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Here, we demonstrate that protein ions can be dissociated prior to mobility analysis in a trapped ion mobility spectrometry device. Generated product ions are mobility separated enabling facile assignment of near isobaric ions.

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4, 22, and 37 °C for 10 days. The concentrations of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA were measured using an RT-ddPCR. Limited decay (1 log

Supplementary Figure S4 from Regulation of Survivin by ErbB2 Signaling: Therapeutic Implications for ErbB2-Overexpressing Breast Cancers

In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of His-tagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors. (Cancer Res 2006; 66(3): 1640-7)

Supplementary Table S2 from Regulation of Survivin by ErbB2 Signaling: Therapeutic Implications for ErbB2-Overexpressing Breast Cancers

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that has been increasingly adopted due to its inherently high resolution and reduced instrumental footprint. In currently deployed commercial instruments, however, typical modes of collisional activation do not precede IMS analysis, and thus, the instruments are incapable of performing CIU. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of native-like protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker.ImportanceHuman exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

Ion mobility separations (IMS) have increasingly been coupled with mass spectrometry to increase peak capacity and deconvolute complex mass spectra in proteomics workflows. IMS separations can be integrated prior to or following the collisional activation step. Post-activation IMS separations have demonstrated many advantages, yet few instrument platforms are capable of this feat. Here, we present the fragmentation of peptide ions within a commercially available trapped-ion mobility spectrometry device. Fragmentation is initiated prior to mobility analysis enabling the separation of generated product ions. The added separation step deconvolutes product ion spectra and permits improved annotation of product ions. Furthermore, we demonstrate the isolation and fragmentation of mobility separated product ions with the downstream quadrupole and collisional cell. When applied to melittin and ubiquitin, this ion mobility assisted pseudo-MS3 fragmentation approach generates sequence coverage ∼50% greater than that of typical MS2 analyses. We envision this ion-mobility-assisted fragmentation technique as the foundation of a powerful new pseudo-MS3 workflow for application toward middle- or top-down proteomics.

Estimating total infection levels, including unreported and asymptomatic infections, is important for understanding community disease transmission. Wastewater can provide a pooled community sample to estimate total infections that is independent of case reporting biases toward individuals with moderate to severe symptoms and by test-seeking behavior and access. We derive three mechanistic models for estimating community infection levels from wastewater measurements based on a description of the processes that generate SARS-CoV-2 RNA signals in wastewater and accounting for the fecal strength of wastewater through endogenous microbial markers, daily flow, and per-capita wastewater generation estimates. The models are illustrated through two case studies of wastewater data collected during 2020–2021 in Virginia Beach, VA, and Santa Clara County, CA. Median simulated infection levels generally were higher than reported cases, but at times, were lower, suggesting a discrepancy between the reported cases and wastewater data, or inaccurate modeling results. Daily simulated infection estimates showed large ranges, in part due to dependence on highly variable clinical viral fecal shedding data. Overall, the wastewater-based mechanistic models are useful for normalization of wastewater measurements and for understanding wastewater-based surveillance data for public health decision-making but are currently limited by lack of robust SARS-CoV-2 fecal shedding data.

Wastewater-based epidemiology during the COVID-19 pandemic has proven useful for public health decision-making but is often hampered by sampling methodology constraints, particularly at the building- or neighborhood-level. Time-weighted composite samples are commonly used; however, autosamplers are expensive and can be affected by intermittent flows in sub-sewershed contexts. In this study, we compared time-weighted composite, grab, and passive sampling via Moore swabs, at four locations across a college campus to understand the utility of passive sampling. After optimizing the methods for sample handling and processing for viral RNA extraction, we quantified SARS-CoV-2 N1 and N2, as well as a fecal strength indicator, PMMoV, by ddRT-PCR and applied tiled amplicon sequencing of the SARS-CoV-2 genome. Passive samples compared favorably with composite samples in our study area: for samples collected concurrently, 42 % of the samples agreed between Moore swab and composite samples and 58 % of the samples were positive for SARS-CoV-2 using Moore swabs while composite samples were below the limit of detection. Variant profiles from Moore swabs showed a shift from variant BA.1 to BA.2, consistent with in-person saliva samples. These data have implications for the broader implementation of sewage surveillance without advanced sampling technologies and for the utilization of passive sampling approaches for other emerging pathogens.

