Hypothalamic Transcriptome Response To Simulated Diel Earthen Pond Hypoxia Cycles In Channel Catfish (Ictalurus punctatus).

Commercial culture of channel catfish (Ictalurus punctatus) occurs in earthen ponds that are characterized by diel swings in dissolved oxygen concentration that can fall to severe levels of hypoxia which can suppress appetite and lead to suboptimal growth. Given the significance of the hypothalamus in regulating these processes in other fishes, an investigation into the hypothalamus transcriptome was conducted to identify specific genes and expression patterns responding to hypoxia. Channel catfish in normoxic water were compared to catfish subjected to 12 hours of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27 °C) followed by 12 hours of recovery in normoxia to mimic 24-hours in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-hour time points, with the 6- and 12-hour samplings occurring during hypoxia. A total of 190 genes were differentially expressed during the experiment, with most occurring during hypoxia and returning to baseline values within 6 hours of normoxia. Differentially expressed genes were sorted by function into Gene Ontology biological processes and revealed that most were categorized as "response to hypoxia", "sprouting angiogenesis", and "cellular response to xenobiotic stimulus". The patterns of gene expression reported here suggest that transcriptome responses to hypoxia are broad and quickly reversibly with the onset of normoxia. Although no genes commonly reported to modulate appetite were found to be differentially expressed in this experiment, several candidates were identified for future studies investigating the interplay between hypoxia and appetite in channel catfish, including adm, igfbp1a, igfbp7, and stc2b.

Rapid differentiation of infectious salmon anemia virus avirulent (HPR0) from virulent (HPRΔ) variants using multiplex RT-qPCR.

Infectious salmon anemia virus (ISAV; Isavirus salaris) causes an economically important disease of Atlantic salmon (Salmo salar L.). ISA outbreaks have resulted in significant losses of farmed salmon globally, often with a sudden onset. However, 2 phenotypically distinct variants of ISAV exist, each with divergent disease outcomes, associated regulations, and control measures. ISAV-HPRΔ, also known as ISAV-HPR deleted, is responsible for ISA outbreaks; ISAV-HPR0, is avirulent and is not known to cause fish mortality. Current detection methodology requires genetic sequencing of ISAV-positive samples to differentiate phenotypes, which may slow responses to disease management. To increase the speed of phenotypic determinations of ISAV, we developed a new, rapid multiplex RT-qPCR method capable of 1) detecting if a sample contains any form of ISAV, 2) discriminating whether positive samples contain HPRΔ or HPR0, and 3) validating RNA extractions with an internal control, all in a single reaction. Following assay development and optimization, we validated this new multiplex on 31 ISAV strains collected from North America and Europe (28 ISAV-HPRΔ, 3 ISAV-HPR0). Finally, we completed an inter-laboratory comparison of this multiplex qPCR with commercial ISAV testing and found that both methods provided equivalent results for ISAV detection.

Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.

Using aircraft tracking data to estimate the geographic scope of noise impacts from low-level overflights above parks and protected areas.

Sightseeing air tours have proven to be a challenging management issue for many tourist destinations around the world, especially at locations meant to protect natural and cultural resources and wilderness character. Two of the primary challenges with managing air tours are a lack of information about their travel patterns and how such patterns result in a measurable noise impact to listeners. Recent studies have highlighted the usefulness of newer technology for tracking aircraft travel patterns, particularly over national parks. In this synthesis, we pair aircraft tracks with acoustic data using a quantitative observer-based audibility modelling software toolkit. The findings delimit the long-term geographic scope of audibility for specific aircraft noise sources above landscapes of Hawai'i Volcanoes and Denali National Parks, U.S. and identify practical, 3-dimensional offset distances that can be used to reduce the functional effects of air tour noise in terms of sound level.

The generation of the first chromosome-level de novo genome assembly and the development and validation of a 50K SNP array for the St. John River aquaculture strain of North American Atlantic salmon.

