such as melanoma. Herein, we fabricated a DPMN patch containing a potent enzyme-nanozyme composite that transforms the upregulated glucose consumption of cancerous cells into lethal reactive oxygen species via a cascade reaction accelerated by endogenous chloride ions and external near-infrared (NIR) irradiation. This was accomplished by combining glucose oxidase (Gox) with a NIR-responsive chloroperoxidase-like copper sulfide (CuS) nanozyme. In contrast with subcutaneous injection, the microneedle system highly localizes the treatment, enhancing nanomedicine uptake by the tumor and reducing its systemic exposure to the kidneys and spleen. NIR irradiation further controls the potency and toxicity of the formulation by thermally disabling Gox. In a mouse melanoma model, this unique combination of photothermal, starvation, and chemodynamic therapies resulted in complete tumor eradication (99.2 ± 0.8 % reduction in tumor volume within 10 d) without producing signs of systemic toxicity. By comparison, other treatment combinations only resulted in a 42-76.5 % reduction in tumor growth. The microneedle patch design is therefore not only highly potent but also with regulated toxicity and improved safety.

Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

Implementing an adaptive, two-tiered SARS-CoV-2 wastewater surveillance program on a university campus using passive sampling.

Building-level wastewater-based surveillance (WBS) has been increasingly applied upstream from wastewater treatment plants to conduct targeted monitoring for SARS-CoV-2. In this study, a two-tiered, trigger-based wastewater surveillance program was developed on a university campus to monitor dormitory wastewater. The objective was to determine if passive sampling with cotton gauze as a sampling medium could be used to support institution-level public health action. Two nucleocapsid gene targets (N1 and N2) of SARS-CoV-2 as well as the endogenous fecal indicator pepper mild mottle virus (PMMoV) were quantified using RT-qPCR. >500 samples were analyzed during two contrasting surveillance periods. In the Fall of 2021 community viral burden was low and a tiered sampling network was able to isolate individual clinical cases at the building-scale. In the Winter of 2022 wastewater signals were quickly elevated by the emergence of the highly transmissible SARS-CoV-2 Omicron (B.1.1.529) variant. Prevalence of SARS-CoV-2 shifted surveillance objectives from isolating cases to monitoring trends, revealing both the benefits and limitations of a tiered surveillance design under different public health situations. Normalization of SARS-CoV-2 by PMMoV was not reflective of upstream population differences, suggesting saturation of the material occurred during the exposure period. The passive sampling method detected nearly all known clinical cases and in one instance was able to identify one pre-symptomatic individual days prior to confirmation by clinical test. Comparisons between campus samplers and municipal wastewater influent suggests that the spread of COVID-19 on the campus was similar to that of the broader community. The results demonstrate that passive sampling is an effective tool that can produce semi-quantitative data capable of tracking temporal trends to guide targeted public health decision-making at an institutional level. Practitioners of WBS can utilize these results to inform surveillance program designs that prioritize efficient resource use and rapid reporting.

Assessment of seasonality and normalization techniques for wastewater-based surveillance in Ontario, Canada.

Introduction: Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021-2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Methods: Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Results: Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener (r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1-N2) concentration in wastewater (n = 99-191) was strongly correlated to the CBED in the communities (r = 0.620-0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization (n = 99-191) at Humber AMF and Kitchener and crAssphage normalization (n = 29-57) correlations at all three sites were not significantly different from raw N1-N2 correlations with CBED. Discussion: Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

Intersex manifestation in the rainbow darter (Etheostoma caeruleum): Are adult male fish susceptible to developing and recovering from intersex after exposure to endocrine active compounds?

For over a decade, intersex has been observed in rainbow darter (RD) (Etheostoma caeruleum) populations living downstream wastewater treatment plants (WWTPs) in the Grand River, Ontario, Canada. To further our understanding of intersex development in adult male fish, the current study addressed three objectives: i) can intersex be induced in adult male fish, ii) is there a specific window of exposure when adult male fish are more susceptible to developing intersex, and iii) can pre-exposed adult male fish recover from intersex? To assess intersex induction in adult male fish, wild male RD were exposed in the laboratory for 22 weeks (during periods of spawning, gonadal regression, and gonadal recrudescence) to environmentally relevant concentrations of 17α-ethinylestradiol (EE2) including nominal 0, 1, and 10 ng/L. Intersex rates and severity at 10 ng/L EE2 were similar to those observed historically in adult male populations living downstream WWTPs in the Grand River and confirmed previous predictions that 1-10 ng/L EE2 would cause these adverse effects. To assess a window of sensitivity in developing intersex, male RD were exposed to nominal 0, 1 or 10 ng/L EE2 for 4 weeks during three different periods of gonadal development, including (i) spawning, (ii) early recrudescence and (iii) late recrudescence. These short-term exposures revealed that intersex incidence and severity were greater when RD were exposed while gonads were fully developed (during spawning) compared to periods of recrudescence. To assess if RD recover from intersex, wild fish were collected downstream WWTPs in the Grand River and assessed for intersex both before and after a 22-week recovery period in clean water that included gonadal regression and recrudescence. Results showed that fish did not recover from intersex, with intersex rates and severity similar to those both before and after the transition to clean water. This study further advances our knowledge on intersex manifestation in adult male fish including their sensitivity to endocrine active compounds during different periods of their annual reproductive cycle and their limited ability to recover from intersex after onset of the condition.

