Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus normalization.

Wastewater surveillance allows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection levels to be tracked in a community. Here, we present a protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using quantitative reverse-transcription PCR (RT-qPCR) and pepper mild mottle virus (PMMoV) normalization. We describe steps for the pasteurization of wastewater samples, solids separation, supernatant filtration, viral precipitation and concentration, and RNA extraction. We then detail procedures for RT-qPCR, viral concentration extrapolation, PMMoV normalization, and longitudinal analysis. This protocol has the potential to be used for surveillance of other microorganisms. For complete details on the use and execution of this protocol, please refer to Sanchez Jimenez et al.1.

Trends of Self-Inflicted Firearm Injuries at a Rural Level 1 Trauma Center.

Firearm injuries are a major public health concern with much focus on injuries due to violent crimes in urban areas. Less focus has been on self-inflicted injuries and rural settings. This study included 201 patients, of which 124 (61.7%) were accidental and 77 (38.3%) were intentional self-inflicted gunshot wounds (GSWs) sustained over 6 years at a rural level 1 trauma center. Injury severity scores (P < .001), hospital days (P < .001), and mortality (P < .001) were significantly higher among intentional self-inflicted GSWs. Injuries to the head were the most common injury among patients with intentional self-inflicted GSWs (P < .001).Accidental and intentional self-inflicted GSWs make up a large portion of firearm injuries seen at our rural level 1 trauma center, and defining these injuries can facilitate the need for targeted gun safety and injury prevention efforts.

Antithrombotics in the Fall Patient: Appropriateness and Risk-Benefit Analysis.

Falls are the leading cause of hospitalizations following trauma nationwide, resulting in over 3 million admissions in 2020. This population is typically aged, and many are prescribed antithrombotic (AT) therapy. In this prospective study, we aimed to analyze fall history while assessing appropriateness of AT regimen relative to fall risk. Patients presenting following ground level fall (GLF) and meeting inclusion criteria during the study period were enrolled. Primary outcome was the relationship between AT therapy necessity (CHA2DS2-VASc) and fall risk (Morse Fall Risk). The cohort of 30 patients had an average age of 77. CHA2DS2-VASc and Morse Fall Risk showed a moderate-positive correlation (r = 0.47; P = 0.012); however, 17% of patients categorized as high fall risk had a <5% 1-year risk of VTE. This study demonstrates that risks of hemorrhage may outweigh thromboembolism prophylaxis in a significant number of patients and sheds light on the astonishing fall volume in this population.

Outcome of Seatbelt Education and Safety Program Among Teenagers.

Despite the effectiveness of seatbelts, concerns persist about compliance, especially among teenagers. Survey data from a local high school and registry data from a level 1 trauma center were used to observe seatbelt and motor vehicle accident trends. The survey data was analyzed to gauge student's sentiments on seatbelt education. The trauma center data was analyzed to identify characteristics and trends among teenage motor vehicle accidents. Social media was the most common strategy selected for seatbelt safety awareness. Random seatbelt checks performed over 4 months revealed seatbelt compliance rates of 90%, 93.55%, and 96.94% after education intervention. Trauma center data showed that lack of seatbelt usage resulted in greater morbidity. These findings emphasize the need for targeted interventions. This study provides insights into creating effective education campaigns that can be used to enhance safety belt compliance and potentially reduce injury.

Design and Evaluation of PROTACs Targeting Acyl Protein Thioesterase 1.

PROTAC linker design remains mostly an empirical task. We employed the PRosettaC computational software in the design of sulfonyl-fluoride-based PROTACs targeting acyl protein thioesterase 1 (APT1). The software efficiently generated ternary complex models from empirically-designed PROTACs and suggested alkyl linkers to be the preferred type of linker to target APT1. Western blotting analysis revealed efficient degradation of APT1 and activity-based protein profiling showed remarkable selectivity of an alkyl linker-based PROTAC amongst serine hydrolases. Collectively, our data suggests that combining PRosettaC and chemoproteomics can effectively assist in triaging PROTACs for synthesis and providing early data on their potency and selectivity.

