Cloning and Functional Characterization of Novel Human Neutralizing Anti-IFN-α and Anti-IFN-ß Antibodies.

Type I IFNs play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-ß Abs from PBMCs of individuals treated with IFN-α or IFN-ß, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-ß1a-specific signaling and able to block lipopolysaccharide or S100 calcium-binding protein A14-induced IFN-ß signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-ß suggests potential for diverse research and clinical applications.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits Cellular Entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7, and B.1.351 Variants.

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 µM, in contrast to an IC50 of 28.3 µM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 µM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 µM). Notably, (-)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.

If we cannot measure it, we cannot improve it: Understanding measurement problems in routine oral/dental assessments in Canadian nursing homes-Part I.

OBJECTIVE: To compare Resident Assessment Instrument-Minimum Data Set 2.0 (RAI) oral/dental items collected by nursing home (NH) care staff to (a) assessments collected by trained research assistants (RAs) and (b) "gold standard" clinical assessments by dental hygienists (DHs). BACKGROUND: Routine collection of RAI oral/dental items is mandatory in most Canadian NHs. However, the performance of these items is less than optimal and oral/dental problems are severely under-reported. Accurate assessment is a prerequisite for preventing, detecting and treating oral health problems. Not knowing the reasons for performance problems is a barrier to improving performance of the RAI oral/dental items. MATERIALS AND METHODS: We included 103 NH residents from 4 NHs in Edmonton, Alberta, Canada. Using Kappa statistics, we compared the agreement of residents' last (no older than 90 days) RAI assessment with RAI assessments completed by trained RAs and "gold standard" clinical assessments by DHs. We also assessed the inter-rater reliability (IRR) of RA and DH assessments. RESULTS: Care staff assessments had poor agreement with RA and DH assessments (Kappa < 0.2 for most items). RAs and DHs identified more oral/dental problems than care staff. However, IRR of RA assessments was low (Kappa < 0.7 for 7/9 items). IRR of DH assessments was acceptable (Kappa > 0.7) for most items. CONCLUSIONS: The quality of RAI oral/dental assessments can be improved by better training care staff and ensuring appropriate time to do the assessments. However, remaining problems-even with trained RAs-suggest that rewording some of the items or supplementing them by more robust tools may be required.

If we cannot measure it, we cannot improve it: Understanding measurement problems in routine oral/dental assessments in Canadian nursing homes-Part II.

OBJECTIVE: To evaluate the response process validity of the Resident Assessment Instrument-Minimum Data Set 2.0 (RAI) oral/dental items and the organisational processes for assessing nursing home (NH) residents' oral/dental status. BACKGROUND: Although care aides provide most direct care to NH residents, including oral care, they are not directly involved in structured care planning activities, including RAI assessments. This most likely affects the accuracy of RAI assessments, as well quality of care. However, we neither know how well regulated and unregulated care staff understand the RAI oral/dental items, nor what processes are used in completing oral/dental assessments. METHODS: We conducted nine focus groups with 44 care aides, nurses, allied health providers, clinical specialists and managers. We discussed randomly selected RAI oral/dental assessments with focus group participants, including participants' understanding of the items and why the options were selected. Participants also explained the communication and process for completing the RAI. RESULTS: Participants' perceptions of the oral/dental items aligned fairly well with the item definitions. However, responses primarily focused on severe oral/dental problems with obvious physical characteristics (eg black teeth denoting caries). For non-visual oral problems, such as pain, staff relied on resident verbalisation. No formal mechanisms were described for care aides to update nurses on residents' oral health needs. CONCLUSIONS: Performance problems of RAI oral/dental items are largely rooted in poor communication between care aides and nurses and not integrating care aides in assessment processes. We need policies that address these problems in order to improve NH residents' poor oral health.

HIV Risk Among Displaced Adolescent Girls in Ethiopia: the Role of Gender Attitudes and Self-Esteem.

Adolescent girls in sub-Saharan Africa have been deemed one of the most critical populations to address in the campaign for an HIV-free generation. Experiences of intimate partner violence (IPV), harmful gender norms, diminished personal agency, and age-disparate sex have been identified as factors in the increasing rate of new infections among this population. Using baseline data from a cluster-randomized controlled trial in three refugee camps in Benishangul-Gumuz Regional State in Ethiopia, our study quantitatively examined the associations between HIV risk factors, attitudes on gender inequality, IPV acceptability, and self-esteem for female adolescent refugees primarily from Sudan and South Sudan (n = 919). In multivariate models, adjusting for age and education, results showed girls who were more accepting of gender inequitable norms and IPV had greater odds of ever experiencing forced (OR 1.40, CI 1.15-1.70; OR 1.66, CI 1.42-1.94) or transactional sex (OR 1.28, CI 1.05-1.55; OR 1.59, CI 1.37-1.85) compared to girls who demonstrated less approval. Higher self-esteem was associated with increased odds of condom use (OR 1.13, CI 1.02-1.24) as well as decreased odds of adolescent marriage (OR 0.93, CI 0.90-0.95), age-disparate sex (OR 0.90, CI 0.86-0.94), and transactional sex (OR 0.96, CI 0.93-0.99). The findings suggest acceptance of inequitable gender norms (including those that perpetuate violence against women) and low self-esteem to be associated with common HIV risk factors among refugee adolescents living in Ethiopia. Greater attention towards the intersections of gender equality and self-valuation is needed when seeking to understand HIV risk among refugee adolescent girls in sub-Saharan Africa.