Green stormwater infrastructure systems, such as biofilters, provide many water quality and other environmental benefits, but their ability to remove human pathogens and antibiotic resistance genes (ARGs) from stormwater runoff is not well documented. In this study, a field scale biofilter in Southern California (USA) was simultaneously evaluated for the breakthrough of a conservative tracer (bromide), conventional fecal indicators, bacterial and viral human-associated fecal source markers (HF183, crAssphage, and PMMoV), ARGs, and bacterial and viral pathogens. When challenged with a 50:50 mixture of untreated sewage and stormwater (to mimic highly contaminated storm flow) the biofilter significantly removed (p0.05) 14 of 17 microbial markers and ARGsin descending order of concentration reduction: ermB (2.5 log(base 10) reduction)Salmonella (2.3)adenovirus (1.9)coliphage (1.5)crAssphage (1.2)E. coli (1.0) ∼ 16S rRNA genes (1.0) ∼ fecal coliform (1.0) ∼ intl1 (1.0)Enterococcus (0.9) ∼ MRSA (0.9) ∼ sul1 (0.9)PMMoV (0.7)Entero1A (0.5). No significant removal was observed for GenBac3, Campylobacter, and HF183. From the bromide data, we infer that 0.5 log-units of attenuation can be attributed to the dilution of incoming stormwater with water stored in the biofilter; removal above this threshold is presumably associated with non-conservative processes, such as physicochemical filtration, die-off, and predation. Our study documents high variability (100-fold) in the removal of different microbial contaminants and ARGs by a field-scale stormwater biofilter operated under transient flow and raises further questions about the utility of human-associated fecal source markers as surrogates for pathogen removal.

Abstract: The evaluation field’s understanding of Indigenous ontologies and epistemologies must improve in ways that do not serve to privilege Western ways of knowing or governmental priorities for accountability. The literature has not identified ways to bridge these in practical ways, or to move the field to balance community and government needs. This article describes some prevailing epistemological and methodological issues related to evaluation and then identifies practical challenges bridging Western and Indigenous approaches, using the example of the Indigenous Youth Futures Partnership project (IYFP), a seven-year SSHRC-sponsored grant. It is suggested that there are approaches that work well in these contexts but that agency is vitally important to establish reciprocity.

Recent genome-wide association studies of Huntington’s disease (HD) primarily highlighted genes involved in DNA damage repair mechanisms as modifiers of age at onset and disease severity, consistent with evidence that more DNA repair genes are being implicated in late age–onset neurodegenerative diseases. This provides an exciting opportunity to advance therapeutic development in HD, as these pathways have already been under intense investigation in cancer research. Also emerging are the roles of other polyglutamine disease proteins in DNA damage repair mechanisms. A potential universal trigger of oxidative DNA damage shared in these late age–onset diseases is the increase of reactive oxygen species (ROS) in human aging, defining an age-related mechanism that has defied other hypotheses of neurodegeneration. We discuss the potential commonality of DNA damage repair pathways in HD and other neurodegenerative diseases. Potential targets for therapy that may prove beneficial across many of these diseases are also identified, defining nodes in the ataxia telangiectasia-mutated (ATM) complex, mismatch repair, and poly ADP-ribose polymerases (PARPs). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00768-7) contains supplementary material, which is available to authorized users.

Published and unpublished reports show that SARS-CoV-2 RNA in publicly owned treatment work (POTW) wastewater influent and solids is associated with new COVID-19 cases or incidence in associated sewersheds, but methods for comparing data collected from diverse POTWs to infer information about the relative incidence of laboratory-confirmed COVID-19 cases, and scaling to allow such comparisons, have not been previously established Here, we show that SARS-CoV-2 N1 and N2 concentrations in solids normalized by concentrations of PMMoV RNA in solids can be used to compare incidence of laboratory confirmed new COVID-19 cases across POTWs Using data collected at seven POTWs along the United States West Coast, Midwest, and East Coast serving ∼3% of the U S population (9 million people), we show that a 1 log change in N gene/PMMoV is associated with a 0 24 (range 0 19 to 0 29) log10 change in incidence of laboratory confirmed COVID-19 Scaling of N1 and N2 by PMMoV is consistent, conceptually, with a mass balance model relating SARS-CoV-2 RNA to the number of infected individuals shedding virus in their stool This information should support the application of wastewater-based epidemiology to inform the response to the COVID-19 pandemic and potentially future viral pandemics ©

