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Objective: To assess the impact of the COVID-19 pandemic on the state-level enteric disease workforce and routine enteric disease surveillance and outbreak investigation activities in the western United States., Design and Setting: Key informant interviews conducted using bidirectional video from March to April 2022., Participants: Enteric disease epidemiologists at state public health agencies in the western states served by the Colorado and Washington Integrated Food Safety Centers of Excellence., Main Outcomes: Key themes were identified using grounded theory., Results: Nine themes were identified including excessive workload, shifts in local and state responsibilities, challenges with retention and hiring, importance of student teams, laboratory supplies shortages, changes to case and outbreak investigation priorities, transitioning back to enterics, adoption of new methods and technology, and current and future needs., Conclusions: The COVID-19 pandemic response had a substantial impact on state-level enteric disease activities in western states, with many staff members diverted from routine responsibilities and a de-prioritization of enteric disease work. There is a need for sustainable solutions to address staffing shortages, prioritize employee mental health, and effectively manage routine workloads when responding to emergencies., Competing Interests: No conflicts of interest to report., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Cardiovascular complications are major clinical hallmarks of acute and post-acute coronavirus disease 2019 (COVID-19). However, the mechanistic details of SARS-CoV-2 infectivity of endothelial cells remain largely unknown. Here, we demonstrate that the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein shares a similarity with the proline-rich binding ena/VASP homology (EVH1) domain and identified the endoplasmic reticulum (ER) resident calreticulin (CALR) as an S-RBD interacting protein. Our biochemical analysis showed that CALR, via its proline-rich (P) domain, interacts with S-RBD and modulates proteostasis of the S protein. Treatment of cells with the proteasomal inhibitor bortezomib increased the expression of the S protein independent of CALR, whereas the lysosomal/autophagy inhibitor bafilomycin 1A, which interferes with the acidification of lysosome, selectively augmented the S protein levels in a CALR-dependent manner. More importantly, the shRNA-mediated knockdown of CALR increased SARS-CoV-2 infection and impaired calcium homeostasis of human endothelial cells. This study provides new insight into the infectivity of SARS-CoV-2, calcium hemostasis, and the role of CALR in the ER-lysosome-dependent proteolysis of the spike protein, which could be associated with cardiovascular complications in COVID-19 patients.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future., (© 2023. Springer Nature Limited.)

Intrauterine growth restriction (IUGR) arises when maternal stressors coincide with peak placental development, leading to placental insufficiency. When the expanding nutrient demands of the growing fetus subsequently exceed the capacity of the stunted placenta, fetal hypoxemia and hypoglycemia result. Poor fetal nutrient status stimulates greater release of inflammatory cytokines and catecholamines, which in turn lead to thrifty growth and metabolic programming that benefits fetal survival but is maladaptive after birth. Specifically, some IUGR fetal tissues develop enriched expression of inflammatory cytokine receptors and other signaling cascade components, which increases inflammatory sensitivity even when circulating inflammatory cytokines are no longer elevated after birth. Recent evidence indicates that greater inflammatory tone contributes to deficits in skeletal muscle growth and metabolism that are characteristic of IUGR offspring. These deficits underlie the metabolic dysfunction that markedly increases risk for metabolic diseases in IUGR-born individuals. The same programming mechanisms yield reduced metabolic efficiency, poor body composition, and inferior carcass quality in IUGR-born livestock. The ω-3 polyunsaturated fatty acids (PUFA) are diet-derived nutraceuticals with anti-inflammatory effects that have been used to improve conditions of chronic systemic inflammation, including intrauterine stress. In this review, we highlight the role of sustained systemic inflammation in the development of IUGR pathologies. We then discuss the potential for ω-3 PUFA supplementation to improve inflammation-mediated growth and metabolic deficits in IUGR offspring, along with potential barriers that must be considered when developing a supplementation strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 White and Yates.)

