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We present measurements of the damping experienced by custom-made quartz tuning forks submerged in 3He covering frequencies from 20 to 600 kHz. Measurements were conducted in the bulk of normal liquid 3He at temperatures from 1.5 K down to 12 mK and in superfluid 3He-B well below the critical temperature. The presented results complement earlier work on tuning fork damping in 3He, removing possible ambiguities associated with acoustic emission within partially enclosed volumes and extend the probed range of frequencies, leading to a clearly established frequency dependence of the acoustic losses. Our results validate existing models of damping and point toward the same mechanism of wave emission of first sound in normal 3He and liquid 4He and zero sound in superfluid 3He. We observe a steep frequency dependence of the damping ≈ f 5.5, which starts to dominate around 100 kHz and restricts the use of tuning forks as efficient sensors in quantum fluids. The acoustic emission model can predict the limiting frequencies for various devices, including micro-electromechanical and nano-electromechanical structures developed for quantum turbulence and single vortex dynamics research.

Wastewater monitoring has shown promise in providing an early warning for new COVID-19 outbreaks, but to date, no approach has been validated to reliably distinguish signal from noise in wastewater data and thereby alert officials to when the data show a need for heightened public health response. We analyzed 62 weeks of data from 19 sites participating in the North Carolina Wastewater Monitoring Network to characterize wastewater metrics before and around the Delta and Omicron surges. We found that, on average, wastewater data identified new outbreaks four to five days before case data (reported based on the earlier of the symptom start date or test collection date). At most sites, correlations between wastewater and case data were similar regardless of how wastewater concentrations were normalized, and correlations were slightly stronger with county-level cases than sewershed-level cases, suggesting that officials may not need to geospatially align case data with sewershed boundaries to gain insights into disease transmission. Wastewater trend lines showed clear differences in the Delta versus Omicron surge trajectories, but no single wastewater metric (detectability, percent change, or flow-population normalized viral concentrations) adequately indicated when these surges started. After iteratively examining different combinations of these three metrics, we developed a simple algorithm that identifies unprecedented signals in the wastewater to help clarify changes in communities’ COVID-19 burden. Our novel algorithm accurately identified the start of both the Delta and Omicron surges in 84% of sites, potentially providing public health officials with an automated way to flag community-level COVID-19 surges.

Real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) methods have revolutionized environmental microbiology, yielding quantitative organism-specific data of nucleic acid targets in the environment. Such data are essential for characterizing interactions and processes of microbial communities, assessing microbial contaminants in the environment (water, air, fomites), and developing interventions (water treatment, surface disinfection, air purification) to curb infectious disease transmission. However, our review of recent qPCR and dPCR literature in our field of health-related environmental microbiology showed that many researchers are not reporting necessary and sufficient controls and methods, which would serve to strengthen their study results and conclusions. Here, we describe the application, utility, and interpretation of the suite of controls needed to make high quality qPCR and dPCR measurements of microorganisms in the environment. Our presentation is organized by the discrete steps and operations typical of this measurement process. We propose systematic terminology to minimize ambiguity and aid comparisons among studies. Example schemes for batching and combining controls for efficient work flow are demonstrated. We describe critical reporting elements for enhancing data credibility, and we provide an element checklist in the Supporting Information. Additionally, we present several key principles in metrology as context for laboratories to devise their own quality assurance and quality control reporting framework. Following the EMMI guidelines will improve comparability and reproducibility among qPCR and dPCR studies in environmental microbiology, better inform engineering and public health actions for preventing disease transmission through environmental pathways, and for the most pressing issues in the discipline, focus the weight of evidence in the direction toward solutions.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be useful for monitoring population-wide coronavirus disease 2019 (COVID-19) infections, especially given asymptomatic infections and limitations in diagnostic testing. We aimed to detect SARS-CoV-2 RNA in wastewater and compare viral concentrations to COVID-19 case numbers in the respective counties and sewersheds. Influent 24-hour composite wastewater samples were collected from July to December 2020 from two municipal wastewater treatment plants serving different population sizes in Orange and Chatham Counties in North Carolina. After a concentration step via HA filtration, SARS-CoV-2 RNA was detected and quantified by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) and quantitative PCR (RT-qPCR), targeting the N1 and N2 nucleocapsid genes. SARS-CoV-2 RNA was detected by RT-ddPCR in 100 % (24/24) and 79 % (19/24) of influent wastewater samples from the larger and smaller plants, respectively. In comparison, viral RNA was detected by RT-qPCR in 41.7 % (10/24) and 8.3 % (2/24) of samples from the larger and smaller plants, respectively. Positivity rates and method agreement further increased for the RT-qPCR assay when samples with positive signals below the limit of detection were counted as positive. The wastewater data from the larger plant generally correlated (⍴ ~0.5, p0.05) with, and even anticipated, the trends in reported COVID-19 cases, with a notable spike in measured viral RNA preceding a spike in cases when students returned to a college campus in the Orange County sewershed. Correlations were generally higher when using estimates of sewershed-level case data rather than county-level data. This work supports use of wastewater surveillance for tracking COVID-19 disease trends, especially in identifying spikes in cases. Wastewater-based epidemiology can be a valuable resource for tracking disease trends, allocating resources, and evaluating policy in the fight against current and future pandemics.