The mechanisms by which bacteria sense and respond to surface attachment have long been a mystery. Our understanding of the structure and dynamics of bacterial appendages, notably type IV pili (T4P), provided new insights into the potential ways that bacteria sense surfaces. T4P are ubiquitous, retractable hair-like adhesins that until recently, due to their nanoscale size, were difficult to image in the absence of fixation. This review focuses on recent microscopy innovations used to visualize T4P in live cells to reveal the dynamics of their retraction and extension. We discuss recently proposed mechanisms by which T4P facilitate bacterial surface sensing, including the role of surface-exposed PilY1, two-component signal transduction pathways, force-induced structural modifications of the major pilin, and altered dynamics of the T4P motor complex.

Type IVa pili (T4aP) are important virulence factors for many bacterial pathogens. Previous studies suggested that the retraction ATPase, PilT, modulates pathogenicity due to its critical role in pilus dynamics and twitching motility. Here we use a Caenorhabditis elegans slow killing model to show that hyperpiliation, not loss of pilus retraction, reduces virulence of Pseudomonas aeruginosa strains PAK and PA14 by interfering with function of the contact-dependent type III secretion system (T3SS). Hyperactivating point mutations in the P. aeruginosa PilSR two-component system that controls transcription of the major pilin gene, pilA, increased levels of surface pili to the same extent as deleting pilT, without impairing twitching motility. These functionally hyperpiliated PilSR mutants had significant defects in pathogenicity that were rescued by deleting pilA or by increasing the length of T3SS needles via deletion of the needle-length regulator, PscP. Hyperpiliated pilT deletion or pilO point mutants showed similar PilA-dependent impairments in virulence, validating the phenotype. Together, our data support a model where a surfeit of pili prevents effective engagement of contact-dependent virulence factors. These findings suggest that the role of T4aP retraction in virulence should be revised.SIGNIFICANCEPseudomonas aeruginosa is a major contributor to hospital-acquired infections and particularly problematic due to its intrinsic resistance to many front-line antibiotics. Strategies to combat this and other important pathogens include development of anti-virulence therapeutics. We show that the pathogenicity of P. aeruginosa is impaired when the amount of type IVa pili (T4aP) expressed on the cell surface increases, independent of the bacteria’s ability to twitch. We propose that having excess T4aP on the cell surface can physically interfere with productive engagement of the contact-dependent type III secretion toxin delivery system. A better understanding of how T4aP modulate interaction of bacteria with target cells will improve the design of therapeutics targeting components involved in regulation of T4aP expression and function, to reduce the clinical burden of P. aeruginosa and other T4aP-expressing bacteria.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Wastewater-based epidemiology (WBE) may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the pre-analytical and analytical approaches, and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus, PMMoV) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlate positively and significantly with COVID-19 clinical confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARs-CoV-2 in influent.Abstract Figure

Analytical and biological validations of blubber hormone measurements were conducted for North Atlantic right whales. A simplified blubber hormone extraction protocol was successfully developed, improving the efficiency of sample preparation. Cortisol was elevated in whales that died from anthropogenic causes, and reproductive hormone concentrations showed meaningful patterns across known reproductive states., As studies quantifying steroid hormones in marine mammal blubber progress, methodological refinements may improve the utility and consistency of blubber hormone measurements. This study advances blubber extraction methodologies by testing a simplified extraction protocol that reduces time and complexity compared to a protocol widely used in cetacean blubber studies. Using blubber samples archived from remote biopsy (n = 21 live whales) and necropsy collection (n = 7 dead whales) of North Atlantic right whales (NARW; Eubalaena glacialis) of known life history states, we performed analytical and biological validations to assess the feasibility of measuring reproductive (testosterone, progesterone) and glucocorticoid (cortisol) hormones in blubber via enzyme immunoassay following the simplified extraction. Analytical validations (parallelism, accuracy, extraction efficiency, repeatability) showed the simplified extraction produced similar results to the extended protocol, offering a more efficient and consistent technique. In live, apparently healthy whales, blubber testosterone concentrations (mean ± SE) were significantly higher in males (2.02 ± 0.36 ng/g) compared to females (0.81 ± 0.15 ng/g). Blubber progesterone was highest in a confirmed pregnant female (60.3 ng/g), which was 12-fold greater than the mean concentration of non-pregnant females (4.56 ± 0.88 ng/g). Blubber cortisol concentrations in whales that died from anthropogenic causes averaged 5.31 ± 2.28 ng/g, whereas most live, healthy whales had cortisol values below 1 ng/g. Among living whales, a whale actively entangled in fishing gear had the highest blubber cortisol measurement (3.51 ng/g), exhibiting levels similar to whales that died from acute entanglement (2.88 ± 0.42 ng/g). Overall, the highest blubber cortisol concentration (18.0 ng/g) was measured in a dead whale with a severe chronic entanglement, approximately 30-fold greater than mean blubber cortisol of apparently healthy whales (0.58 ± 0.11 ng/g). The methodological approach presented here provides a reference for researchers interested in an alternative, streamlined technique for hormone extraction of cetacean blubber and contributes to the diverse tool set for stress and reproductive assessments of endangered NARWs.