Wastewater surveillance monitoring of SARS-CoV-2 variants of concern and dynamics of transmission and community burden of COVID-19.

Wastewater-based surveillance is a valuable approach for monitoring COVID-19 at community level. Monitoring SARS-CoV-2 variants of concern (VOC) in wastewater has become increasingly relevant when clinical testing capacity and case-based surveillance are limited. In this study, we ascertained the turnover of six VOC in Alberta wastewater from May 2020 to May 2022. Wastewater samples from nine wastewater treatment plants across Alberta were analysed using VOC-specific RT-qPCR assays. The performance of the RT-qPCR assays in identifying VOC in wastewater was evaluated against next generation sequencing. The relative abundance of each VOC in wastewater was compared to positivity rate in COVID-19 testing. VOC-specific RT-qPCR assays performed comparatively well against next generation sequencing; concordance rates ranged from 89% to 98% for detection of Alpha, Beta, Gamma, Omicron BA.1 and Omicron BA.2, with a slightly lower rate of 85% for Delta (p < 0.01). Elevated relative abundance of Alpha, Delta, Omicron BA.1 and BA.2 were each associated with increased COVID-19 positivity rate. Alpha, Delta and Omicron BA.2 reached 90% relative abundance in wastewater within 80, 111 and 62 days after their initial detection, respectively. Omicron BA.1 increased more rapidly, reaching a 90% relative abundance in wastewater after 35 days. Our results from VOC surveillance in wastewater correspond with clinical observations that Omicron is the VOC with highest disease burden over the shortest period in Alberta to date. The findings suggest that changes in relative abundance of a VOC in wastewater can be used as a supplementary indicator to track and perhaps predict COVID-19 burden in a population.

Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada.

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater.

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

Understanding common population markers for SARS-CoV-2 RNA normalization in wastewater - A review.

Wastewater monitoring and epidemiology have seen renewed interest during the recent COVID-19 pandemic. As a result, there is an increasing need to normalize wastewater-derived viral loads in local populations. Chemical tracers, both exogenous and endogenous compounds, have proven to be more stable and reliable for normalization than biological indicators. However, differing instrumentation and extraction methods can make it difficult to compare results. This review examines current extraction and quantification methods for ten common population indicators: creatinine, coprostanol, nicotine, cotinine, sucralose, acesulfame, androstenedione 5-hydroindoleacetic acid (5-HIAA), caffeine, and 1,7-dimethyluric acid. Some wastewater parameters such as ammonia, total nitrogen, total phosphorus, and daily flowrate were also evaluated. The analytical methods included direct injection, dilute and shoot, liquid/liquid, and solid phase extraction (SPE). Creatine, acesulfame, nicotine, 5-HIAA and androstenedione have been analysed by direct injection into LC-MS; however, most authors prefer to include SPE steps to avoid matrix effects. Both LC-MS and GC-MS have been successfully used to quantify coprostanol in wastewater, and the other selected indicators have been quantified successfully with LC-MS. Acidification to stabilize the sample before freezing to maintain the integrity of samples has been reported to be beneficial. However, there are arguments both for and against working at acidic pHs. Wastewater parameters mentioned earlier are quick and easy to quantify, but the data does not always represent the human population effectively. A preference for population indicators originating solely from humans is apparent. This review summarises methods employed for chemical indicators in wastewater, provides a basis for choosing an appropriate extraction and analysis method, and highlights the utility of accurate chemical tracer data for wastewater-based epidemiology.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Realizing the value in "non-standard" parts of the qPCR standard curve by integrating fundamentals of quantitative microbiology.