Hundreds of peptides can be synthesized by automated parallel synthesizers in a single run. In contrast, the most widely used peptide purification method – high-pressure liquid chromatography (HPLC) – only allows one-by-one processing of each sample. The chromatographic purification of many peptides, therefore, remains a time-consuming and costly effort. Catch-and-release methods can be processed in parallel and potentially provide a remedy. However, no such system has yet provided a true alternative to HPLC. Herein we present the development of a side-reaction free, reductively cleavable linker. The linker is added to the target peptide as the last building block during peptide synthesis. After acidic cleavage from synthetic resin, the linker-tagged full-length peptide is caught onto an aldehyde-modified solid support by rapid oxime ligation, allowing removal of all impurities lacking the linker by washing. Reducing the aryl azide to an aniline sensitizes the linker for cleavage. However, scission does not occur at non-acidic pH enabling wash out of reducing agent. Final acidic treatment safely liberates the peptide by an acid-catalysed 1,6-elimination. We showcase this first-in-class reductively cleavable linker system in the parallel purification of a personalized neoantigen cocktail, containing 20 peptides for cancer immunotherapy within six hours., A first-in-class reductively cleavable linker system that enables parallel and traceless purification of peptides through a safety-release is introduced with three linker types and showcased by rapid production of 20 personalized neoantigen peptides.

Destroying superfluidity is a fundamental process and in fermionic superfluid such as He3-B it splits Cooper pairs into thermal excitations, quasiparticles. At the lowest temperatures, a gas of these quasiparticle excitations is tenuous enough for the propagation to be ballistic. We describe here an exploitation of the ballistic quasiparticles as the "photons"to observe the local destruction of superfluid He3-B by a mechanical resonator. We use a 5 by 5 pixel quasiparticle camera to image an emergence of quasiparticle excitations and a tangle of quantized vortices accompanying the pair-breaking. The detected quantum tangle is asymmetric around the mechanical resonator and is governed by the stability of vortices on the resonator surface. The vortex distribution shows that a conventional production of a quantum tangle via repetitive emission of vortex rings starts on the top surface of the generator and spreads around whole surface at high velocity when escaping vortex rings get retrapped by the moving resonator.

Novel halogen ring-trisubstituted 2-methoxyethyl phenylcyanoacrylates, RPhCH=C(CN)CO2CH2CH2OCH3 (where R is 5-chloro-2,3-dimetoxy, 3-bromo-4,5-dimethoxy, 5-bromo-2,3-dimethoxy, 2-bromo-3-hydroxy-4-methoxy, 4-bromo-2,6-difluoro, 3-chloro-2,6-difluoro, 4-chloro-2,6-difluoro, 2-hydroxy-3,5-diiodo, 2,3,6-trichloro, 2,3,4-trifluoro, 2,4,6-trifluoro) were prepared and copolymerized with styrene. The acrylates were synthesized by the piperidine catalyzed Knoevenagel condensation of ring-trisubstituted benzaldehydes and 2-methoxyethyl cyanoacetate, and characterized by CHN analysis, IR, 1H and 13C NMR. All the acrylates were copolymerized with styrene in solution with radical initiation (ABCN) at 70C. The compositions of the copolymers were calculated from nitrogen analysis.

Traumatic spinal cord injury (SCI) above the major spinal sympathetic outflow (T6 level) disinhibits sympathetic neurons from supraspinal control, causing systems-wide “dysautonomia.” We recently showed that remarkable structural remodeling and plasticity occurs within spinal sympathetic circuitry, creating abnormal sympathetic reflexes that exacerbate dysautonomia over time. As an example, thoracic VGluT2+spinal interneurons (SpINs) become structurally and functionally integrated with neurons that comprise the spinal–splenic sympathetic network and immunological dysfunction becomes progressively worse after SCI. To test whether the onset and progression of SCI-induced sympathetic plasticity is neuron activity dependent, we selectively inhibited (or excited) thoracic VGluT2+interneurons using chemogenetics. New data show that silencing VGluT2+interneurons in female and male mice with a T3 SCI, using hM4Di designer receptors exclusively activated by designer drugs (GiDREADDs), blocks structural plasticity and the development of dysautonomia. Specifically, silencing VGluT2+interneurons prevents the structural remodeling of spinal sympathetic networks that project to lymphoid and endocrine organs, reduces the frequency of spontaneous autonomic dysreflexia (AD), and reduces the severity of experimentally induced AD. Features of SCI-induced structural plasticity can be recapitulated in the intact spinal cord by activating excitatory hM3Dq-DREADDs in VGluT2+interneurons. Collectively, these data implicate VGluT2+excitatory SpINs in the onset and propagation of SCI-induced structural plasticity and dysautonomia, and reveal the potential for neuromodulation to block or reduce dysautonomia after severe high-level SCI.SIGNIFICANCE STATEMENTIn response to stress or dangerous stimuli, autonomic spinal neurons coordinate a “fight or flight” response marked by increased cardiac output and release of stress hormones. After a spinal cord injury (SCI), normally harmless stimuli like bladder filling can result in a “false” fight or flight response, causing pathological changes throughout the body. We show that progressive hypertension and immune suppression develop after SCI because thoracic excitatory VGluT2+spinal interneurons (SpINs) provoke structural remodeling in autonomic networks within below-lesion spinal levels. These pathological changes can be prevented in SCI mice or phenocopied in uninjured mice using chemogenetics to selectively manipulate activity in VGluT2+SpINs. Targeted neuromodulation of SpINs could prevent structural plasticity and subsequent autonomic dysfunction in people with SCI.