TfpW is an oligosaccharyltransferase that modifies the subunits of type IV pili from group IV strains of Pseudomonas aeruginosa with oligomers of α-1,5-linked-D-arabinofuranose (D-Araf). Besides its oligosaccharyltransferase activity, TfpW may be responsible for periplasmic translocation and polymerization of D-Araf. Here we investigated these potential roles of TfpW in Pa5196 pilin glycosylation. Topology studies confirmed the periplasmic location of loop 1 and the large C-terminus domain, however the central portion of TfpW had an indeterminate configuration. Reconstitution of the Pa5196 pilin glycosylation system by providing pilA, tfpW +/- tfpX and the D-Araf biosynthesis genes PsPA7_6246-6249 showed that TfpW is sufficient for glycan polymerization and transfer to pilins in P. aeruginosa PAO1, while TfpX is also necessary in Escherichia coli. In addition to PsPA7_6246, DprE1 (PsPA7_6248) and DprE2 (PsPA7_6249), the GtrA-like component PsPA7_6247 was required for pilin glycosylation in E. coli versus PAO1. In a PAO1 ΔarnE/F mutant, loss of PsPA7_6247 expression decreased the level of pilin glycosylation, suggesting that arnE/F may play a role in pilin glycosylation when PsPA7_6247 is absent. Bacterial two-hybrid studies showed interactions of TfpW with itself, TfpX, PsPA7_6247 and DprE2, suggesting the formation of a complex that enables efficient pilin glycosylation. Fluorescence microscopy of E. coli and Pa5196ΔdprE1 expressing a DprE1-sGFP fusion showed that the protein is expressed in the cytoplasm, supporting our model that includes cytoplasmic biosynthesis of the lipid carrier-linked D-Araf precursor prior to its periplasmic translocation. Together these data suggest that TfpW may be the first example of a trifunctional flippase, glycosyltransferase, and oligosaccharyltransferase.

This study investigated the risk of gastrointestinal illness associated with swimming in surface waters with aged sewage contamination. First, a systematic review compiled 333 first order decay rate constants ( k) for human norovirus and its surrogates feline calicivirus and murine norovirus, Salmonella, Campylobacter, Escherichia coli O157:H7, Giardia, and Cryptosporidium, and human-associated indicators in surface water. A meta-analysis investigated effects of sunlight, temperature, and water matrix on k. There was a relatively large number of k for bacterial pathogens and some human-associated indicators ( n40), fewer for protozoans ( n = 14-22), and few for human norovirus and its Caliciviridae surrogates ( n = 2-4). Average k ranked: Campylobacterhuman-associated markersSalmonellaE. coli O157:H7norovirus and its surrogatesGiardiaCryptosporidium. Compiled k values were used in a quantitative microbial risk assessment (QMRA) to simulate gastrointestinal illness risk associated with swimming in water with aged sewage contamination. The QMRA used human-associated fecal indicator HF183 as an index for the amount of sewage present and thereby provided insight into how risk relates to HF183 concentrations in surface water. Because exposure to norovirus contributed the majority of risk, and HF183 k is greater than norovirus k, the risk associated with exposure to a fixed HF183 concentration increases with the age of contamination. Swimmer exposure to sewage after it has aged ∼3 days results in median risks less than 30/1000. A risk-based water quality threshold for HF183 in surface waters that takes into account uncertainty in contamination age is derived to be 4100 copies/100 mL.