Atlantic salmon (Salmo salar) in Northeastern US and Eastern Canada has high economic value for the sport fishing and aquaculture industries. Large differences exist between the genomes of Atlantic salmon of European origin and North American (N.A.) origin. Given the genetic and genomic differences between the 2 lineages, it is crucial to develop unique genomic resources for N.A. Atlantic salmon. Here, we describe the resources that we recently developed for genomic and genetic research in N.A. Atlantic salmon aquaculture. Firstly, a new single nucleotide polymorphism (SNP) database for N.A. Atlantic salmon consisting of 3.1 million putative SNPs was generated using data from whole-genome resequencing of 80 N.A. Atlantic salmon individuals. Secondly, a high-density 50K SNP array enriched for the genic regions of the genome and containing 3 sex determination and 61 putative continent of origin markers was developed and validated. Thirdly, a genetic map composed of 27 linkage groups with 36K SNP markers was generated from 2,512 individuals in 141 full-sib families. Finally, a chromosome-level de novo genome assembly from a male N.A. Atlantic salmon from the St. John River aquaculture strain was generated using PacBio long reads. Information from Hi-C proximity ligation sequences and Bionano optical mapping was used to concatenate the contigs into scaffolds. The assembly contains 1,755 scaffolds and only 1,253 gaps, with a total length of 2.83 Gb and N50 of 17.2 Mb. A BUSCO analysis detected 96.2% of the conserved Actinopterygii genes in the assembly, and the genetic linkage information was used to guide the formation of 27 chromosome sequences. Comparative analysis with the reference genome assembly of the European Atlantic salmon confirmed that the karyotype differences between the 2 lineages are caused by a fission in chromosome Ssa01 and 3 chromosome fusions including the p arm of chromosome Ssa01 with Ssa23, Ssa08 with Ssa29, and Ssa26 with Ssa28. The genomic resources we have generated for Atlantic salmon provide a crucial boost for genetic research and for management of farmed and wild populations in this highly valued species.

Using Forensic Toxicology to Enhance Medicolegal Death Investigations.

ABSTRACT: The Milwaukee County Medical Examiner's Office performed an evidence-based review of natural deaths certified without autopsy or toxicology testing. Three hundred fifteen such cases from 2020 and 2021 were selected. This study reviewed the deaths to determine if the cause was drug-related instead due to natural disease.Blood samples were screened by liquid chromatography-quadrupole mass spectrometry with time-of-flight detector. The analytes detected were evaluated for contribution to the cause resulting in a change in manner from natural to accident or suicide. Confirmatory analysis was performed where appropriate, and results were reported to the forensic pathologist for evaluation. Where appropriate, the death certificate was amended.As a result of the screening and confirmatory work, 18 cases were identified where significant drugs were detected, and thus the cause and manner of death were amended. One case was amended from natural to suicide after a conversation with the family about the toxicology findings. The remaining cases were amended to a manner of accident. The scope of drugs responsible for the deaths included prescription and illicit.

Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1's ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.

Assessment and application of wavelet-based optical flow velocimetry (wOFV) to wall-bounded turbulent flows.

The performance of a wavelet-based optical flow velocimetry (wOFV) algorithm in extracting high accuracy and high-resolution velocity fields from tracer particle images in wall-bounded turbulent flows is assessed. wOFV is first evaluated using synthetic particle images generated from a channel flow DNS of a turbulent boundary layer. The sensitivity of wOFV to the regularization parameter ( λ ) is quantified and results are compared to cross-correlation-based PIV. Results on synthetic particle images indicated different sensitivity to under-regularization or over-regularization depending on which region of the boundary layer is being analyzed. Nonetheless, tests on synthetic data revealed that wOFV can modestly outperform PIV in vector accuracy across a broad λ range. wOFV showed clear advantages over PIV in resolving the viscous sublayer and obtaining highly accurate estimates of the wall shear stress and thus normalizing boundary layer variables. wOFV was also applied to experimental data of a developing turbulent boundary layer. Overall, wOFV revealed good agreement with both PIV and a combined PIV + PTV method. However, wOFV was able to successfully resolve the wall shear stress and correctly normalize the boundary layer streamwise velocity to wall units where PIV and PIV + PTV showed larger deviations. Analysis of the turbulent velocity fluctuations revealed spurious results for PIV in close proximity to the wall, leading to significantly exaggerated and non-physical turbulence intensity in the viscous sublayer region. PIV + PTV showed only a minor improvement in this aspect. wOFV did not exhibit this same effect, revealing that it is more accurate in capturing small-scale turbulent motion in the vicinity of boundaries. The enhanced vector resolution of wOFV enabled improved estimation of instantaneous derivative quantities and intricate flow structure both closer to the wall and more accurately than the other velocimetry methods. These aspects show that, within a reasonable λ range that can be verified using physical principles, wOFV can provide improvements in diagnostics capability in resolving turbulent motion occurring in the vicinity of physical boundaries.