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

EWS is a member of the FET family of RNA/DNA binding proteins that regulate crucial phases of nucleic acid metabolism. EWS comprises an N-terminal low-complexity domain (LCD) and a C-terminal RNA-binding domain (RBD). The RBD is further divided into three RG-rich regions, which flank an RNA-recognition motif (RRM) and a zinc finger (ZnF) domain. Recently, EWS was shown to regulate R-loops in Ewing sarcoma, a pediatric bone and soft-tissue cancer in which a chromosomal translocation fuses the N-terminal LCD of EWS to the C-terminal DNA binding domain of the transcription factor FLI1. Though EWS was shown to directly bind R-loops, the binding mechanism was not elucidated. In the current study, the RBD of EWS was divided into several constructs, which were subsequently assayed for binding to various nucleic acid structures expected to form at R-loops, including RNA stem-loops, DNA G-quadruplexes, and RNA:DNA hybrids. EWS interacted with all three nucleic acid structures with varying affinities and multiple domains contributed to binding each substrate. The RRM and RG2 region appear to bind nucleic acids promiscuously while the ZnF displayed more selectivity for single-stranded structures. With these results, the structural underpinnings of EWS recognition and binding of R-loops and other nucleic acid structures is better understood., (© 2023 Wiley Periodicals LLC.)

Background: In 2015, the American College of Surgeons (ACS) created a new hospital improvement program to enhance the performance of pediatric care in US hospitals. The Children's Surgery Verification (CSV) Quality Improvement Program is predicated on the idea that pediatric surgical patients have improved outcomes when treated at children's hospitals with optimal resources. Achieving ACS level I CSV designation at pediatric trauma centers may lead to greater benefits for pediatric trauma patients; however, the specific benefits have yet to be identified. We hypothesize that achieving the additional designation of ACS level I CSV is associated with decreased narcotic use perioperatively and improved efficiency when managing pediatric patients with femur fractures., Study Design: This study is a retrospective analysis of traumatic pediatric orthopaedic femur fractures treated at a verified level I pediatric trauma center before and after CSV designation (2010 to 2014 vs 2015 to 2019). Efficiency parameters, defined as time from admission to surgery, duration of surgery, and duration of hospital stay, and narcotic administration in oral morphine equivalents (OMEs) were compared., Results: Of 185 traumatic femur fractures analyzed, 80 occurred before meeting ACS level I CSV criteria, and 105 occurred after. Post-CSV, there was a significant decrease in mean wait time from admission to surgery (16.64 hours pre-CSV, 12.52 hours post-CSV [p < 0.01]) and duration of hospital stay (103.49 hours pre-CSV, 71.61 hours post-CSV [p < 0.01]). Narcotic usage was significantly decreased in both the preoperative period (40.61 OMEs pre-CSV, 23.77 OMEs post-CSV [p < 0.01]) and postoperative period (126.67 OMEs pre-CSV, 45.72 OMEs post-CSV [p < 0.01])., Conclusions: Achieving ACS level I CSV designation is associated with increased efficiency and decreased preoperative and postoperative narcotic use when treating pediatric trauma patients., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Surgeons.)

Mild traumatic brain injuries (mTBI) constitute a significant health concern with clinical symptoms ranging from headaches to cognitive deficits. Despite the myriad of symptoms commonly reported following this injury, there is still a lack of knowledge on the various pathophysiological changes that occur. Preclinical studies are at the forefront of discovery delineating the changes that occur within this heterogeneous injury, with the emergence of translational models such as closed-head impact models allowing for further exploration of this injury mechanism. In the current study, male rats were subjected to a closed-head controlled cortical impact (cCCI), producing a concussion (mTBI). The pathological effects of this injury were then evaluated using immunoflourescence seven days following. The results exhibited a unique glial-specific inflammatory response, with both the ipsilateral and contralateral sides of the cortex and hippocampus showing pathological changes following impact. Overall these findings are consistent with glial changes reported following concussions and may contribute to subsequent symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 White and VandeVord.)

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs., Significance: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873., (©2021 The Authors; Published by the American Association for Cancer Research.)