Wastewater surveillance in the COVID-19 post-emergency pandemic period: A promising approach to monitor and predict SARS-CoV-2 surges and evolution.

On May 24, 2023, approximately 3.5 years into the pandemic, the World Health Organization (WHO) declared the end of the COVID-19 global health emergency. However, as there are still ∼3000 COVID-19 deaths per day in May 2023, robust surveillance systems are still warranted to return to normalcy in times of low risk and respond appropriately in times of high risk. The different phases of the pandemic have been defined by infection numbers and variants, both of which have been determined through clinical tests that are subject to many biases. Unfortunately, the end of the COVID-19 emergency threatens to exasperate these biases, thereby warranting alternative tracking methods. We hypothesized that wastewater surveillance could be used as a more accurate and comprehensive method to track SARS-CoV-2 in the post-emergency pandemic period (PEPP). SARS-CoV-2 was quantified and sequenced from wastewater between June 2022 and March 2023 to research the anticipated 2022/23 winter surge. However, in the 2022/23 winter, there was lower-than-expected SARS-CoV-2 circulation, which was hypothesized to be due to diagnostic testing biases but was confirmed by our wastewater analysis, thereby emphasizing the unpredictable nature of SARS-CoV-2 surges while also questioning its winter seasonality. Even in times of low baseline circulation, we found wastewater surveillance to be sensitive enough to detect minor changes in circulation levels ∼30-46 days prior to diagnostic tests, suggesting that wastewater surveillance may be a more appropriate early warning system to prepare for unpredictable surges in the PEPP. Furthermore, sequencing of wastewater detected variants of concern that were positively correlated with clinical samples and also provided a method to identify mutations with a high likelihood of appearing in future variants, necessary for updating vaccines and therapeutics prior to novel variant circulation. Together, these data highlight the effectiveness of wastewater surveillance in the PEPP to limit the global health burden of SARS-CoV-2 due to increases in circulation and/or viral evolution.

A multi-study analysis of gut microbiome data from the blue mussel (Mytilus edulis) emphasises the impact of depuration on biological interpretation.

The blue mussel (Mytilus edulis) is a suspension feeder which has been used in gut-microbiome surveys. Although raw 16S sequence data are often publicly available, unifying secondary analyses are lacking. The present work analysed raw data from seven projects conducted by one group over 7 years. Although each project had different motivations, experimental designs and conclusions, all selected samples were from the guts of M. edulis collected from a single location in Long Island Sound. The goal of this analysis was to determine which independent factors (e.g., collection date, depuration status) were responsible for governing composition and diversity in the gut microbiomes. Results indicated that whether mussels had undergone depuration, defined here as voidance of faeces in a controlled, no-food period, was the primary factor that governed gut microbiome composition. Gut microbiomes from non-depurated mussels were mixtures of resident and transient communities and were influenced by temporal factors. Resident communities from depurated mussels were influenced by the final food source and length of time host mussels were held under laboratory conditions. These findings reinforce the paradigm that gut microbiota are divided into resident and transient components and suggest that depuration status should be taken into consideration when designing and interpreting future experiments.

Overdose and mortality risk following a non-fatal opioid overdose treated by Emergency Medical Services in King County, Washington.

BACKGROUND: Emergency Medical Services (EMS) agencies respond to hundreds of thousands of acute overdose events each year. We conducted a retrospective cohort study of EMS patients who survived a prior opioid overdose in 2019-2021 in King County, Washington. METHODS: A novel record linkage algorithm was applied to EMS electronic health records and the state vital statistics registry to identify repeat overdoses and deaths that occurred up to 3 years following the index opioid overdose. We measured overdose incidence rates and applied survival analysis techniques to assess all-cause and overdose-specific mortality risks. RESULTS: In the year following the index opioid overdose, the overdose (fatal or non-fatal) incidence rate was 23.3 per 100 person-year, overdose mortality rate was 2.7 per 100 person-year, and all-cause mortality rate was 5.2 per 100 person-year in this cohort of overdose survivors (n=4234). Overdose incidence was highest in the first 30 days following the index overdose (43 opioid overdoses and 4 fatal overdoses per 1000 person-months), declined precipitously, and then plateaued from the third month onwards (10-15 opioid overdoses and 1-2 fatal overdoses per 1000 person-months). Overdose incidence rates, measured at 30 days, were highest among overdose survivors who were young, male, and experienced a low severity index opioid overdose, but these differences diminished when measured at 12 months. CONCLUSIONS: Among EMS patients who survived an opioid overdose, the risk of subsequent overdose is high, especially in the weeks following the index opioid overdose. Non-fatal overdose may represent a pivotal time to connect patients with harm-reduction, treatment, and other support services.