This article advances the thesis that Hagar’s statement in Gen. 21.16, ‘Let me not look upon the death of the child’, is not so much a despairing whimper of resignation as it is a cohortative prayer for divine intervention. Accordingly, the ‘casting’ of her son under a bush is not an act of exposure, but a signal of the child’s availability for adoption. Attending to the vocabulary and syntax of Hagar’s ordeal, then, we understand the scene to represent the enactment of Ishmael’s name, ‘God hears’.

Eating raw oysters can come with serious health risks, as oysters can potentially contain bacteria of the Vibrio genus that cause food-borne infections. Vibrio bacteria are concentrated by oysters and, when consumed, infections can result with severe symptoms such as diarrhoea, lesions on the extremities, or even death. Vibrio spp. concentrations are strongly affected by season, location, and other factors such as temperature and salinity. Previous research in North Carolina oysters has been conducted on wild and farmed oysters but not at the same time. Farmed, or aquaculture raised, oysters are considerably different from wild oysters and could possibly pose different health risks. Farmed oysters are handled, raised from seed, and often grown using suspended grow-out systems called 'floating cages'. Therefore, farmed oysters can be grown at the surface of the estuary, while wild oysters typically grow at the bottom of the water column. This project compared the concentrations of Vibrio spp. in suspended, farm-grown oysters and wild oysters at three sites, using a paired approach with farmed and wild oysters sampled in proximity. An important part of this comparison was identifying pathogenicity of the bacteria isolated from the samples. Distinction was made between off- and on-bottom farming. Interestingly, on-bottom oysters had more pathogenic V. vulnificus than off-bottom oysters.

We report measurements of the thermal conductance of a structure made from commercial Acrylonitrile Butadiene Styrene (ABS) modules, known as LEGO® blocks, in the temperature range from 70 mK to 1.8 K. A power law for the sample’s thermal conductivity κ = (8.7 ± 0.3) × 10−5T 1.75±0.02 WK−1 m−1 was determined. We conclude that this ABS/void compound material provides better thermal isolation than well-known bulk insulator materials in the explored temperature range, whilst maintaining solid support. LEGO blocks represent a cheap and superlative alternative to materials such as Macor or Vespel. In our setup, 2 to over 1 K, without any significant change to the base temperature of the dilution refrigerator. This work suggests that custom-built modular materials with even better thermal performance could be readily and cheaply produced by 3D printing.

Since we still lack a theory of classical turbulence, attention has focused on the conceptually simpler turbulence in quantum fluids. Reaching a better understanding of the quantum case may provide additional insight into the classical counterpart. That said, we have hitherto lacked detectors capable of the real-time, non-invasive probing of the wide range of length scales involved in quantum turbulence. Here we demonstrate the real-time detection of quantum vortices by a nanoscale resonant beam in superfluid 4He at 10 mK. Essentially, we trap a single vortex along the length of a nanobeam and observe the transitions as a vortex is either trapped or released, detected through the shift in the beam resonant frequency. By exciting a tuning fork, we control the ambient vortex density and follow its influence on the vortex capture and release rates demonstrating that these devices are capable of probing turbulence on the micron scale. Previous work has shown the detection of quantum turbulence with mechanical resonators but with limited spatial and temporal resolution. Here, the authors demonstrate real-time detection of single quantum vortices in superfluid 4He with millisecond and micron resolution at temperatures of 10 millikelvin.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Wastewater surveillance for SARS-CoV-2 has garnered extensive public attention during the COVID-19 pandemic as a proposed complement to existing disease surveillance systems. Over the past year, environmental microbiology and engineering researchers have advanced methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage and demonstrated that the trends in wastewater are correlated with trends in cases reported days to weeks later depending on the location. At the start of the pandemic, the virus was also detected in wastewater in locations prior to known cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, it is necessary to bridge the barriers between researchers and the public health responders who will ultimately use the data. Here we describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions. This perspective was formed from a multidisciplinary group of environmental microbiology, engineering, wastewater, and public health experts, as well as from opinions shared during three focus group discussions with officials from ten state and local public health agencies. The key barriers to use of wastewater surveillance data identified were: (1) As a new data source, most public health agencies are not yet comfortable interpreting wastewater data; (2) Public health agencies want to see SARS-CoV-2 wastewater data in their own communities to gain confidence in its utility; (3) New institutional knowledge and increased capacity is likely needed to sustain wastewater surveillance systems; and (4) The ethics of wastewater surveillance data collection, sharing, and use are not yet established. Overall, while wastewater surveillance to assess community infections is not a new idea, by addressing these barriers, the COVID-19 pandemic may be the initiating event that turns this emerging public health tool into a sustainable nationwide surveillance system.