Information sharing, a component of patient and family engagement (PFE), is an important process that may contribute to intensive care unit (ICU) quality of care. Yet, virtually no studies explore how the process of information sharing unfolds in the ICU from the interprofessional team and family member perspectives. To better understand the process of information sharing, we conducted ethnographic fieldwork in a 20-bed medical ICU, focusing on behaviors and interactions of the interprofessional team and family members (May 2016 - October 2016). We completed 17.5 observation hours, 6 shadowing sessions, and 12 semi-structured interviews with 17 total participants. We used thematic content analysis and iterative inductive coding to identify three themes about the information sharing process: 1) family factors (health literacy and past experience with the ICU environment) influence information sharing; 2) clinicians strategies can support engagement in the process of information sharing (assessing families' need for information, understanding a families' hope, using rounds as an opportunity for information sharing); 3) the process of information sharing allows for trust building between families and the ICU team. Our findings suggest that information sharing is a crucial process that may serve as a catalyst for effective patient and family engagement in the ICU.

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness., Competing Interests: The authors have declared that no competing interests exist.

Mannose-binding lectins effectively inhibit most seasonal strains of influenza A virus and contribute to the innate host defense vs. these viruses. In contrast, pandemic IAV strains are largely resistant to these lectins, likely contributing to increased spread and worse outcomes. In this paper, we evaluated the inhibition of IAV by mannose-binding lectins of human, bacterial, and fungal origin to understand and possibly increase activity vs. the pandemic IAV. A modified version of the human surfactant protein D (SP-D) neck and carbohydrate recognition domain (NCRD) with combinatorial substitutions at the 325 and 343 positions, previously shown to inhibit pandemic H3N2 IAV in vitro and in vivo , and to inhibit pandemic H1N1 in vitro , failed to protect mice from pandemic H1N1 in vivo in the current study. We attempted a variety of maneuvers to improve the activity of the mutant NCRDs vs. the 2009 pandemic H1N1, including the formation of full-length SP-D molecules containing the mutant NCRD, cross-linking of NCRDs through the use of antibodies, combining SP-D or NCRDs with alpha-2-macroglobulin, and introducing an additional mutation to the double mutant NCRD. None of these substantially increased the antiviral activity for the pandemic H1N1. We also tested the activity of bacterial and algal mannose-binding lectins, cyanovirin, and griffithsin, against IAV. These had strong activity against seasonal IAV, which was largely retained against pandemic H1N1. We propose mechanisms to account for differences in activity of SP-D constructs against pandemic H3N2 and H1N1, and for differences in activity of cyanovirin vs. SP-D constructs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 White, Nikolaidis, McCormack, Crouch and Hartshorn.)