Fluorescence lifetime of injected indocyanine green as a universal marker of solid tumours in patients.

The surgical resection of solid tumours can be enhanced by fluorescence-guided imaging. However, variable tumour uptake and incomplete clearance of fluorescent dyes reduces the accuracy of distinguishing tumour from normal tissue via conventional fluorescence intensity-based imaging. Here we show that, after systemic injection of the near-infrared dye indocyanine green in patients with various types of solid tumour, the fluorescence lifetime (FLT) of tumour tissue is longer than the FLT of non-cancerous tissue. This tumour-specific shift in FLT can be used to distinguish tumours from normal tissue with an accuracy of over 97% across tumour types, and can be visualized at the cellular level using microscopy and in larger specimens through wide-field imaging. Unlike fluorescence intensity, which depends on imaging-system parameters, tissue depth and the amount of dye taken up by tumours, FLT is a photophysical property that is largely independent of these factors. FLT imaging with indocyanine green may improve the accuracy of cancer surgeries.

Nylon microfibers develop a distinct plastisphere but have no apparent effects on the gut microbiome or gut tissue status in the blue mussel, Mytilus edulis.

Ingestion of microplastics (MP) by suspension-feeding bivalves has been well-documented. However, it is unclear whether exposure to MP could damage the stomach and digestive gland (gut) of these animals, causing ramifications for organism and ecosystem health. Here, we show no apparent effects of nylon microfiber (MF) ingestion on the gut microbiome or digestive tissues of the blue mussel, Mytilus edulis. We exposed mussels to two low concentrations (50 and 100 particles/L) of either nylon MF or Spartina spp. particles (dried, ground marsh grass), ca. 250-500 µm in length, or a no particle control laboratory treatment for 21 days. Results showed that nylon MF, when aged in coarsely filtered seawater, developed a different microbial community than Spartina spp. particles and seawater, however, even after exposure to this different community, mussel gut microbial communities resisted disturbance from nylon MF. The microbial communities of experimental mussels clustered together in ordination and were similar in taxonomic composition and measures of alpha diversity. Additionally, there was no evidence of damage to gut tissues after ingestion of nylon MF or Spartina spp. Post-ingestive particle processing likely mediated a short gut retention time of these relatively large particles, contributing to the negligible treatment effects.

Impact of Trauma Resuscitation Emergency Care Nurse Deployment in Trauma Activations in a Rural Trauma Center.

BACKGROUND: Although the role of a dedicated trauma nurse has been implemented in an urban setting, it has not been studied in the rural trauma setting. We instituted a trauma resuscitation emergency care (TREC) nurse role to respond to trauma activations at our rural trauma center. OBJECTIVE: This study aims to determine the impact of TREC nurse deployment on the timeliness of resuscitation interventions in trauma activations. METHODS: This pre- and postintervention study at a rural Level I trauma center compared the time to resuscitation interventions before (August 2018 to July 2019) and after (August 2019 to July 2020) deploying TREC nurses to trauma activations. RESULTS: A total of 2,593 participants were studied, of which 1,153 (44%) were in the pre-TREC group and 1,440 (56%) in the post-TREC group. After TREC deployment, the median (interquartile range [IQR]) emergency department times within the first hour decreased from 45 (31.23-53) to 35 (16-51) min ( p = .013). The median (IQR) time to the operating room within the first hour decreased from 46 (37-52) to 29 (12-46) min ( p = .001), and within the first 2 hr, decreased from 59 (43.8-86) to 48 (23-72) min ( p = .014). CONCLUSION: Our study found that TREC nurse deployment improved resuscitation intervention timeliness during the first 2 hr (early phase) of trauma activations.