Examinations of stormwater delivery in the context of tidal inundation are lacking. Along the coastal plains of the southeastern United States, tidal inundation is increasing in frequency and severity, often with dramatic adverse impacts on timely stormwater discharge, coastal flooding hazards, and even "sunny day flooding". Therefore, a comprehensive study was conducted to examine tidally-influenced stormwater outfalls discharging to Taylor's Creek, an estuary proximal to Beaufort, NC used regularly for recreation and tourism. Over a wide range of meteorological conditions, water samples were collected and analyzed for fecal indicator bacteria (FIB, used for water quality management) and previously published quantitative microbial source tracking (qMST) markers. Nineteen sampling events were conducted from July 2017-June 2018 with samples classified according to tidal state and defined as either inundated, receding, or transition. A first-of-its-kind multiple linear regression model was developed to predict concentrations of Enterococcus sp. by tidal cycle, salinity and antecedent rainfall. We demonstrated that the majority of variability associated with the concentration of Enterococcus sp. could be predicted by E. coli concentration and tidal phase. FIB concentrations were significantly (0.05) influenced by tide with higher concentrations observed in samples collected during receding (low) tides (EC: log 3.12 MPN/100 mL; ENT: 2.67 MPN/100 mL) compared to those collected during inundated (high) (EC: log 2.62 MPN/100 mL; ENT: 2.11 MPN/100 mL) or transition (EC: log 2.74 MPN/100 mL; ENT: 2.53 MPN/100 mL) tidal periods. Salinity, was also found to significantly (0.05) correlate with Enterococcus sp. concentrations during inundated (high) tidal conditions (sal: 17 ppt; ENT: 2.04 MPN/100 mL). Tide, not precipitation, was shown to be a significant driver in explaining the variability in Enterococcus sp. concentrations. Precipitation has previously been shown to be a driver of Enterococcus sp. concentrations, but our project demonstrates the need for tidal parameters to be included in the future development of water quality monitoring programs.

People infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shed the virus and its genetic material via their sputum, nasopharyngeal secretions, saliva, urine and feces (Cevik et al.2021). Hence, public health and water quality scientists throughout the world have been monitoring untreated and/or primary treated wastewater and sludge for the surveillance of SARS-CoV-2 in communities (https://arcg.is/1aummW). Numerous reviews have discussed the possibility of SARS-CoV-2 transmission to humans from exposure to wastewater or waters receiving untreated or inadequately treated wastewater based on limited empirical evidence (Adelodun et al. 2020;Bilal et al. 2020;Olusola-Makinde and Reuben 2020;Elsamadony et al. 2021;Khorram-Manesh, Goniewicz and Burkle 2021;Shutler et al. 2021). Multiple transmission routes have been suggested, including waterborne transmission, airborne transmission, contact with contaminated surfaces (fomites) and subsequent touching of mucous membranes such as the mouth, nose, or eyes. Herein, we briefly summarize the empirical evidence pertaining to the transmission of SARS-CoV-2 associated with wastewater exposure.

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus to be the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to 105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases when lagging 5 to 12 days (ρ = 0.52–0.92, p, Graphical abstract Unlabelled Image

Traumatic spinal cord injury (SCI) causes widespread damage to neurons, glia and endothelia located throughout the spinal parenchyma. In response to the injury, resident and blood-derived leukocytes orchestrate an intraspinal inflammatory response that propagates secondary neuropathology and also promotes tissue repair. SCI also negatively affects autonomic control over peripheral immune organs, notably the spleen. The spleen is the largest secondary lymphoid organ in mammals, with major roles in blood filtration and host defense. Splenic function is carefully regulated by neuroendocrine mechanisms that ensure that the immune responses to infection or injury are proportionate to the initiating stimulus, and can be terminated when the stimulus is cleared. After SCI, control over the viscera, including endocrine and lymphoid tissues is lost due to damage to spinal autonomic (sympathetic) circuitry. This review begins by examining the normal structure and function of the spleen including patterns of innervation and the role played by the nervous system in regulating spleen function. We then describe how after SCI, loss of proper neural control over splenic function leads to systems-wide neuropathology, immune suppression and autoimmunity. We conclude by discussing opportunities for targeting the spleen to restore immune homeostasis, reduce morbidity and mortality, and improve functional recovery after SCI.

Since the onset of fisheries science, monitoring programs have been implemented to support stock assessments and fisheries management. Here, we take inventory of the monitoring programs of the U.S. Gulf of Mexico (GOM) surveying fish and invertebrates and conduct a gap analysis of these programs. We also compile a large monitoring database encompassing much of the monitoring data collected in the U.S. GOM using random sampling schemes and employ this database to fit statistical models to then map the spatial distributions of 61 fish and invertebrate functional groups, species and life stages of the U.S. GOM. Finally, we provide recommendations for improving current monitoring programs and designing new programs, and guidance for more comprehensive use and sharing of monitoring data, with the ultimate goal of enhancing the inputs provided to stock assessments and ecosystem-based fisheries management (EBFM) projects in the U.S. GOM. Our inventory revealed that 73 fisheries-independent and fisheries-dependent programs have been conducted in the U.S. GOM, most of which (85%) are still active. One distinctive feature of monitoring programs of the U.S. GOM is that they include many fisheries-independent surveys conducted almost year-round, contrasting with most other marine regions. A major sampling recommendation is the development of a coordinated strategy for collecting diet information by existing U.S. GOM monitoring programs for advancing EBFM.

Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult. Here, we use droplet digital Polymerase Chain Reaction (digital PCR) and digital reverse transcriptase PCR (digital RT-PCR) assays for direct quantification of pathogenic viruses, pathogenic bacteria, and source-specific markers of fecal contamination in the stormwater discharges. We applied these assays across multiple storm events from two different watersheds that discharge to popular surfing beaches in San Diego, CA. Stormwater discharges had higher FIB concentrations as compared to proximal beaches, often by ten-fold or more during wet weather. Multiple lines of evidence indicated that the stormwater discharges contained human fecal contamination, despite the presence of separate storm sewer and sanitary sewer systems in both watersheds. Human fecal source markers (up to 100% of samples, 20-12440 HF183 copies per 100 ml) and human norovirus (up to 96% of samples, 25-495 NoV copies per 100 ml) were routinely detected in stormwater discharge samples. Potential bacterial pathogens were also detected and quantified: Campylobacter spp. (up to 100% of samples, 16-504 gene copies per 100 ml) and Salmonella (up to 25% of samples, 6-86 gene copies per 100 ml). Other viral human pathogens were also measured, but occurred at generally lower concentrations: adenovirus (detected in up to 22% of samples, 14-41 AdV copies per 100 ml); no enterovirus was detected in any stormwater discharge sample. Higher concentrations of avian source markers were noted in the stormwater discharge located immediately downstream of a large bird sanctuary along with increased Campylobacter concentrations and notably different Campylobacter species composition than the watershed that had no bird sanctuary. This study is one of the few to directly measure an array of important bacterial and viral pathogens in stormwater discharges to recreational beaches, and provides context for stormwater-based management of beaches during high risk wet-weather periods. Furthermore, the combination of culture-based and digital PCR-derived data is demonstrated to be valuable for assessing hydrographic relationships, considering delivery mechanisms, and providing foundational exposure information for risk assessment.

Graphical abstract, Throughout the COVID-19 global pandemic there has been significant interest and investment in using Wastewater-Based Epidemiology (WBE) for surveillance of viral pathogen presence and infections at the community level. There has been a push for widescale implementation of standardized protocols to quantify viral loads in a range of wastewater systems. To address concerns regarding sensitivity, limits of quantification, and large-scale reproducibility, a comparison of two similar workflows using RT-qPCR and RT-ddPCR was conducted. Sixty raw wastewater influent samples were acquired from nine distinct wastewater treatment plants (WWTP’s) served by the Hampton Roads Sanitation District (HRSD, Virginia Beach, Virginia) over a 6-month period beginning March 9th, 2020. Common reagents, controls, master mixes and nucleic acid extracts were shared between two individual processing groups based out of HRSD and the UNC Chapel Hill Institute of Marine Sciences (IMS, Morehead City, North Carolina). Samples were analyzed in parallel using One-Step RT-qPCR and One-Step RT-ddPCR with Nucleocapsid Protein 2 (N2) specific primers and probe. Influent SARS-CoV-2 N2 concentrations steadily increased over time spanning a range from non-detectable to 2.13E + 05 copies/L. Systematic dilution of the extracts indicated that inhibitory components in the wastewater matrices did not significantly impede the detection of a positive N2 signal for either workflow. The RT-ddPCR workflow had a greater analytical sensitivity with a lower Limit of Detection (LOD) at 0.066 copies/μl of template compared to RT-qPCR with a calculated LOD of 12.0 copies/μL of template. Interlaboratory comparisons using non-parametric correlation analysis demonstrated that there was a strong, significant, positive correlation between split extracts when employing RT-ddPCR for analysis with a ρ value of 0.86.

Vibrio parahaemolyticus is a ubiquitous and abundant member of native microbial assemblages in coastal waters and shellfish. Though V. parahaemolyticus is predominantly environmental, some strains have infected human hosts and caused outbreaks of seafood-related gastroenteritis. In order to understand differences among clinical and environmental V. parahaemolyticus strains, we used high quality DNA sequencing data to compare the genomes of V. parahaemolyticus isolates (n = 43) from a variety of geographic locations and clinical and environmental sample matrices. We used phylogenetic trees inferred from multilocus sequence typing (MLST) and whole-genome (WG) alignments, as well as a novel classification and genome clustering approach that relies on protein motif fingerprints (MFs), to assess relationships between V. parahaemolyticus strains and identify novel molecular targets associated with virulence. Differences in strain clustering at more than one position were observed between the MLST and WG phylogenetic trees. The WG phylogeny had higher support values and strain resolution since isolates of the same sequence type could be differentiated. The MF analysis revealed groups of protein motifs that were associated with the pathogenic MLST type ST36 and a large group of clinical strains isolated from human stool. A subset of the stool and ST36-associated protein motifs were selected for further analysis and the motif sequences were found in genes with a variety of functions, including transposases, secretion system components and effectors, and hypothetical proteins. DNA sequences associated with these protein motifs are candidate targets for future molecular assays in order to improve surveys of pathogenic V. parahaemolyticus in the environment and seafood.