Initial recommendations for surface disinfection to prevent SARS-CoV-2 transmission were developed using previous evidence from potential surrogates. To the best of our knowledge, no appropriate surrogate for SARS-CoV-2 has been identified or confirmed for chlorine and antimicrobial surface disinfection. We completed a study to evaluate the efficacy of two hypothesized antimicrobial surfaces, and four chlorine solutions on nonporous and porous surfaces, against SARS-CoV-2 and three potential SARS-CoV-2 surrogates [coronavirus mouse hepatitis virus (MHV) and bacteriophages Phi6 and MS2], to identify a BSL-1 or BSL-2 virus to use in future studies. We found SARS-CoV-2 can be reduced >4 log 10 on porous and nonporous surfaces within 30 s upon exposure to 0.5% NaOCl. The results indicate coronavirus MHV-GFP is inactivated faster than SARS-CoV-2 (MHV-GFP ≥ 6.08 log 10 ; SARS-CoV-2 = 0.66 log 10 at 30 s with 0.05% NaOCl on steel) and MS2 is inactivated more slowly. Phi6 is inactivated like SARS-CoV-2, and we propose Phi6 as a slightly conservative surrogate for SARS-CoV-2 chlorine disinfection. Additionally, disinfection of bacteriophages on wood was challenging, and exposure to antimicrobial surfaces had no disinfection efficacy as tested. We recommend using 0.5% chlorine on surfaces for a minimum of 30 s of contact to disinfect SARS-CoV-2 and recommend additional research on Phi6 disinfection with varied surfaces and conditions., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Innate immunity is vital for the early control of influenza A virus (IAV) infection. Serum amyloid A (SAA1) is an acute phase reactant produced in the liver and lung that rises dramatically during IAV infection. The potential role of SAA1 in host defense against IAV is unknown. SAA1 has been reported to directly activate neutrophils and to recruit them to the lung during infectious and inflammatory processes. Neutrophils are the most abundant cell recruited to the lung in the early phase of IAV infection. There are different forms and preparations of SAA1 that have found to have different effects on phagocyte responses, through various receptors. In this paper, we test the direct effects of various preparations of serum derived or recombinant SAA on IAV and how it modulates the interactions of IAV with neutrophils. All SAA preparations bound to IAV in vitro but caused minimal hemagglutination inhibition or viral aggregation. The human serum-derived SAA1 or the complex of SAA1 with HDL did have IAV neutralizing activity in vitro, whereas the recombinant SAA1 preparations did not. We found that different SAA preparations also had markedly different effects on neutrophil functions, with E. coli-derived SAA1 triggering some responses in neutrophils on its own or in presence of IAV whereas mammalian cell-derived SAA1 did not. This discrepancy could be explained by the reported contamination of the former preparation with bacterial components. Of interest, however, serum SAA alone, serum SAA complexed with HDL, or HDL alone potentiated some neutrophil responses to IAV. Our results suggest that SAA may play some role in host response to IAV, but further work needs to be done to clarify the role of different variants of SAA alone or complexed with HDL., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung, and liver diseases. However, little is known about their interactions with neutrophils and how this might contribute to injury. Here, it is shown that histone H4 acts as neutrophil activator by inducing hydrogen peroxide production, degranulation, cell adhesion, and IL-8 generation. Histone H4 caused permeabilization of the neutrophil membrane (a phenomenon described in other cell types) leading to accelerated cell death. H4 caused sustained rise in neutrophil intracellular calcium that is necessary for respiratory burst activation and degranulation. Convincing evidence was not found for TLRs or ATP receptors in H4 mediated activation. However, pertussis toxin and wortmannin (inhibitors of G protein and PI3K) inhibited H4-induced hydrogen peroxide production and degranulation. These studies suggest that release of histone H4 in sites of infection or inflammation may potentiate neutrophil activation and promote additional inflammatory responses. These studies may provide a better basis for developing novel therapeutic strategies to block neutrophil extracellular trap (NET) and H4-related pathology in sepsis and various forms of lung injury including that induced by viruses like influenza or SAR-CoV2., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Small-molecule inhibitors of transforming growth factor beta receptor 1 (TGFβRI) have a history of significant class-based toxicities (eg, cardiac valvulopathy) in preclinical species that have limited their development as new medicines. Nevertheless, some TGFβRI inhibitors have entered into clinical trials using intermittent-dosing schedules and exposure limits in an attempt to avoid these toxicities. This report describes the toxicity profile of the small-molecule TGFβRI inhibitor, BMS-986260, in rats and dogs. Daily oral dosing for 10 days resulted in valvulopathy and/or aortic pathology at systemic exposures that would have been targeted clinically, preventing further development with this dosing schedule. These toxicities were not observed in either species in 1-month studies using the same doses on an intermittent-dosing schedule of 3 days on and 4 days off (QDx3 once weekly). Subsequently, 3-month studies were conducted (QDx3 once weekly), and while there were no cardiovascular findings in dogs, valvulopathy and mortality occurred early in rats. The only difference compared to the 1-month study was that the rats in the 3-month study were 2 weeks younger at the start of dosing. Therefore, a follow-up 1-month study was conducted to evaluate whether the age of rats influences sensitivity to target-mediated toxicity. Using the same dosing schedule and similar doses as in the 3-month study, there was no difference in the toxicity of BMS-986260 in young (8 weeks) or adult (8 months) rats. In summary, an intermittent-dosing schedule mitigated target-based cardiovascular toxicity in dogs but did not prevent valvulopathy in rats, and thus the development of BMS-986260 was terminated., (© 2020 John Wiley & Sons, Ltd.)