Urban runoff is one of the greatest sources of microbial pollution to surface waters. Biofilters can limit the impact of stormwater runoff on surface water quality by diverting runoff from receiving waters. However, our understanding of how biofilter design choices, including the addition of vegetation and geomedia, may impact the removal of pathogens is lacking. In this study, we characterized viruses (adenovirus, enterovirus, norovirus GII, crAssphage) in San Francisco Bay area urban runoff and assessed the removal of lab-cultured viruses (MS2, adenovirus 2, coxsackievirus B5) from biochar-amended biofilter mesocosms during challenge testing. We quantified viruses using (RT-)qPCR and F+ coliphage plaque assays. We found that all the pathogenic viruses targeted were found at low concentrations (adenovirus: all positive samples werelimit of quantification, enterovirus:limit of quantification-1.9 × 10

BackgroundDissemination of accurate health research information to patients and families has become increasingly important with the rise of the internet as a means of finding health information. However, the public faces several barriers to accessing research information; including paywalls and technical jargon. One method to bridge this gap between patients, families, and research is using lay summaries. SCAsource is an online knowledge translation platform where peer-reviewed research papers on ataxia are translated into lay summaries. This online platform was launched in September 2018, with the goal of making ataxia research more accessible and understandable to patients and families. A secondary goal is to provide opportunities for ataxia researchers to develop and hone their knowledge translation skills, altogether improving the quality of patient communication in the ataxia community.AimThe aim of this study was to measure the impact of SCAsource on its readers and volunteer contributors after one year of activity. This is to ensure SCAsource is meeting its goals of (1) improving access and understanding of ataxia research to lay audiences, and (2) improving knowledge translation skills of volunteer contributors.MethodsTwo online surveys were launched, one for readers and one for volunteers. Each survey had a combination of multiple-choice, Likert-scale type, and open-ended short-answer questions. Descriptive quantitative analysis was used for respondent characteristics and Likert-type data. A grounded theory coding approach was used to analyze narrative feedback data.ResultsWe found that SCAsource has mutually beneficial outcomes for both lay person readers and volunteer contributors. Readers have an increased understanding of ataxia research and access to up-to-date information on recent publications. Volunteers develop knowledge translation skills and have increased confidence in communicating results to lay audiences. Areas of improvement were identified to be incorporated into the platform.ConclusionWe demonstrated that SCAsource improves access to information and understanding of research to lay audiences, while providing opportunities for researchers to develop knowledge translation skills. This framework can potentially be used by other rare disease organizations to launch and evaluate their own knowledge translation websites.

Municipal wastewater treatment plants in coastal areas are facing numerous challenges, including the need to provide a cost-effective approach for removing nutrients and trace organic contaminants from wastewater, as well as adapting to the effects of climate change. The horizontal levee is a multi-benefit response to these issues that consists of a sloped subsurface treatment wetland built between a coastal levee and tidal marshes. The wetland attenuates storm surges and can provide space for wetland transgression to higher elevations as sea levels rise, while simultaneously removing contaminants from treated wastewater effluent. To assess the ability of the horizontal levee to improve water quality and to identify optimal operating conditions, a 0.7-ha experimental system was studied over a two-year period. The removal of nitrate and trace organic contaminants was particularly sensitive to hydrology; rapid and near complete removal (>97%) of these contaminants was observed in water flowing through the subsurface, whereas surface flows did not exhibit measurable contaminant removal. Removal of F+ coliphage also appeared to be sensitive to hydrology, with up to 99% removal of these indicator viruses in subsurface flow. For phosphate, removal was not as sensitive to hydrology, but significant removal (>83%) was still observed when overland flow was eliminated. Although removal of contaminants did not appear to be sensitive to other design considerations, parameters such as soil texture and planting regimes affected the maximum subsurface flows, which in turn controlled contaminant mass loadings. Rapid subsurface removal of contaminants suggests that water quality benefits of these systems are limited by physical constraints (i.e., the ability of the system to maintain subsurface flow) and not chemical or biological conditions in the subsurface., Graphical abstract Image 1, Highlights • Horizontal levees remove a wide range of contaminants from wastewater effluent. • Contaminant removal is controlled primarily by subsurface flow. • Design parameters (e.g., planting regime) have significant impacts on water balance.