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to “new” water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the “new” laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials.

Role abandonment once was considered unlikely by research scientists; however emergency management officials have experienced catastrophic events that counter prior assumptions. Event types such as deluges and pandemics surface as scenarios supporting one set of examples. The authors explore a different angle, focusing on individual practitioners including: (1) fire, (2) police and (3) emergency medical services. Surveys were taken by the various practitioner group types. Results suggested that there may be role abandonment issues, differing from one practitioner type to another, each with unique reasons given the event type. Although communities and individual emergency officials may never encounter such situations, it's imperative that this event type be taken into account during the design and implementation of disaster management systems. Systems developed should be designed to support and modify needs given the size and magnitude of the event, be it a routine emergency, a larger disaster or a 'once in a lifetime' catastrophic event. In this case, the authors focus on human resources. It is for this reason that the authors believe that algorithms be identified, developed and implemented so that such information be accessible to emergency officials, should this rare situation arise.

Spinal cord injury (SCI) damages supraspinal control over spinal autonomic circuitry. As a result, normally innocuous visceral or somatic stimuli (e.g. distended bladder or bowel) trigger pathological dysautonomia caused by uncontrolled reflex activation of sympathetic circuitry. In this report we show that remarkable structural remodeling and synaptic plasticity occurs within spinal autonomic circuitry and that this increases the frequency of autonomic dysreflexia (AD), a life-threatening condition of episodic vascular hypertension that develops progressively in SCI animals and people. We also prove that maladaptive plasticity can be prevented if SCI mice are treated with human equivalent doses of the FDA-approved drug, gabapentin (GBP) before the onset of plasticity. Although GBP is used clinically to treat pain or spasticity after symptom onset, we tested the hypothesis that GBP would prevent maladaptive plasticity if it were used as a prophylactic therapy, starting before the onset of a critical period of enhanced synaptogenesis with maintenance dosing continued for up to 35d post-injury. Using this novel treatment regimen, we show that prophylactic GBP successfully blocks multi-segmental excitatory synaptogenesis within spinal autonomic lamina, abolishes sprouting of autonomic neurons that innervate immune organs and also the sensory afferents that trigger pain and AD. Functionally, this treatment protocol delays the onset and decreases the frequency of spontaneous AD, confers resistance to experimentally induced AD, and protects mice from SCI-induced immune suppression. These data show that GBP could be repurposed as a prophylactic therapy in at-risk individuals with a high-level SCI to prevent the pathological effects of autonomic dysfunction.

Flexural mode vibrations of miniature piezoelectric tuning forks (TFs) are known to be highly sensitive to superfluid excitations and quantum turbulence in 3He and 4He quantum fluids, as well as to the elastic properties of solid 4He, complementing studies by large scale torsional resonators. Here, we explore the sensitivity of a TF, capable of simultaneously operating in both the flexural and torsional modes, to excitations in the normal and superfluid 4He. The torsional mode is predominantly sensitive to shear forces at the sensor–fluid interface and much less sensitive to changes in the density of the surrounding fluid when compared to the flexural mode. Although we did not reach the critical velocity for the quantum turbulence onset in the torsional mode, due to its order of magnitude higher frequency and increased acoustic damping, the torsional mode was directly sensitive to fluid excitations, linked to quantum turbulence created by the flexural mode. The combination of two dissimilar modes in a single TF sensor can provide a means to study the details of elementary excitations in quantum liquids and at interfaces between solids and quantum fluid.Flexural mode vibrations of miniature piezoelectric tuning forks (TFs) are known to be highly sensitive to superfluid excitations and quantum turbulence in 3He and 4He quantum fluids, as well as to the elastic properties of solid 4He, complementing studies by large scale torsional resonators. Here, we explore the sensitivity of a TF, capable of simultaneously operating in both the flexural and torsional modes, to excitations in the normal and superfluid 4He. The torsional mode is predominantly sensitive to shear forces at the sensor–fluid interface and much less sensitive to changes in the density of the surrounding fluid when compared to the flexural mode. Although we did not reach the critical velocity for the quantum turbulence onset in the torsional mode, due to its order of magnitude higher frequency and increased acoustic damping, the torsional mode was directly sensitive to fluid excitations, linked to quantum turbulence created by the flexural mode. The combination of two dissimilar modes in a sin...

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made.

Summary Ormen Lange is Norway's second largest gas field. The key uncertainties for field management and late life development are aquifer influx and reservoir compressibility. Seismic data quality over the field is extremely challenging and expected seismic changes due to production were predicted to be small. Therefore, a multi-faceted program of reservoir surveillance was planned, consisting of seismic, gravity and seafloor geodesy. Combining and calibrating all the recorded surveillance data has given us a rich dataset for interpretation and has provided crucial information on lateral connectivity, compressibility and water movement.