The real-time polymerase chain reaction (PCR), commonly known as quantitative PCR (qPCR), is increasingly common in environmental microbiology applications. During the COVID-19 pandemic, qPCR combined with reverse transcription (RT-qPCR) has been used to detect and quantify SARS-CoV-2 in clinical diagnoses and wastewater monitoring of local trends. Estimation of concentrations using qPCR often features a log-linear standard curve model calibrating quantification cycle (Cq) values obtained from underlying fluorescence measurements to standard concentrations. This process works well at high concentrations within a linear dynamic range but has diminishing reliability at low concentrations because it cannot explain "non-standard" data such as Cq values reflecting increasing variability at low concentrations or non-detects that do not yield Cq values at all. Here, fundamental probabilistic modeling concepts from classical quantitative microbiology were integrated into standard curve modeling approaches by reflecting well-understood mechanisms for random error in microbial data. This work showed that data diverging from the log-linear regression model at low concentrations as well as non-detects can be seamlessly integrated into enhanced standard curve analysis. The newly developed model provides improved representation of standard curve data at low concentrations while converging asymptotically upon conventional log-linear regression at high concentrations and adding no fitting parameters. Such modeling facilitates exploration of the effects of various random error mechanisms in experiments generating standard curve data, enables quantification of uncertainty in standard curve parameters, and is an important step toward quantifying uncertainty in qPCR-based concentration estimates. Improving understanding of the random error in qPCR data and standard curve modeling is especially important when low concentrations are of particular interest and inappropriate analysis can unduly affect interpretation, conclusions regarding lab performance, reported concentration estimates, and associated decision-making.

A wastewater-based risk index for SARS-CoV-2 infections among three cities on the Canadian Prairie.

Wastewater surveillance (WWS) is useful to better understand the spreading of coronavirus disease 2019 (COVID-19) in communities, which can help design and implement suitable mitigation measures. The main objective of this study was to develop the Wastewater Viral Load Risk Index (WWVLRI) for three Saskatchewan cities to offer a simple metric to interpret WWS. The index was developed by considering relationships between reproduction number, clinical data, daily per capita concentrations of virus particles in wastewater, and weekly viral load change rate. Trends of daily per capita concentrations of SARS-CoV-2 in wastewater for Saskatoon, Prince Albert, and North Battleford were similar during the pandemic, suggesting that per capita viral load can be useful to quantitatively compare wastewater signals among cities and develop an effective and comprehensible WWVLRI. The effective reproduction number (Rt) and the daily per capita efficiency adjusted viral load thresholds of 85 × 106 and 200 × 106 N2 gene counts (gc)/population day (pd) were determined. These values with rates of change were used to categorize the potential for COVID-19 outbreaks and subsequent declines. The weekly average was considered 'low risk' when the per capita viral load was 85 × 106 N2 gc/pd. A 'medium risk' occurs when the per capita copies were between 85 × 106 and 200 × 106 N2 gc/pd. with a rate of change <100 %. The start of an outbreak is indicated by a 'medium-high' risk classification when the week-over-week rate of change was >100 %, and the absolute magnitude of concentrations of viral particles was >85 × 106 N2 gc/pd. Lastly, a 'high risk' occurs when the viral load exceeds 200 × 106 N2 gc/pd. This methodology provides a valuable resource for decision-makers and health authorities, specifically given the limitation of COVID-19 surveillance based on clinical data.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

Photoresponsive polymeric microneedles: An innovative way to monitor and treat diseases.

Microneedles (MN) technology is an emerging technology for the transdermal delivery of therapeutics. When combined with photoresponsive (PR) materials, MNs can deliver therapeutics precisely and effectively with enhanced efficacy or synergistic effects. This review systematically summarizes the therapeutic applications of PRMNs in cancer therapy, wound healing, diabetes treatment, and diagnostics. Different PR approaches to activate and control the release of therapeutic agents from MNs are also discussed. Overall, PRMNs are a powerful tool for stimuli-responsive controlled-release therapeutic delivery to treat various diseases.

A Nanomedicine Structure-Activity Framework for Research, Development, and Regulation of Future Cancer Therapies.

Despite their clinical success in drug delivery applications, the potential of theranostic nanomedicines is hampered by mechanistic uncertainty and a lack of science-informed regulatory guidance. Both the therapeutic efficacy and the toxicity of nanoformulations are tightly controlled by the complex interplay of the nanoparticle's physicochemical properties and the individual patient/tumor biology; however, it can be difficult to correlate such information with observed outcomes. Additionally, as nanomedicine research attempts to gradually move away from large-scale animal testing, the need for computer-assisted solutions for evaluation will increase. Such models will depend on a clear understanding of structure-activity relationships. This review provides a comprehensive overview of the field of cancer nanomedicine and provides a knowledge framework and foundational interaction maps that can facilitate future research, assessments, and regulation. By forming three complementary maps profiling nanobio interactions and pathways at different levels of biological complexity, a clear picture of a nanoparticle's journey through the body and the therapeutic and adverse consequences of each potential interaction are presented.