The ERAD system is restricted by elevated ceramides.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

Five-year outcomes from a prospective, multicenter study of endovascular repair of iliac artery aneurysms using an iliac branch device.

OBJECTIVE: We have reported the 5-year results of a pivotal prospective, multicenter study conducted in the United States of a specifically designed iliac branch endoprosthesis (IBE; W.L. Gore & Associates, Flagstaff, AZ) for endovascular repair of aortoiliac aneurysms and common iliac artery aneurysms. METHODS: A total of 63 patients (98.4% male; mean age, 70 years) with aortoiliac or common iliac artery aneurysms had undergone implantation of a single IBE device and a bifurcated aortoiliac stent graft. Patients with bilateral common iliac artery aneurysms (n = 22; 34.9%) had undergone either staged occlusion or surgical revascularization of the contralateral internal iliac artery before study enrollment. At 5 years, 36 of the 63 patients had completed the final study follow-up examinations, including clinical examinations (n = 35) and computed tomography (n = 32), with the results evaluated by an independent core laboratory and adverse events adjudicated by a clinical events committee. RESULTS: At 5 years, freedom from all-cause mortality was 85.7% and freedom from aneurysm-related mortality was 100%. The nine deaths that had occurred (range, 132-1898 days) were adjudicated as unrelated to the aneurysm or procedure. Primary patency of the internal and external iliac artery IBE limbs was 95.1% and 100%, respectively. No patients had experienced new-onset buttock claudication on the IBE side or self-reported new-onset erectile dysfunction. The common iliac artery diameter on the IBE side was either unchanged or had decreased by ≥5 mm in 30 of the 31 patients (96.8%) with a baseline (1 month) and 5-year (range, 1641-2006 days) computed tomography scan available. Of the 31 evaluable patients, 9 (29.0%) had had an increase of ≥5 mm in the aortic diameter, 5 of whom had had a concurrent type II endoleak. No type I or type III endoleaks or device migration were identified by the core laboratory. Six patients had undergone eight secondary interventions, including five interventions for a type II endoleak. The freedom from secondary intervention was 90.5%. CONCLUSIONS: The 5-year results of our prospective, multicenter study have confirmed the safety, efficacy, and durability of the IBE device for the treatment of aortoiliac and iliac artery aneurysms. The device effectively prevented common iliac artery aneurysm rupture, maintained the patency of the internal iliac artery, and avoided the complications associated with internal iliac artery sacrifice. Although common iliac artery aneurysm enlargement was rare, abdominal aortic enlargement was more common, suggesting that the outcomes of endovascular aneurysm repair might be different for patients with or without associated common iliac artery aneurysms.

REACT: Rapid Evaluation Assessment of Clinical Reasoning Tool.