Objective: Quantify work loss and costs associated with prescription opioid use disorder (OUD) from the employer perspective., Methods: Retrospective claims analysis to compare missed work days and associated costs between employees with and without an OUD diagnosis in a 12-month period., Results: Two thousand three hundred eleven matched-pairs of employees were compared. The mean (SD) number of days missed while waiting for disability benefits (0.24 [1.4] vs 0.17 [1.0]; P = 0.035), absenteeism due to disability claims (9.5 [40.9] vs 5.6 [30.0]; P < 0.001), and medical visits (17.8 [18.5] vs 10.0 [12.4]; P < 0.001) was higher for employees with OUD compared with those without, resulting in higher mean (SD) indirect cost estimates of $8193 ($14,694) per employee (OUD) versus $5438 ($13,683) per employee (no OUD) (P < 0.001)., Conclusions: Prescription OUD is associated with significant work loss and may pose considerable economic burden on employers.

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low-pathogenicity avian influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortment and emergence in human populations. Since the presence of head region high-mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here, we investigate the activities of two recombinant human SP-D forms against representative LPAIV strains, including H2N1, H5N1, H6N1, H11N9, an avian H3N8, and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPAIV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between the protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high-mannose glycans in H3 HA but by complex glycans in all LPAIV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high-mannose glycan on the head region, our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three-dimensional structural analysis. IMPORTANCE Low-pathogenicity avian influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations, as was seen in the 1957 pandemic, in which an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first-line innate immunity defense, removes influenza type A virus (IAV) through interaction with hemagglutinin (HA) head region high-mannose glycan(s). While it is known that both H1 and H3 HAs have one or more key high-mannose glycosites in the head region, little is known about similar glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high-mannose glycans and do not interact with SP-D, and that sequence analysis can predict glycan subtype, thus predicting the presence or absence of this virulence marker., (Copyright © 2020 American Society for Microbiology.)

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans., (Copyright © 2019 van Eijk, Hillaire, Rimmelzwaan, Rynkiewicz, White, Hartshorn, Hessing, Koolmees, Tersteeg, van Es, Meijerhof, Huckriede and Haagsman.)

Fetal hemoglobin (HbF) expression is partially governed by the trans-acting quantitative trait loci BCL11A and MYB and a cis-acting locus linked to the HBB gene cluster. Our previous analysis of the Genotype-Tissue Expression database suggested that BCL2L1 was associated with HbF gene expression. In erythroid progenitors from patients with sickle cell disease, BCL2L1 messenger RNA (mRNA) levels were positively correlated with HBG mRNA and total HbF concentration (r2 = 0.72, P = .047 and r2 = 0.68, P = .01, respectively). Inhibition of BCL2L1 protein activity in HbF-expressing HUDEP-1 cells decreased HBG expression in a dose-dependent manner. Overexpression of BCL2L1 in these cells increased HBG expression fourfold (P < .05) and increased F cells by 13% (P < .05). Overexpression of BCL2L1 in erythroid progenitors derived from primary adult CD34+ cells upregulated HBG expression 11-fold (P < .05), increased F cells by 18% (P < .01), did not significantly affect cell differentiation or proliferation, and had a minor effect on survival. Although the mechanism remains unknown, our results suggest that BCL2L1 is associated with HbF gene activation., (© 2019 by The American Society of Hematology.)

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mental health consumers who hear voices frequently experience distress and express a desire to discuss their voice-hearing experience. Nurses do not regularly demonstrate a willingness to engage in such discussions. With the introduction of educational strategies that develop empathy and an understanding of voice-hearing experiences, it is anticipated that early career nurses will be able to translate such understanding into their professional nursing practice. To explore early career nurses' understanding of providing care to mental health consumers who hear voices, a qualitative exploratory descriptive study was conducted in which nine early career Registered Nurses were interviewed regarding their experiences of caring for people who hear voices. Thematic analysis was employed to analyse the data and generate themes. Participants reported difficulty in developing relationships with consumers who hear voices, due to a workplace culture that was focussed on risk and lacking professional support. Nurses need specific education to develop the skills necessary to respond to consumers who hear voices and engage in dialogue that assists consumers to relate to the voices in a meaningful way. However, for this to succeed in practice, changes need to be supported by addressing the cultural barriers, such as risk-focussed environments, that prevent nurses implementing best practice., (© 2018 Australian College of Mental Health Nurses Inc.)