Through optimization of the linker region and key stabilizing mutations, it has been possible to improve the stability of the circularly permuted (cp) Trp-cage miniprotein. However, even the most stable Trp-cage circular permutants are still less stable than the analogous standard topology (std) Trp-cages. Extending mutational studies of Trp-cage fold stability to cp-species, including analogs lacking chain terminal charges, has uncovered and quantitated some additional stabilizing and destabilizing interactions. Upon protonation, the circular permutants are destabilized to a much greater extent than the standard topology series. End effects, particularly Coulombic interactions, appear to be more important for the cp-series while the Y10/P4 interaction in the cp-series is not as significant a stabilizing feature as the corresponding Y3/P19 in the standard topology series.

The name of author Samuel Dorevitch was mispelled in the original paper. © 2021 American Chemical Society. All rights reserved.

Many of the devastating pandemics and outbreaks of the 20th and 21st centuries have involved enveloped viruses, including influenza, HIV, SARS, MERS, and Ebola. However, little is known about the presence and fate of enveloped viruses in municipal wastewater. Here, we compared the survival and partitioning behavior of two model enveloped viruses (MHV and ϕ6) and two nonenveloped bacteriophages (MS2 and T3) in raw wastewater samples. We showed that MHV and ϕ6 remained infective on the time scale of days. Up to 26% of the two enveloped viruses adsorbed to the solid fraction of wastewater compared to 6% of the two nonenveloped viruses. Based on this partitioning behavior, we assessed and optimized methods for recovering enveloped viruses from wastewater. Our optimized ultrafiltration method resulted in mean recoveries (±SD) of 25.1% (±3.6%) and 18.2% (±9.5%) for the enveloped MHV and ϕ6, respectively, and mean recoveries of 55.6% (±16.7%) and 85.5% (±24.5%) for the nonenveloped MS2 and T3, respectively. A maximum of 3.7% of MHV and 2% of MS2 could be recovered from the solids. These results shed light on the environmental fate of an important group of viruses and the presented methods will enable future research on enveloped viruses in water environments.

We characterized the microbial communities in sea spray aerosols (SSA), water and sand of three beaches in central California (Cowell Beach, Baker Beach and Lovers Point) by sequencing the V4 region of the 16S rRNA gene. Average concentrations of 16S rRNA genes in SSA ranged from 2.4 × 104 to 1.4 × 105 gene copies per m3 of air. A total of 9781 distinct OTUs were identified in SSA and of these, 1042 OTUs were found in SSA of all beaches. SSA microbial communities included marine taxa, as well as some associated with the terrestrial environment. SSA taxa included organisms that play important roles in biogeochemical cycling of elements such as Planctomyces and Synechococcus, as well as those representing potential pathogens and fecal indicator bacteria including Staphylococcus epidermidis and Enterococcus spp. There were a large number of shared OTUs among SSA and water, and there was relatively high similarity between SSA and water communities. Results are consistent with a conceptual model where SSA is generated by breaking waves and bubble bursting in marine waters and that enables the transport of microorganisms from the sea to sand or other environments.

Hormone therapies were tested for efficacy to induce ovulation in the dusky gopher frog. The number of eggs produced, and cleavage rates following IVF were compared between treatments. Ultrasound was explored as a complementary tool to hormone therapy. Findings are critical to enhancing conservation and broodstock production for reintroduction programs. Editor: Steven Cooke, Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs (n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater (P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher (P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1–5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant (P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3–4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate (P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.

Beaches often receive fecal contamination from more than one source. Human sources include untreated sewage as well as treated wastewater effluent, and animal sources include wildlife such as gulls. Different contamination sources are expected to pose different health risks to swimmers. Genetic microbial source tracking (MST) markers can be used to detect bacteria that are associated with different animal sources, but the health risks associated with a mixture of MST markers are unknown. This study presents a method for predicting these health risks, using human- and gull-associated markers as an example. Quantitative Microbial Risk Assessment (QMRA) is conducted with MST markers as indicators. We find that risks associated with exposure to a specific concentration of a human-associated MST marker (HF) are greater if the HF source is untreated sewage rather than treated wastewater effluent. We also provide a risk-based threshold of HF from untreated sewage at a beach, to stay below a predicted illness risk of 3 per 100 swimmers, that is a function of gull-associated MST marker (CAT) concentration.