INTRODUCTION: Clinical reasoning encompasses the process of data collection, synthesis, and interpretation to generate a working diagnosis and make management decisions. Situated cognition theory suggests that knowledge is relative to contextual factors, and clinical reasoning in urgent situations is framed by pressure of consequential, time-sensitive decision-making for diagnosis and management. These unique aspects of urgent clinical care may limit the effectiveness of traditional tools to assess, teach, and remediate clinical reasoning. METHODS: Using two validated frameworks, a multidisciplinary group of clinicians trained to remediate clinical reasoning and with experience in urgent clinical care encounters designed the novel Rapid Evaluation Assessment of Clinical Reasoning Tool (REACT). REACT is a behaviorally anchored assessment tool scoring five domains used to provide formative feedback to learners evaluating patients during urgent clinical situations. A pilot study was performed to assess fourth-year medical students during simulated urgent clinical scenarios. Learners were scored using REACT by a separate, multidisciplinary group of clinician educators with no additional training in the clinical reasoning process. REACT scores were analyzed for internal consistency across raters and observations. RESULTS: Overall internal consistency for the 41 patient simulations as measured by Cronbach's alpha was 0.86. A weighted kappa statistic was used to assess the overall score inter-rater reliability. Moderate reliability was observed at 0.56. DISCUSSION: To our knowledge, REACT is the first tool designed specifically for formative assessment of a learner's clinical reasoning performance during simulated urgent clinical situations. With evidence of reliability and content validity, this tool guides feedback to learners during high-risk urgent clinical scenarios, with the goal of reducing diagnostic and management errors to limit patient harm.

Functional feeds marginally alter immune expression and microbiota of Atlantic salmon (Salmo salar) gut, gill, and skin mucosa though evidence of tissue-specific signatures and host-microbe coadaptation remain.

BACKGROUND: Mucosal surfaces of fish provide cardinal defense against environmental pathogens and toxins, yet these external mucosae are also responsible for maintaining and regulating beneficial microbiota. To better our understanding of interactions between host, diet, and microbiota in finfish and how those interactions may vary across mucosal tissue, we used an integrative approach to characterize and compare immune biomarkers and microbiota across three mucosal tissues (skin, gill, and gut) in Atlantic salmon receiving a control diet or diets supplemented with mannan-oligosaccharides, coconut oil, or both. Dietary impacts on mucosal immunity were further evaluated by experimental ectoparasitic sea lice (Lepeophtheirus salmonis) challenge. RESULTS: Fish grew to a final size of 646.5 g ± 35.8 during the 12-week trial, with no dietary effects on growth or sea lice resistance. Bacterial richness differed among the three tissues with the highest richness detected in the gill, followed by skin, then gut, although dietary effects on richness were only detected within skin and gill. Shannon diversity was reduced in the gut compared to skin and gill but was not influenced by diet. Microbiota communities clustered separately by tissue, with dietary impacts on phylogenetic composition only detected in the skin, although skin and gill communities showed greater overlap compared to the gut according to overall composition, differential abundance, and covariance networks. Inferred metagenomic functions revealed preliminary evidence for tissue-specific host-microbiota coadaptation, as putative microbiota functions showed ties to the physiology of each tissue. Immune gene expression profiles displayed tissue-specific signatures, yet dietary effects were also detected within each tissue and peripheral blood leukocytes. Procrustes analysis comparing sample-matched multivariate variation in microbiota composition to that of immune expression profiles indicated a highly significant correlation between datasets. CONCLUSIONS: Diets supplemented with functional ingredients, namely mannan-oligosaccharide, coconut oil, or a both, resulted in no difference in Atlantic salmon growth or resistance to sea lice infection. However, at the molecular level, functional ingredients caused physiologically relevant changes to mucosal microbiota and host immune expression. Putative tissue-specific metagenomic functions and the high correlation between expression profiles and microbiota composition suggest host and microbiota are interdependent and coadapted in a tissue-specific manner.

Examining the Examiners: How Medical Death Investigators Describe Suicidal, Homicidal, and Accidental Death.

This study describes differences in medicolegal death investigators' written descriptions for people who died by homicide, suicide, or accident. We evaluated 17 years of death descriptions from a midsized metropolitan midwestern county in the United States to assess how death investigators psychologically respond to different manners of death (N = 10,408 cases). Automated text analyses suggest investigators describe accidental deaths with more immediacy relative to homicides, while they also described suicidal deaths in less emotional terms than homicides as well. These data suggest medicolegal death investigators have different psychological reactions to circumstances and manners of death as indicated by their professional writing. Future research may surface context-specific psychological reactions to vicarious trauma that could inform the design or personalization of workplace-coping interventions.

