Transcriptional profiling of mouse projection neurons with VECTORseq.

Existing techniques for transcriptional profiling of projection neurons could be applied to only one neuronal population per experiment. To increase throughput, we developed VECTORseq, which repurposes retrogradely infecting viruses to deliver multiplexable RNA barcodes, enabling projection anatomy to be read out in single-cell datasets. In this protocol, we describe the delivery of viral barcodes to mouse brain to label different projection neurons. We then detail single-cell or nuclei isolation for sequencing, followed by the analysis of single-cell sequencing data. For complete details on the use and execution of this protocol, please refer to Cheung et al. (2021).

Occurrence of COVID-19 personal protective equipment (PPE) litters along the eastern coast of Palawan Island, Philippines.

The emergence of the COVID-19 pandemic has caused worldwide health constraints. This study was conducted to establish a baseline monitoring survey to describe the distribution of PPE litters during the COVID-19 pandemic in the province of Palawan, Philippines. A total of 386 COVID-19-related PPE items were present in 83 % of coastal sampling sites with over a cumulative area of 48,200 m2, with a density of 8 × 10-3 items m-2. The facemask (98 %; n = 377) was the primary type of PPE, followed by face shield (2 %; n = 9). Meanwhile, the daily density of PPE litters in San Manuel, Puerto Princesa ranged from 0 to 9.9 × 10-2 items m-2, with a mean density of 8 × 10-3 items m-2. The accumulation rates of PPE items ranged from 3.27 × 10-1 items to 1.143 items d-1, with an average rate of 7.29 × 10-1 items d-1.

Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism.

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds nuclear pore component Nup98-Rae1 to inhibit nuclear translocation of activated STAT1 and IRF3 transcription factors. However, this direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6-Nup98 interaction domain robustly blocked nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral membrane protein, as opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication. This article has an associated First Person interview with the first author of the paper.

Association of locus coeruleus integrity with Braak stage and neuropsychiatric symptom severity in Alzheimer's disease.

The clinical and pathophysiological correlates of locus coeruleus (LC) degeneration in Alzheimer's disease (AD) could be clarified using a method to index LC integrity in vivo, neuromelanin-sensitive MRI (NM-MRI). We examined whether integrity of the LC-norepinephrine system, assessed with NM-MRI, is associated with stage of AD and with neuropsychiatric symptoms (NPS), independent of cortical pathophysiology (amyloid-ß and tau burden). Cognitively normal older adults (n = 118), and individuals with mild cognitive impairment (MCI, n = 44), and AD (n = 28) underwent MR imaging and tau and amyloid-ß positron emission tomography (with [18F]MK6240 and [18F]AZD4694, respectively). Integrity of the LC-norepinephrine system was assessed based on contrast-to-noise ratio of the LC on NM-MRI images. Braak stage of AD was derived from regional binding of [18F]MK6240. NPS were assessed with the Mild Behavioral Impairment Checklist (MBI-C). LC signal contrast was decreased in tau-positive participants (t186 = -4.00, p = 0.0001) and negatively correlated to Braak stage (Spearman ρ = -0.31, p = 0.00006). In tau-positive participants (n = 51), higher LC signal predicted NPS severity (ρ = 0.35, p = 0.019) independently of tau burden, amyloid-ß burden, and cortical gray matter volume. This relationship appeared to be driven by the impulse dyscontrol domain of NPS, which was highly correlated to LC signal (ρ = 0.44, p = 0.0027). NM-MRI reveals loss of LC integrity that correlates to severity of AD. However, LC preservation in AD may also have negative consequences by conferring risk for impulse control symptoms. NM-MRI shows promise as a practical biomarker that could have utility in predicting the risk of NPS or guiding their treatment in AD.

Improving the Pediatric Floor Discharge Process Following Tonsillectomy.

OBJECTIVES/HYPOTHESIS: Over 300,000 tonsillectomies are performed nationwide every year. In 2017, half of children undergoing tonsillectomy at our institution were admitted to the pediatric floor, with only 10.4% being discharged before 11 AM on postoperative day 1 (POD1). Our primary objective was to increase the percentage of patients discharged before 11 AM on POD1 to at least 50% within 1 year. STUDY DESIGN: Prospective observational (quality improvement). METHODS: A multidisciplinary quality improvement (QI) team was assembled. The primary outcome was "timely discharges," defined as percentage of patients discharged before 11 AM on POD1; secondary outcomes were percentage of patients discharged before 1 PM and mean length of stay (hours). Seven-day readmission rate served as our balancing measure. Prior year data served as baseline. A process map, Ishikawa diagram, and Pareto chart were utilized to identify specific target areas for improvement. Key interventions included announcement of our initiative, an electronic health record-based handoff text prompt, discharge checklist, automated discharge instructions, encouragement to place discharge orders by 9 AM and implementation of early POD1 rounds. Data were collected on a biweekly basis and the primary and secondary outcomes were plotted on control charts and analyzed using rules for special cause variation. RESULTS: Within 12 months, POD1 discharges before 11 AM and before 1 PM increased to 44.9% and 83.8%, respectively, with sustained improvement for the first 6 months of the subsequent year. Mean length of stay decreased, and 7-day readmission rates were unchanged. CONCLUSIONS: By understanding the factors influencing timely POD1 discharges after tonsillectomy, key interventions were implemented to achieve an increase in timely discharges. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:225-233, 2022.

Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration.

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

Looking Through the COVID-19 Window of Opportunity: Future Scenarios Arising From the COVID-19 Pandemic Across Five Case Study Sites.

The COVID-19 pandemic has caused (and continues to cause) severe disruption in global and local economies and has forced countries, societies, and individuals to adapt quickly to the unprecedented and unpredictable situations. Despite the obvious negative consequences of the pandemic, many have called for efforts to identify transformative opportunities for sustainable development throughout this disorderly time. In the present paper, we explore such potential opportunities in the context of an interdisciplinary, international research project, which is focusing on sustainable marine management in biosphere reserves and marine parks in Southeast Asia. During a virtual workshop conducted as part of the GCRF (Government's Global Challenges Research Fund) Blue Communities Project, future scenarios were developed depicting the potential effects of the COVID-19 pandemic on five case study sites. All of these sites are in areas of internationally recognized outstanding ecological value (Taka Bonerate Kepulauan-Selayar Biosphere Reserve, Indonesia; Tun Mustapha Park, Sabah, Malaysia; Palawan Biosphere Reserve, Philippines; North Devon Biosphere Reserve, United Kingdom; Cu Lao Cham-Hoi An Biosphere Reserve, Vietnam). At the macro-level, economies, governance structures, and societal norms are undergoing big changes. At the micro-level, the livelihoods, lifestyles, and backyards of local residents have to adapt. Collaboratively, we explored how COVID-19 posed challenges in our five case study sites, but we also focused on the potential COVID-19-related windows of opportunity for future sustainable development. Opportunities could be identified in all three pillars of sustainable development: the environment, the society, and the economy. Although remarkable similarities can be found across all five sites, we conclude that there cannot be a "one-size-fits-all" solution to turn the tide toward achieving sustainable development. Just as before the pandemic, sustainable development starts with engaging with and understanding local environments, challenges, and situations; building on local knowledge; and developing tailor-made solutions for the communities in situ.

Oesophageal rupture from a pneumatic blast injury: an unusual mechanism of blunt oesophageal trauma.

Oesophageal perforation is a serious condition associated with significant morbidity and mortality. Clinical suspicion of oesophageal injury in patients sustaining neck or torso trauma is essential as early diagnosis and management are associated with better outcomes. Oesophageal perforation resulting from blunt trauma is uncommon, and traumatic oesophageal perforation following blast injury is exceedingly rare. We present two cases of patients developing oesophageal perforation from this rare mechanism, review basic principles of management, and key learning points.

Evidence for Dopamine Abnormalities in the Substantia Nigra in Cocaine Addiction Revealed by Neuromelanin-Sensitive MRI.

OBJECTIVE: Recent evidence supports the use of neuromelanin-sensitive MRI (NM-MRI) as a novel tool to investigate dopamine function in the human brain. The authors investigated the NM-MRI signal in individuals with cocaine use disorder, compared with age- and sex-matched control subjects, based on previous imaging studies showing that this disorder is associated with blunted presynaptic striatal dopamine. METHODS: NM-MRI and T1-weighted images were acquired from 20 participants with cocaine use disorder and 35 control subjects. Diagnostic group effects in NM-MRI signal were determined using a voxelwise analysis within the substantia nigra. A subset of 20 cocaine users and 17 control subjects also underwent functional MRI imaging using the monetary incentive delay task, in order to investigate whether NM-MRI signal was associated with alterations in reward processing. RESULTS: Compared with control subjects, cocaine users showed significantly increased NM-MRI signal in ventrolateral regions of the substantia nigra (area under the receiver operating characteristic curve=0.83). Exploratory analyses did not find a significant correlation of NM-MRI signal to activation of the ventral striatum during anticipation of monetary reward. CONCLUSIONS: Given that previous imaging studies show decreased dopamine signaling in the striatum, the finding of increased NM-MRI signal in the substantia nigra provides additional insight into the pathophysiology of cocaine use disorder. One interpretation is that cocaine use disorder is associated with a redistribution of dopamine between cytosolic and vesicular pools, leading to increased accumulation of neuromelanin. The study findings thus suggest that NM-MRI can serve as a practical imaging tool for interrogating the dopamine system in addiction.