Background Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations. Methods Body size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively. Results Body size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p

This study tests the immediate impact of two interventions for intimate partner violent (IPV) men in affecting behavioral and emotional change during arguments with their partners. Couples with an abusive male partner (N=100) discussed an area of conflict twice, interrupted by a brief intervention. Men were randomly assigned to receive (a) an editing-out-the-negative skills training, (b) an accepting influence skills training, or (c) a time-out. IPV men in both skills-training conditions showed greater decreases in aggressive feelings than IPV men in the time-out condition based on their self-report and observed affective behavior. Women also reported feeling less aggressive when their husbands were assigned to one of the skills-training conditions as compared to the control (time-out) condition. Results suggest that IPV men can learn to adopt new communication skills and that they do appear to have a positive impact on the emotional tone of their arguments. Clinically, communication skills training may be a useful addition to battering intervention programs, although these skills may need to be taught to both men and women involved in violent relationships.

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.

This article examines the distribution of federal public servants across Canada and asks the question “Does the distribution of the federal public service across Canada matter in the formulation of federal policies related to municipalities?” The article takes a two-part approach. First, it examines the history of and commentary on decentralization and de-concentration of the federal public service. It then proceeds to examine the data on the distribution of federal employment over time and uses regression analysis to explain the variations in the relative federal employee presence across local areas and through time. Analysis of initiatives to reform the way in which the public service operates and of the data regarding the location of public servants across the country suggest that broad political, economic and regional factors influence the size and distribution of federal employment across the country, perhaps more than any conscious intent about improving the responsiveness of the federal public service to local conditions. At present, the “knowledge functions” of the federal government are concentrated in the National Capital Region. It is an open question whether this will serve the federal government and Canadians well in the future. Sommaire : Cet article etudie la repartition des fonctionnaires federaux a travers le Canada et pose la question suivante : «Est-ce que la repartition des fonctionnaires federaux au Canada a un role a jouer dans la formulation des politiques federales ayant trait aux municipalites?» L'article comporte une approche en deux parties. Dans la premiere, les auteurs examinent l'histoire et les commentaires sur la decentralisation et la deconcentration de la fonction publique federale. Ensuite, ils etudient les donnees sur la repartition des employes federaux au fil des annees et se servent de l'analyse de regression pour expliquer les variations dans la presence relative des fonctionnaires federaux dans les secteurs municipaux et dans le temps. L'analyse des initiatives visant a reformer la maniere dont la fonction publique fonctionne et des donnees concernant la localisation des fonctionnaires a travers le pays laisse entendre que d'importants facteurs politiques, economiques et regionaux influencent la taille et la repartition des emplois federaux a travers le pays, peut-etre plus que toute intention deliberee d'ameliorer la receptivite de la fonction publique federale aux conditions locales. A l'heure actuelle, les «fonctions de connaissance» du gouvernement federal sont concentrees dans la region de la capitale nationale. La question est de savoir si cela servira la cause du gouvernement federal et du peuple canadien a l'avenir.

Drawing on research that is part of the five-year Major Collaborative Re- search Initiative project examining public policy in Canadian municipalities, this article provides an overview of the federal-municipal machinery developed to facil- itate urban policy and program development. The ''federal-municipal machinery'' refers to the many programs and initiatives that have been used to structure federal- municipal relations and influence urban policy and development. The research time frame begins with the period leading up to the creation of the Ministry of State for Urban Affairs, in 1971, and continues through four decades to include recent events of note such as the creation of the Prime Minister's Caucus Task Force on Urban Is- sues, the subsequent announcements of the New Deal, and the Gas Tax Fund for Canada's cities and communities. The authors begin with a discussion of why federal governments involve themselves in municipal and urban affairs and then provide an overview of their analysis by identifying patterns and trends in the evolution of fed- eral-municipal machinery. They then discuss the effectiveness of the machinery, over three periods, and conclude by considering the implications of their findings for the future of federal-municipal relations in the context of the growing pressures facing urban Canada. Sommaire : Fondesur la recherche entreprise sur cinq ans dans le cadre du projet intitule´ sGrands travaux de recherche concerteequi examine la politique gou- vernementale dans les municipalites canadiennes, le present article donne un apercu de l'appareil federal-municipal mis au point pour faciliter l'elaboration de pro- grammes et de politiques dans le secteur urbain. L' sappareil federal-municipal � fait reference aux nombreux programmes et initiatives qui ont servi astructurer les relations federales-municipales et ainfluencer la politique d'urbanisme et son deve- loppement. Le cadre de reference de la recherche commence avec la periode menant a` la creation du departement d'Etat chargedes Affaires urbaines en 1971 et se poursuit