Does Economic Strengthening Improve Viral Suppression Among Adolescents Living with HIV? Results From a Cluster Randomized Trial in Uganda.

To assess the effect of a savings-led economic empowerment intervention on viral suppression among adolescents living with HIV. Using data from Suubi + Adherence, a longitudinal, cluster randomized trial in southern Uganda (2012-2017), we examine the effect of the intervention on HIV RNA viral load, dichotomized between undetectable (< 40 copies/ml) and detectable (≥ 40 copies/ml). Cluster-adjusted comparisons of means and proportions were used to descriptively analyze changes in viral load between study arms while multi-level modelling was used to estimate treatment efficacy after adjusting for fixed and random effects. At 24-months post intervention initiation, the proportion of virally suppressed participants in the intervention cohort increased tenfold (ΔT2-T0 = + 10.0, p = 0.001) relative to the control group (ΔT2-T0 = + 1.1, p = 0.733). In adjusted mixed models, simple main effects tests identified significantly lower odds of intervention adolescents having a detectable viral load at both 12- and 24-months. Interventions addressing economic insecurity have the potential to bolster health outcomes, such as HIV viral suppression, by improving ART adherence among vulnerable adolescents living in low-resource environments. Further research and policy dialogue on the intersections of financial security and HIV treatment are warranted.

MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus.

Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5' UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784-789]. On the other hand, the 5q translocation breakpoint disrupts the 3' UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3' UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3(Gt-ex13) gene trap allele that disrupted the 3' portion of the gene. Matr3(Gt-ex13) homozygotes are early embryo lethal, but Matr3(Gt-ex13) heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3(Gt-ex13) gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.

Von Willebrand factor regulates complement on endothelial cells.

Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury.

Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy.

Hematopoietic stem cell transplant (HSCT)-associated thrombotic microangiopathy (TMA) is a complication that occurs in 25% to 35% of HSCT recipients and shares histomorphologic similarities with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The hallmark of all thrombotic microangiopathies is vascular endothelial cell injury of various origins, resulting in microangiopathic hemolytic anemia, platelet consumption, fibrin deposition in the microcirculation, and tissue damage. Although significant advances have been made in understanding the pathogenesis of other thrombotic microangiopathies, post-HSCT TMA remains poorly understood. We report an analysis of the complement alternative pathway, which has recently been linked to the pathogenesis of both the Shiga toxin mediated and the atypical forms of HUS, with a focus on genetic variations in the complement Factor H (CFH) gene cluster and CFH autoantibodies in six children with post-HSCT TMA. We identified a high prevalence of deletions in CFH-related genes 3 and 1 (delCFHR3-CFHR1) and CFH autoantibodies in these patients with HSCT-TMA. Conversely, CFH autoantibodies were not detected in 18 children undergoing HSCT who did not develop TMA. Our observations suggest that complement alternative pathway dysregulation may be involved in the pathogenesis of post-HSCT TMA. These findings shed light on a novel mechanism of endothelial injury in transplant-TMA and may therefore guide the development of targeted treatment interventions.

Eculizumab therapy in children with severe hematopoietic stem cell transplantation-associated thrombotic microangiopathy.

We recently observed that dysregulation of the complement system may be involved in the pathogenesis of hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA). These findings suggest that the complement inhibitor eculizumab could be a therapeutic option for this severe HSCT complication with high mortality. However, the efficacy of eculizumab in children with HSCT-TMA and its dosing requirements are not known. We treated 6 children with severe HSCT-TMA using eculizumab and adjusted the dose to achieve a therapeutic level >99 µg/mL. HSCT-TMA resolved over time in 4 of 6 children after achieving therapeutic eculizumab levels and complete complement blockade, as measured by low total hemolytic complement activity (CH50). To achieve therapeutic drug levels and a clinical response, children with HSCT-TMA required higher doses or more frequent eculizumab infusions than currently recommended for children with atypical hemolytic uremic syndrome. Two critically ill patients failed to reach therapeutic eculizumab levels, even after dose escalation, and subsequently died. Our data indicate that eculizumab may be a therapeutic option for HSCT-TMA, but HSCT patients appear to require higher medication dosing than recommended for other conditions. We also observed that a CH50 level ≤ 4 complement activity enzyme units correlated with therapeutic eculizumab levels and clinical response, and therefore CH50 may be useful to guide eculizumab dosing in HSCT patients as drug level monitoring is not readily available.