Pexacerfont is a corticotropin-releasing factor subtype 1 receptor antagonist that was developed for the treatment of anxiety- and stress-related disorders. This report describes the results of repeat-dose oral toxicity studies in rats (3 and 6 months) and dogs (3 months and 1 year). Pexacerfont was well tolerated in all of these studies at exposures equal to or greater than areas under the curve in humans (clinical dose of 100 mg). Microscopic changes in the liver (hepatocellular hypertrophy), thyroid glands (hypertrophy/hyperplasia and adenomas of follicular cells), and pituitary (hypertrophy/hyperplasia and vacuolation of thyrotrophs) were only observed in rats and were considered adaptive changes in response to hepatic enzyme induction and subsequent alterations in serum thyroid hormone levels. Evidence for hepatic enzyme induction in dogs was limited to increased liver weights and reduced thyroxine (T 4 ) levels. Mammary gland hyperplasia and altered female estrous cycling were only observed in rats, whereas adverse testicular effects (consistent with minimal to moderate degeneration of the germinal epithelium) were only noted following chronic dosing in dogs. The testicular effects were reversible changes with exposure margins of 8× at the no observed adverse effect level. It is not clear whether the changes in mammary gland, estrous cycling, and testes represent secondary hormonal changes due to perturbation of the hypothalamic-pituitary-adrenal axis or are off-target effects. In conclusion, the results of chronic toxicity studies in rats and dogs show that pexacerfont has an acceptable safety profile to support further clinical testing.

Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells., (Copyright © 2018. Published by Elsevier Ltd.)

Inflorescence capacity plays a crucial role in reproductive fitness in plants, and in production of hybrid crops. Maize is a monoecious species bearing separate male and female flowers (tassel and ear, respectively). The switch from open-pollinated populations of maize to hybrid-based breeding schemes in the early 20th century was accompanied by a dramatic reduction in tassel size, and the trend has continued with modern breeding over the recent decades. The goal of this study was to identify selection signatures in genes that may underlie this dramatic transformation. Using a population of 942 diverse inbred maize accessions and a nested association mapping population comprising three 200-line biparental populations, we measured 15 tassel morphological characteristics by manual and image-based methods. Genome-wide association studies identified 242 single nucleotide polymorphisms significantly associated with measured traits. We compared 41 unselected lines from the Iowa Stiff Stalk Synthetic (BSSS) population to 21 highly selected lines developed by modern commercial breeding programs, and found that tassel size and weight were reduced significantly. We assayed genetic differences between the two groups using three selection statistics: cross population extended haplotype homozogysity, cross-population composite likelihood ratio, and fixation index. All three statistics show evidence of selection at genomic regions associated with tassel morphology relative to genome-wide null distributions. These results support the tremendous effect, both phenotypic and genotypic, that selection has had on maize male inflorescence morphology., (Copyright © 2018 J. L. Gage et al.)

In experimental contexts, affect-related word lists have been widely applied when examining how cognitive processes interact with emotional processes. These lists, however, present limitations when studying the relation between emotion and cognitive processes such as time and number processing because affective words do not inherently contain time or quantity information. Live events, in contrast, are experienced by an observer and therefore inherently carry affect information. Unfortunately, existing life-event lists and inventories have been largely applied within clinical contexts as diagnostic tools, and therefore are not suitable for many experimental contexts because they do not contain a balanced number of reliably positive, negative, and neutral life events. In Experiment 1, we create a standardized affect-related life-events list with 171 positive, negative, and neutral affect-related life events. In Experiment 2, we show that strength of affect and significance of the event are integral dimensions, suggesting that these two features are difficult to separate perceptually. The implications of these findings and some potential future applications of the created life-events list are discussed.