Differences in reported testosterone concentrations in male sea turtle blood samples are common in the veterinary literature, but may be accounted for by differences in sample handling and processing prior to assay. Therefore, our study was performed to determine best practices for testosterone analysis in male sea turtles ( Caretta caretta and Chelonia mydas). Blood samples were collected into 5 collection tube types, and assay validation and measured testosterone concentrations were compared across different sample storage (fresh, refrigerated 1 week, or frozen), extraction (unextracted or ether-extracted), and processing treatment (untreated, homogenized, or dissociation reagent) conditions. Ether-extracted and dissociation reagent–treated samples validated in all conditions tested and are recommended for use, as unextracted samples validated only if assayed fresh. Dissociation reagent treatment was simpler to perform than ether extraction and resulted in total testosterone concentrations ~2.7–3.5 times greater than free testosterone measured in ether-extracted samples. Sample homogenization did not affect measured testosterone concentrations, and could be used to increase volume in gelled samples. An annual seasonal testosterone increase was observed in both species when ether extraction or dissociation reagent treatment was used. Annual deslorelin implant treatments in a Chelonia mydas male resulted in suppression of seasonal testosterone following the fourth treatment. Seasonal testosterone patterns resumed following discontinuation of deslorelin. Comparison of in-house and commercially available enzyme immunoassay kits revealed similar patterns of seasonal testosterone increases and deslorelin-induced suppression. Our study highlights the importance of methodological validation and provides laboratorians with best practices for testosterone enzyme immunoassay in sea turtles.

In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of His-tagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors. (Cancer Res 2006; 66(3): 1640-7)

The present study attempts to replicate Gottman et al.'s (1995; see record 1995-44075-001; and Jacobson & Gottman, 1998) psychophysiological study that classifies partner assaultive men into two distinct groups: heart rate (HR) decelerators (Type 1 batterers) and HR accelerators (Type 2 batterers). Current results indicate no significant differences between Type 1 and Type 2 batterers on the antisocial spectrum of behaviors. Resting HR, rather than HR change, was negatively related to the antisocial spectrum of behavior for batterers with severe, clinical levels of violence only. Reasons for subsequent failures to replicate the Type 1 versus Type 2 distinctions may be attributable to unusually high autonomic arousal during baseline in the original study. Consideration of resting HR and the use of dimensional as opposed to categorical approaches in analyzing the heterogeneity of batterers are proposed as possible solutions to clarifying inconsistencies across laboratories. Language: en

This article examines the saga of local government restructuring in Canada's capital city. Specifically, it analyzes the interplay between provincial and local agendas for local government reform over many years, which culminated in provincial legislation and a one-year transition process to establish one municipality for the Ottawa city region. In doing so, the article addresses the extent to which the Ottawa transition demonstrates learning from other major urban restructuring efforts and the extent to which the Ottawa case provides new insights for future local government reform efforts. Key conclusions are that the key motivation for provincially initiated reform—cost saving through simplification of the local government structure in Ottawa—does not fully coincide with local needs and interests. Furthermore, the promise of financial savings has proven difficult to realize as a result of the local politics surrounding existing municipal debt and unresolved human resource management costs. Instead, future benefits from the amalgamation may lie in improved capacity to manage physical development, environmental sustainability, and cultural diversity.

We have isolated a rat intracisternal-A particle element (IAP)-like element (IAP-LE) from ovarian granulosa cells that appears to be identical to the rat EST clone AA964260. The compiled cDNA sequences contain several putative in-frame translation initiation codons with the largest capable of encoding a 365 amino acid protein with a reverse transcriptase domain in the N-terminus as well as a bipartite nuclear localization signal sequence in the middle. Northern blotting shows a major approximately 7 Kb transcript and a minor approximately 5 Kb transcript that are abundantly expressed in the ovary. In situ hybridization histochemistry using ovaries from gonadotropin-treated immature rats and regularly cycling adult rats show that this transcript is predominantly localized to granulosa cells of all healthy follicles, including primary follicles, and to newly-formed and healthy corpora lutea. This cell-specific expression pattern of the IAP-LE gene is distinct from those of the several known retroviral elements, suggesting the potentially novel functional importance of the IAP-LE gene. Taken together, our results demonstrate abundant and cell-specific expression of a novel IAP-LE in rat granulosa cells.