SUMMARY After spinal cord injury (SCI), normally innocuous visceral or somatic stimuli can trigger uncontrolled reflex activation of sympathetic circuitry, causing pathological dysautonomia. We show that remarkable structural remodeling and plasticity occur within spinal autonomic circuitry, creating abnormal sympathetic reflexes that promote dysautonomia. However, when mice are treated early after SCI with human-equivalent doses of the US Food and Drug Administration (FDA)-approved drug gabapentin (GBP), it is possible to block multi-segmental excitatory synaptogenesis and abolish sprouting of autonomic neurons that innervate immune organs and sensory afferents that trigger pain and autonomic dysreflexia (AD). This “prophylactic GBP” regimen decreases the frequency and severity of AD and protects against SCI-induced immune suppression. These benefits persist even 1 month after stopping treatment. GBP could be repurposed to prevent dysautonomia in at-risk individuals with high-level SCI., In Brief Brennan et al. show that α2δ−1 calcium channel subunits drive remarkable structural reorganization of autonomic circuitry and autonomic dysfunction after spinal cord injury. Early (prophylactic) post-injury treatment with gabapentin, an FDA-approved drug, prevents α2δ−1-dependent structural changes and autonomic dysfunction. Prophylactic gabapentin could be repurposed clinically for at-risk individuals., Graphical Abstract

Copyright © 2020 The Author(s). Background: Pollution – unwanted waste released to air, water, and land by human activity – is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources – coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children’s risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals – phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste – can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South – environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth’s resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted. Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored. Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health. Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress. Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries. Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas. The Centre Scientifique de Monaco, the Prince Albert II of Monaco Foundation and the Government of the Principality of Monaco John J. Stegeman is supported by U.S. Oceans and Human Health Program (NIH grant P01ES028938 and National Science Foundation grant OCE-1840381). Lora E. Fleming is supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 774567 (H2020 SOPHIE Project) and No 666773 (H2020 BlueHealth Project). Plastic toxicity research for Dimitri Deheyn is supported by the BEST Initiative (https://deheynlab.ucsd.edu/best-2/). Barbara Demeneix is supported by grants from the program H2020. Charles J. Dorman is supported by Science Foundation Ireland Investigator Award 13/IA/1875. William H. Gaze is supported by a Natural Environment Research Council Knowledge Exchange Fellowship NE/S006257/1 on the environmental dimension of antimicrobial resistance. Philippe Grandjean is supported by National Institute of Environmental Health Sciences (NIEHS) of the NIH (grant No. ES027706), a Superfund center grant for the Sources, Transport, Exposure and Effects of Perfluoroalkyl Substances (STEEP) Center. Mark E. Hahn is supported by U.S. Oceans and Human Health Program (NIH grant P01ES028938 and National Science Foundation grant OCE-1840381). Amro Hamdoun is supported by NIH and NSF Program on Oceans and Human Health Grants NIH ES030318 and NSF 1840844. Philipp Hess is supported by the IAEA Core Research Project K41014, by the European H2020 program for funding the EMERTOX project (grant number 778069), by the Atlantic Interreg (grant number Alertox-Net EAPA-317-2016) and by EFSA for the project EUROCIGUA (framework partnership agreement GP/EFSA/AFSCO/2015/03). Rachel T. Noble was supported by the US National Science Foundation Accelerating Innovations in Research #1602023 and the NOAA NERRS Science Collaborative. Maria Luiza Pedrotti is supported by Centre National de la Recherche Scientifique (CNRS). Luigi Vezzulli is supported by the following grants: European FP7 Program Grant AQUAVALENS 311846 and European Union’s Horizon 2020 Research and Innovation Program Grant VIVALDI 678589. Pál Weihe is supported by the Danish EPA programme: Danish Cooperation for Environment in the Arctic and by the Faroese Research Council.

Fecal contamination is observed downstream of municipal separate storm sewer systems in coastal North Carolina. While it is well accepted that wet weather contributes to this phenomenon, less is understood about the contribution of the complex hydrology in this low-lying coastal plain. A quantitative microbial assessment was conducted in Beaufort, North Carolina to identify trends and potential sources of fecal contamination in stormwater receiving waters. Fecal indicator concentrations were significantly higher in receiving water downstream of a tidally submerged outfall compared to an outfall that was permanently submerged (p 0.001), though tidal height was not predictive of human-specific microbial source tracking (MST) marker concentrations at the tidally submerged site. Short-term rainfall (i.e.12 h) was predictive of E. coli, Enterococcus spp., and human-specific MST marker concentrations (Fecal Bacteroides, BacHum, and HF183) in receiving waters. The strong correlation between 12-hr antecedent rainfall and Enterococcus spp. (r = 0.57, p 0.001, n = 92) suggests a predictive model could be developed based on rainfall to communicate risk for bathers. Additional molecular marker data indicates that the delivery of fecal sources is complex and highly variable, likely due to the influence of tidal influx (saltwater intrusion from the estuary) into the low-lying stormwater pipes. In particular, elevated MST marker concentrations (up to 2.56 × 10

This article seeks to elucidate the nature and significance of the enigmatic mantle transfer rite involving Elijah and Elisha in 1 Kgs 19.19. When Yahweh instructs Elijah to anoint Elisha to be prophet in his place, Yahweh has a type of symbolic adoption in mind to take Elisha as an heir through the rite of mantle transfer. This article argues that Elijah's mantle is a marker of his identity, and that the conferment of his clothing is a means of claiming Elisha, who subsequently takes leave of his natural father (v. 20). Elijah and Elisha function in the northern kingdom as cultic prophets, performing duties typical of both prophet and priest, not unlike Samuel. This double role gives context for an analogy—or possible typology—for Elisha's inheritance with that of the Levitical priest in Deuteronomy.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms.