Shifting Sociodemographic Characteristics of a Phase I Clinical Trial Population at an NCI-Designated Comprehensive Cancer Center in the Southeast.

Racial and ethnic minority populations are consistently under-represented in oncology clinical trials despite comprising a disproportionate share of a cancer burden. Phase I oncology clinical trials pose a unique challenge and opportunity for minority inclusion. Here we compared the sociodemographic characteristics of patients participating in phase 1 clinical trials a National Cancer Institute ( NCI)-designated comprehensive center to all patients at the center, patients with new cancer diagnosis in metropolitan Atlanta and patients with new cancer diagnoses in the state of Georgia. From 2015 to 2020, 2325 patients (43.4% female, 56.6% male) consented to participate in a phase I trial. Grouped self-reported race distribution was 70.3% White, 26.2% Black, and 3.5% other. Of new patient registrations at Winship Cancer Institute (N = 107 497) (50% F, 50% M), grouped race distribution was 63.3% White, 32.0% Black, and 4.7% other. Patients with new cancer diagnoses in metro Atlanta from 2015 to 2016 (N = 31101) were 58.4% White, 37.2% Black, and 4.3% other. Race and sex distribution of phase I patients was significantly different than Winship patients (P < .001). Over time, percent of White patients decreased in both phase I and Winship groups (P = .009 and P < .001, respectively); percentage of females did not change in either group (P = .54 phase I, P = .063 Winship). Although phase I patients were more likely to be White, male, and privately ensured than the Winship cohort, from 2015 to 2020 the percentage of White patients in phase I trials and among all new patients treated at Winship decreased. The intent of characterizing existing disparities is to improve the representation of patients from racial and ethnic minority backgrounds in phase I clinical trials.

Outcomes after stairway falls in a rural Appalachian trauma center.

BACKGROUND: Injuries due to falls represent one of the most common etiologies of traumatic injury in the United States. Stairway-related falls in particular can lead to significant morbidity, mortality, and concomitant long-term disability and economic costs. Our study aims to evaluate the outcomes of patients presenting to a rural academic trauma center after experiencing a fall down stairs. METHODS: This was a single institution retrospective analysis of data extracted from our trauma registry. The study was considered exempt by Ballad Health Institutional Review Board. The data included patients aged 18 years or older who presented to the emergency department after a fall down stairs between January 1, 2017, and June 17, 2022. Patients who experienced falls other than those involving stairs were excluded. RESULTS: Of the 439 patients evaluated for falls down stairs, 259 (58.9%) were aged ≥65 years. Compared with younger patients, older patients required significantly longer hospital admissions (4.8 vs 3.6 days, P < .003), had significantly higher injury severity scores (9.1 vs 6.8, P < .05), and were more likely to be discharged to a posthospital care facility (51% vs 14.9%, P < .05). There was no difference in length of intensive care unit stay (3.8 vs 3.6 days, P < .72), ventilator days (3.3 vs 3.3 days, P < .97), or mortality (7% vs 3%, P < .08). When considering sex, male patients had significantly worse outcomes in injury severity score (9.0 vs 7.6, P < .02) and mortality (10% vs 2%, P < .0002) but no difference in hospital (4.5 vs 4.0 days, P < .20), intensive care unit (3.8 vs 3.5 days, P < .59) or ventilator days (2.8 vs 4.3 days, P < .27) when compared with female patients. CONCLUSION: Patients aged 65 years or older who experience a fall down stairs are more severely injured and require more posthospital care. Our findings demonstrate that males have an elevated risk of mortality and increased injury severity compared to female patients. Previous findings from our institution examining injuries from falls, including a sub-analysis on ground-level falls, have shown similar sex disparity. This study shows the necessity of preventing stair-related falls, especially in the older population.

Monosynaptic restriction of the anterograde herpes simplex virus strain H129 for neural circuit tracing.

Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129. Given that this knockout blocks viral genome packaging and thus viral spread, we reasoned that co-infection of a HSV H129 ΔUL6 virus with a recombinant adeno-associated virus expressing UL6 in a cre-dependent manner would result in monosynaptic spread from target cre-expressing neuronal populations. Application of this system to five nonreciprocal neural circuits resulted in labeling of neurons in expected projection areas. While some caveats may preclude certain applications, this system provides a reliable method to label postsynaptic partners in a brain-wide fashion.

A Selective SARS-CoV-2 Host-Directed Antiviral Targeting Stress Response to Reactive Oxygen Species.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) catalyzed the development of vaccines and antivirals. Clinically approved drugs against SARS-CoV-2 target the virus directly, which makes them susceptible to viral mutations, which in turn can attenuate their antiviral activity. Here we report a host-directed antiviral (HDA), piperlongumine (PL), which exhibits robust antiviral activity as a result of selective induction of reactive oxygen species in infected cells by GSTP1 inhibition. Using a transgenic K18-hACE2 mouse model, we benchmarked PL against plitidepsin, a HDA undergoing phase III clinical trials. We observed that intranasal administration of PL is superior in delaying disease progression and reducing lung inflammation. Importantly, we showed that PL is effective against several variants of concern (VOCs), making it an ideal pan-variant antiviral. PL may display a critical role as an intranasal treatment or prophylaxis against a range of viruses, expanding the arsenal of tools to fight future outbreaks.

Differences in personal care product use by race/ethnicity among women in California: implications for chemical exposures.

BACKGROUND: Personal care products may contain many chemicals, some of which are suspected endocrine disrupters. This is an important source of chemical exposure for women, but little is known about how chemical exposure differs among different races/ethnicities. OBJECTIVE: This study examines differences in personal care product use among Black, Latina, Vietnamese, Mixed Race, and White women in California. METHODS: We used a community-based participatory process to create and administer a personal care product usage survey to 321 Black, Latina, Vietnamese, Mixed Race, and White women. We used multivariate regression models with pairwise comparisons to examine the frequency of product use by race/ethnicity. RESULTS: We found distinct trends of personal care product use by race/ethnicity: Latina women typically used makeup most frequently; Black women used certain hair products or styles most frequently; and Vietnamese women were most likely to use facial cleansing products compared to other races/ethnicities. Latina and Vietnamese women were less likely to try to avoid certain ingredients in their products. SIGNIFICANCE: These findings can help estimate disparities in chemical exposure from personal care product use and complement future research on health inequities due to chemical exposures in the larger environmental and social context.

Fishing for a balance: Toward promyelinating drug discovery.

In this issue, Häberlein et al. demonstrate a role for GPR17 in regulating zebrafish oligodendrocyte differentiation. Zebrafish expressing a humanized GPR17 respond to modulators, which are inactive against the endogenous zebrafish receptor. These findings highlight the potential for humanized zebrafish as an in vivo platform for targeted remyelination drug screens.

Beyond sound: bimodal acoustic calls used in mate-choice and aggression by red-eyed treefrogs.

Airborne sound signals function as key mediators of mate-choice, aggression and other social interactions in a wide range of vertebrate and invertebrate animals. Calling animals produce more than sound, however. When displaying on or near a solid substrate, such as vegetation or soil, they also unavoidably excite substrate vibrations because of the physics of sound production and of acoustic propagation, and these vibrations can propagate to receivers. Despite their near ubiquity, these vibrational signal components have received very little research attention and in vertebrates it is unknown whether they are relevant to mate-choice, an important driver of evolutionary divergence. Here, we show that female red-eyed treefrogs are more than twice as likely to choose a male mating call when airborne sound is paired with its corresponding substrate vibrations. Furthermore, males of the same species are more aggressive towards and display a greater range of aggressive behaviors in response to bimodal (sound and vibration) versus unimodal (sound or vibration alone) calls. In aggressive contexts, at least, air- and substrate-borne signal components function non-redundantly. These results are a clear demonstration that vibrations produced by a calling animal can function together with airborne sound to markedly enhance the function of a signal. If this phenomenon proves widespread, this finding has the potential to substantially influence our understanding of the function and evolution of acoustic signals.