Study Objective: Crisis resource management skills are integral to leading the resuscitation of a critically ill patient. Despite their importance, crisis resource management skills (and their associated cognitive processes) have traditionally been difficult to study in the real world. The objective of this study was to derive key cognitive processes underpinning expert performance in resuscitation medicine, using a new eye-tracking-based video capture method during clinical cases., Methods: During an 18-month period, a sample of 10 trauma resuscitations led by 4 expert trauma team leaders was analyzed. The physician team leaders were outfitted with mobile eye-tracking glasses for each case. After each resuscitation, participants were debriefed with a modified cognitive task analysis, based on a cued-recall protocol, augmented by viewing their own first-person perspective eye-tracking video from the clinical encounter., Results: Eye-tracking technology was successfully applied as a tool to aid in the qualitative analysis of expert performance in a clinical setting. All participants stated that using these methods helped uncover previously unconscious aspects of their cognition. Overall, 5 major themes were derived from the interviews: logistic awareness, managing uncertainty, visual fixation behaviors, selective attendance to information, and anticipatory behaviors., Conclusion: The novel approach of cognitive task analysis augmented by eye tracking allowed the derivation of 5 unique cognitive processes underpinning expert performance in leading a resuscitation. An understanding of these cognitive processes has the potential to enhance educational methods and to create new assessment modalities of these previously tacit aspects of expertise in this field., (Copyright © 2018 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV. An N -linked CRD glycosylation provides interactions with the sialic acid-binding site of IAV, and a tripeptide loop at the lectin-binding site facilitates enhanced interactions with IAV glycans. Here, to investigate both mechanisms of IAV neutralization in greater detail, we produced an N -glycosylated neck-CRD fragment of porcine SP-D (RpNCRD) in HEK293 cells. X-ray crystallography disclosed that the N -glycan did not alter the CRD backbone structure, including the lectin site conformation, but revealed a potential second nonlectin-binding site for glycans. IAV hemagglutination inhibition, IAV aggregation, and neutralization of IAV infection studies showed that RpNCRD, unlike the human analogue RhNCRD, exhibits potent neutralizing activity against pandemic A/Aichi/68 (H3N2), enabled by both porcine-specific structural features of its CRD. MS analysis revealed an N -glycan site-occupancy of >98% at Asn-303 of RpNCRD with complex-type, heterogeneously branched and predominantly α(2,3)-sialylated oligosaccharides. Glycan-binding array data characterized both RpNCRD and RhNCRD as mannose-type lectins. RpNCRD also bound Lewis Y structures, whereas RhNCRD bound polylactosamine-containing glycans. The presence of the N -glycan in the CRD increases the glycan-binding specificity of RpNCRD. These insights increase our understanding of porcine-specific innate defense against pandemic IAV and may inform the design of recombinant SP-D-based antiviral drugs., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Influenza A viruses (IAVs) continue to pose major risks of morbidity and mortality during yearly epidemics and periodic pandemics. The genomic instability of IAV allows it to evade adaptive immune responses developed during prior infection. Of particular concern are pandemics which result from wholesale incorporation of viral genome sections from animal sources. These pandemic strains are radically different from circulating human strains and pose great risk for the human population. For these reasons, innate immunity plays a strong role in the initial containment of IAV infection. Soluble inhibitors present in respiratory lining fluids and blood provide a level of early protection against IAV. In general, these inhibitors act by binding to the viral hemagglutinin (HA). Surfactant protein D (SP-D) and mannose-binding lectin (MBL) attach to mannosylated glycans on the HA in a calcium dependent manner. In contrast, surfactant protein A, ficolins, and other inhibitors present sialic acid rich ligands to which the HA can bind. Among these inhibitors, SP-D seems to be the most potent due to its specific mode of binding to viral carbohydrates and its ability to strongly aggregate viral particles. We have studied specific properties of the N-terminal and collagen domain of SP-D that enable formation of highly multimerized molecules and cooperative binding among the multiple trimeric lectin domains in the protein. In addition, we have studied in depth the lectin activity of SP-D through expression of isolated lectin domains and targeted mutations of the SP-D lectin binding site. Through modifying specific residues around the saccharide binding pocket, antiviral activity of isolated lectin domains of SP-D can be markedly increased for seasonal strains of IAV. Wild-type SP-D causes little inhibition of pandemic IAV, but mutated versions of SP-D were able to inhibit pandemic IAV through enhanced binding to the reduced number of mannosylated glycans present on the HA of these strains. Through collaborative studies involving crystallography of isolated lectin domains of SP-D, glycomics analysis of the HA, and molecular modeling, the mechanism of binding of wild type and mutant forms of SP-D have been determined. These studies could guide investigation of the interactions of SP-D with other pathogens.