Breast Equalization Augmentation: The Use of Ultrasonic Assisted Liposuction for Correction of Primary Breast Asymmetry with Bilateral Augmentation.

BACKGROUND: The purpose of this study was to determine the effectiveness of using ultrasonic assisted liposuction (UAL) to achieve sufficient breast symmetry allowing for the use of same sized implants in patients presenting for elective breast augmentation surgery. METHODS: A retrospective review was performed of patients presenting for augmentation mammoplasty with breast asymmetry who underwent ultrasonic assisted liposuction in combination with implant augmentation. Pre-operative differences in breast volumes were determined using water displacement, and these measurements were compared to final lipoaspirates required to achieve symmetry. To estimate the volume of lipoaspirate required, we suggested to aim for twice the volume difference obtained by water displacement. The success of the procedure was measured by the ability to use the same size implants bilaterally. RESULTS: A total of 35 patients were included in this review. In 85% of patients, UAL was sufficient to permit the use of equally sized implants bilaterally. However, when different sized implants were required, the size difference between implants was not greater than 25 cc. The UAL was also effective in correcting minor ptosis and nipple position. The main observed disadvantage was prolonged bruising and swelling. CONCLUSION: The use of UAL for correction of primary breast asymmetry with bilateral breast augmentation allows balancing of breast tissue volumes and the use of same size implants and eliminates the need for balancing mastopexy procedures. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Simplified Technique for Creating an Umbilicus with Scarless Caudal Aspect and Superior Hooding.

A non-aesthetic post-abdominoplasty umbilicus is known to be a significant concern for many patients who consider this procedure, due to its central and visible location. The goal of this method is to minimize the visible scar and create a natural-looking and aesthetically pleasing umbilicus. In this multimedia article, we illustrate our technique that is both reproducible and easy to perform. It produces a scarless caudal aspect, pleasant depth, and natural superior hooding appearance to the post-op umbilicus. Limitations of this technique are discussed.Level of Evidence IV This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Ultrafast multi-photon excitation of ScVO4:Bi3+ for luminescence thermometry.

We demonstrate a multi-photon excitation (MPE) scheme for luminescence thermometry using ScVO4:Bi3+. MPE is performed using a 37 fs Ti:sapphire laser pulse centered at 800 nm. Log-log plots of the phosphorescence intensity versus excitation power show that the 800 nm MPE of ScVO4:Bi3+ involves a 2- and 3-photon absorption process in comparison with a single-photon excitation (SPE) process at 266 and 400 nm. Spectroscopic investigation shows that with the 800 nm MPE and 266 nm SPE schemes, the emission spectra of ScVO4:Bi3+ are similarly characterized by emissions of the VO4 3- groups and Bi3+. MPE is advantageous to suppress fluorescence that interferes with the phosphorescence signal. We demonstrate this aspect for a ScVO4:Bi3+ coating applied on an alumina substrate. The luminescence lifetime is calibrated with temperature in the range of 294-334 K; the MPE scheme has an equally impressive temperature sensitivity (3.4-1.7%/K) and precision (0.2-0.7 K) compared with the SPE schemes. The MPE scheme can be applied to a variety of phosphors and is valuable for precise temperature measurements, even in applications where isolating interfering background emissions is challenging.

Face Coverings, Aerosol Dispersion and Mitigation of Virus Transmission Risk.

The SARS-CoV-2 virus is primarily transmitted through virus-laden fluid particles ejected from the mouth of infected people. Face covers can mitigate the risk of virus transmission but their outward effectiveness is not fully ascertained. Objective: by using a background oriented schlieren technique, we aim to investigate the air flow ejected by a person while quietly and heavily breathing, while coughing, and with different face covers. Results: we found that all face covers without an outlet valve reduce the front flow through by at least 63% and perhaps as high as 86% if the unfiltered cough jet distance was resolved to the anticipated maximum distance of 2-3 m. However, surgical and handmade masks, and face shields, generate significant leakage jets that may present major hazards. Conclusions: the effectiveness of the masks should mostly be considered based on the generation of secondary jets rather than on the ability to mitigate the front throughflow.