Rapid qPCR methods for enumerating enterococci can provide results in a few hours, thereby enhancing public health protection. Analysis of 140 samples collected from 11 beaches in Hawaii for enterococci using EPA Method 1611 revealed that a majority (70%) of samples yielded unusable data using the recommended protocol due to DNA losses during extraction. The DNA loss was correlated to the presence of suspended coralline sand. Acidification of samples alleviated or removed the interference, enabling successful method application. There were significant correlations across the three methods evaluated (Enterolert®/Method 1600 R2 = 0.85, Enterolert®/ Method 1611 R2 = 0.78 and Method 1600/1611 R2 = 0.82). When samples were not compromised, there was also good agreement among methods for beach management decisions. This study presents a protocol for beach areas with coralline sands, and re-emphasizes the need to use appropriate controls to prevent underestimation of bacterial concentrations at recreational beaches.

The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously. The method is from: Patel A, Noble RT, Steele JA, Schwalbach MS, Hewson I, Fuhrman JA.Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nat Protoc. 2007;2(2):269-76. Please see the full manuscript for more details.

Of marine eubacteria, the genus Vibrio is intriguing because member species are relevant to both marine ecology and human health. Many studies have touted the relationships of Vibrio to environmental factors, especially temperature and salinity, to predict total Vibrio abundance but lacked the taxonomic resolution to identify the relationships among species and the key drivers of Vibrio dynamics. To improve next-generation sequencing (NGS) surveys of Vibrio , we have conducted both 16S small subunit rRNA and heat shock protein 60 ( hsp60 ) amplicon sequencing of water samples collected at two well-studied locations in the Neuse River Estuary, NC. Samples were collected between May and December 2016 with enhanced sampling efforts in response to two named storms. Using hsp60 sequences, 21 Vibrio species were identified, including the potential human pathogens V. cholerae , V. parahaemolyticus , and V. vulnificus . Changes in the Vibrio community mirrored seasonal and storm-related changes in the water column, especially in response to an influx of nutrient-rich freshwater to the estuary after Hurricane Matthew, which initiated dramatic changes in the overall Vibrio community. Individual species dynamics were wide ranging, indicating that individual Vibrio taxa have unique ecologies and that total Vibrio abundance predictors are insufficient for risk assessments of potentially pathogenic species. Positive relationships between Vibrio , dinoflagellates, and Cyanobacteria were identified, as were intraspecies associations, which further illuminated the interactions of cooccurring Vibrio taxa along environmental gradients. IMPORTANCE The objectives of this research were to utilize a novel approach to improve sequence-based surveys of Vibrio communities and to demonstrate the usefulness of this approach by presenting an analysis of Vibrio dynamics in the context of environmental conditions, with a particular focus on species that cause disease in humans and on storm effects. The methods presented here enabled the analysis of Vibrio dynamics with excellent taxonomic resolution and could be incorporated into future ecological studies and risk prediction strategies for potentially pathogenic species. Next-generation sequencing of hsp60 and other innovative sequence-based approaches are valuable tools and show great promise for studying Vibrio ecology and associated public health risks.

Of marine eubacteria, the genus Vibrio is intriguing because member species are relevant to both marine ecology and human health. Many studies have touted the relationships of Vibrio to environmental factors, especially temperature and salinity, to predict total Vibrio abundance but lacked the taxonomic resolution to identify the relationships among species and the key drivers of Vibrio dynamics. To improve next-generation sequencing (NGS) surveys of Vibrio, we have conducted both 16S small subunit rRNA and heat shock protein 60 (hsp60) amplicon sequencing of water samples collected at two well-studied locations in the Neuse River Estuary, NC. Samples were collected between May and December 2016 with enhanced sampling efforts in response to two named storms. Using hsp60 sequences, 21 Vibrio species were identified, including the potential human pathogens V. cholerae, V. parahaemolyticus, and V. vulnificus. Changes in the Vibrio community mirrored seasonal and storm-related changes in the water column, especially in response to an influx of nutrient-rich freshwater to the estuary after Hurricane Matthew, which initiated dramatic changes in the overall Vibrio community. Individual species dynamics were wide ranging, indicating that individual Vibrio taxa have unique ecologies and that total Vibrio abundance predictors are insufficient for risk assessments of potentially pathogenic species. Positive relationships between Vibrio, dinoflagellates, and Cyanobacteria were identified, as were intraspecies associations, which further illuminated the interactions of cooccurring Vibrio taxa along environmental gradients.

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.