Boerhaave's syndrome in an ultra-distance runner.

A runner competing in a 100 mile trail race presented with severe lower chest pain and right upper abdominal pain. His pain started immediately after he took an over the counter non steroidal anti-inflammatory pill a few hundred metres after leaving the aid station. When he took the pill, he immediately had to vomit and spit out the pill. On arriving back at the aid station, he was noted to have severe left-sided chest pain that worsened with reclining. He also had profound dyspnoea. Initial vital signs were unremarkable. The runner was immediately transported to an emergency room and eventually found to have an oesophageal rupture. After surgical intervention and a lengthy recovery, the runner is back to participating in sport.

Utility of MRI versus tumor markers for post-treatment surveillance of marker-positive CNS germ cell tumors.

Patients with marker-positive central nervous system (CNS) germ cell tumors are typically monitored for tumor recurrence with both tumor markers (AFP and b-hCG) and MRI. We hypothesize that the recurrence of these tumors will always be accompanied by an elevation in tumor markers, and that surveillance MRI may not be necessary. We retrospectively identified 28 patients with CNS germ cell tumors treated at our institution that presented with an elevated serum or cerebrospinal fluid (CSF) tumor marker at the time of diagnosis. We then identified those who had a tumor recurrence after having been in remission and whether each recurrence was detected via MRI changes, elevated tumor markers, or both. Four patients suffered a tumor recurrence. Only one patient had simultaneously elevated tumor markers and MRI evidence of recurrence. Two patients had evidence of recurrence on MRI without corresponding elevations in serum or CSF tumor markers. One patient had abnormal tumor markers with no evidence of recurrence on MRI until 6 months later. We conclude that in patients with marker-positive CNS germ cell tumors who achieve complete remission, continued surveillance imaging in addition to measurement of tumor markers is indicated to detect recurrences.

Restless Legs Syndrome in Pediatric Patients With Nephrotic Syndrome.

Background. Restless legs syndrome (RLS) is a sleep disorder characterized by an urge to move or the presence of unpleasant sensations in the extremities. The prevalence of RLS is higher in children and adults with chronic kidney disease and in adults with glomerular disease. Objective. To determine the prevalence of RLS in children with nephrotic syndrome. Methods. We studied 50 children with nephrotic syndrome and 22 controls. The following surveys were administered: Pediatric Emory RLS questionnaire, Pediatric Daytime Sleepiness Scale, and Pediatric Sleep Questionnaire. Results. Children with nephrotic syndrome were 9.0 ± 4.4 years old, 27 were male, and 27 were in remission. The prevalence of RLS was similar in the nephrotic syndrome cases and controls, whether or not indeterminate cases were considered positive: 14.0% versus 13.6% including indeterminate cases, and 8.0% versus 9.1% excluding indeterminate cases. Conclusion. RLS is not more common in children with glomerular disease compared to healthy controls.

Hemolytic uremic syndrome: toxins, vessels, and inflammation.

Hemolytic uremic syndrome (HUS) is characterized by thrombotic microangiopathy of the glomerular microcirculation and other vascular beds. Its defining clinical phenotype is acute kidney injury (AKI), microangiopathic anemia, and thrombocytopenia. There are many etiologies of HUS including infection by Shiga toxin-producing bacterial strains, medications, viral infections, malignancy, and mutations of genes coding for proteins involved in the alternative pathway of complement. In the aggregate, although HUS is a rare disease, it is one of the most common causes of AKI in previously healthy children and accounts for a sizable number of pediatric and adult patients who progress to end stage kidney disease. There has been great progress over the past 20 years in understanding the pathophysiology of HUS and its related disorders. There has been intense focus on vascular injury in HUS as the major mechanism of disease and target for effective therapies for this acute illness. In all forms of HUS, there is evidence of both systemic and intra-glomerular inflammation and perturbations in the immune system. Renewed investigation into these aspects of HUS may prove helpful in developing new interventions that can attenuate glomerular and tubular injury and improve clinical outcomes in patients with HUS.

Relative sensitivity of two marine bivalves for detection of genotoxic and cytotoxic effects: a field assessment in the Tamar Estuary, South West England.

The input of anthropogenic contaminants to the aquatic environment is a major concern for scientists, regulators and the public. This is especially relevant in areas such as the Tamar valley in SW England, which has a legacy of contamination from industrial activity in the nineteenth and twentieth centuries. Following on from previous laboratory validation studies, this study aimed to assess the relationship between genotoxic and cytotoxic responses and heavy metal concentrations in two bivalve species sampled from locations along the Tamar estuary. Adult cockles, Cerastoderma edule, and blue mussels, Mytilus edulis, were sampled from five locations in the Tamar and one reference location on the south Devon coast. Bivalve haemocytes were processed for comet and neutral red retention (NRR) assays to determine potential genotoxic and cytotoxic effects, respectively. Sediment and soft tissue samples were analysed for metal content by inductively coupled plasma mass spectrometry. Sediment concentrations were consistent with the physico-chemical nature of the Tamar estuary. A significant correlation (P = 0.05) was found between total metal concentration in sediment and C. edule soft tissues, but no such correlation was found for M. edulis samples. DNA damage was elevated at the site with highest Cr concentrations for M. edulis and at the site with highest Ni and Pb concentrations for C. edule. Analysis of NRR revealed a slight increase in retention time at one site, in contrast to comet data. We conclude that the comet assay is a reliable indicator of genotoxic damage in the field for both M. edulis and C. edule and discuss reasons for the apparent discrepancy with NRR.

Core competencies for gynecologic endoscopy in residency training: a national consensus project.

Residents and educators in obstetrics and gynecology have identified the need to improve endoscopic surgical education. The Canadian Endoscopy Education Project aims to create a national standardized endoscopy curriculum. The objective of the current project was to identify the core competencies for a gynecologic endoscopy (GE) curriculum in residency training programs. This expert consensus project (Canadian Task Force Classification III) included all 16 academic obstetrics and gynecology residency programs in Canada. Each university program selected their leading endoscopy educator to participate in the consensus process. Competencies for proficiency in GE were identified and then reviewed in 3 sequential rounds of consensus building using the Delphi technique. Overall, 213 objectives were reviewed and 199 (93%) of the items achieved consensus agreement. Competencies that were deemed outside the realm of general residency education were also collated and may represent a guide to subspecialty fellowship training in the future. The core competencies for GE training in obstetrics and gynecology residency were determined through national expert consensus. This provides the basis for a national standardized endoscopy curriculum for general obstetrics and gynecology training.

An introduction to metabolomics and its potential application in veterinary science.

Metabolomics has been found to be applicable to a wide range of fields, including the study of gene function, toxicology, plant sciences, environmental analysis, clinical diagnostics, nutrition, and the discrimination of organism genotypes. This approach combines high-throughput sample analysis with computer-assisted multivariate pattern-recognition techniques. It is increasingly being deployed in toxico- and pharmacokinetic studies in the pharmaceutical industry, especially during the safety assessment of candidate drugs in human medicine. However, despite the potential of this technique to reduce both costs and the numbers of animals used for research, examples of the application of metabolomics in veterinary research are, thus far, rare. Here we give an introduction to metabolomics and discuss its potential in the field of veterinary science.

Development of the in vivo chromosome aberration assay in oyster (Crassostrea gigas) embryo-larvae for genotoxicity assessment.

Bioassay methods currently used to assess the toxicity of effluents, transitional and marine waters measure endpoints such as larval immobilisation or mortality, however, they offer limited data regarding important sub-lethal effects, including genotoxicity. The metaphase chromosome aberration (CAb) assay is routinely used in mammalian systems for testing samples for genotoxicity. In the current study, an in vivo CAb test system has been developed and optimised for use with the early (embryo-larval) life stages of the Pacific oyster Crassostrea gigas, a species used routinely in both effluent hazard assessment and marine environmental monitoring programmes. The method was validated with two reference mutagens: Methylmethanesulfonate, a direct acting mutagen; and benzo[a]pyrene an indirect acting reference mutagen.

An evaluation of the relative sensitivity of two marine bivalve mollusc species using the Comet assay.

The aim of this study was to (a) evaluate the potential for the 'Comet assay' to be used as a method for detecting genetic damage in the common cockle Cerastoderma edule; and (b) to compare the relative sensitivity with Mytilus edulis as the bivalve widely used as a sentinel species in biomonitoring studies. In vitro validation studies were carried out on haemocytes from each species using hydrogen peroxide (H2O2), a known oxidant and the induced DNA damage was measured using the Comet assay. On exposure to 0, 100, 500 and 1000 microM H2O2, a significant concentration-dependent increase was observed in both species. Use of an additional concentration of 5000 microM H2O2 showed that while DNA damage could be assessed in M. edulis at this concentration, only a few cells from C. edule were amenable to measurements owing to extensive DNA damage ("hedgehog cells"). The evidence also suggested that the cells from C. edule are more sensitive to oxidative damage induced by H2O2 when compared with M. edulis. Bearing in mind that sediments are the ultimate sink for many contaminants, this study demonstrates the potential application of sediment-dwelling C. edule as a useful biomonitoring species.