Cloning and functional characterization of novel human neutralizing anti-interferon-alpha and anti-interferon-beta antibodies.

Type I interferons (IFNs) play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-ß antibodies (Abs) from peripheral blood mononuclear cells of individuals treated with IFN-α or IFN-ß, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-ß1a-specific signaling, and able to block Lipopolysaccharide or S100 calcium binding protein A14-induced IFN-ß signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-ß suggests potential for diverse research and clinical applications.

Targeting LxCxE cleft pocket of retinoblastoma protein in M2 macrophages inhibits ovarian cancer progression.

Ovarian cancer remains a major health threat with limited treatment options available. It is characterized by immunosuppressive tumor microenvironment (TME) maintained by tumor- associated macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. Here we show that targeting retinoblastoma protein (Rb) by disruption of its LxCxE cleft pocket, causes cell death in TAMs by induction of ER stress, p53 and mitochondria-related cell death pathways. A reduction of pro-tumor Rb high M2-type macrophages from TME in vivo enhanced T cell infiltration and inhibited cancer progression. We demonstrate an increased Rb expression in TAMs in women with ovarian cancer is associated with poorer prognosis. Ex vivo, we show analogous cell death induction by therapeutic Rb targeting in TAMs in post-surgery ascites from ovarian cancer patients. Overall, our data elucidates therapeutic targeting of the Rb LxCxE cleft pocket as a novel promising approach for ovarian cancer treatment through depletion of TAMs and re-shaping TME immune landscape. Statement of significance: Currently, targeting immunosuppressive myeloid cells in ovarian cancer microenvironment is the first priority need to enable successful immunotherapy, but no effective solutions are clinically available. We show that targeting LxCxE cleft pocket of Retinoblastoma protein unexpectedly induces preferential cell death in M2 tumor-associated macrophages. Depletion of immunosuppressive M2 tumor-associated macrophages reshapes tumor microenvironment, enhances anti-tumor T cell responses, and inhibits ovarian cancer. Thus, we identify a novel paradoxical function of Retinoblastoma protein in regulating macrophage viability as well as a promising target to enhance immunotherapy efficacy in ovarian cancer.

Depletion of autoreactive plasma cells and treatment of lupus nephritis in mice using CEP-33779, a novel, orally active, selective inhibitor of JAK2.

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC(50) = 1.3 nM; JAK3 enzyme IC(50)/JAK2 enzyme IC(50) = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1ß, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138(+) plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

The In-Cell Western immunofluorescence assay to monitor PROTAC mediated protein degradation.

The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT6 receptor antagonist showing activity in rat social recognition test.

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ∼30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.

Novel brain penetrant benzofuropiperidine 5-HT6 receptor antagonists.

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.

Development of a Unified National Database of Burn Centers With Colocated Emergency Departments, 2020.

The care of severely burned patients comes with unique requirements for specialized burn centers. The American Burn Association sets guidelines for burn centers and provides a voluntary program to verify their quality of care. However, not all burn centers are verified, and it is unclear which nonverified centers have met requirements set by their state health departments. To compile a complete database of all U.S. emergency departments in facilities with confirmed burn centers, we investigated state requirements to supplement data from the American Burn Association verification process. In 2020, only 13 states set requirements for burn centers; 3 states explicitly required American Burn Association verification, 4 used modified American Burn Association criteria, and 6 used alternate criteria. Only two states had separate requirements for pediatric burn centers. Based on adherence to state and American Burn Association criteria, we identified 90 confirmed burn centers in 2020, 85 of which had emergency departments. Of these 85, 45 (53%) were only verified, 17 (20%) were only state-confirmed, and 23 (27%) were both. Emergency departments in a confirmed burn center were more likely-than those without-to have higher adult and pediatric visit volumes, be academic, be a stroke or trauma (adult or pediatric) center, have a dedicated pediatric area, and have a pediatric emergency care coordinator. We compiled the first unified burn center database that incorporates state and American Burn Association lists. This database can be utilized in future health services research and is available to the public through a smartphone application.

A Geographical Analysis of Access to Trauma Centers from US National Parks in 2018.

INTRODUCTION: Millions of people visit US national parks annually to engage in recreational wilderness activities, which can occasionally result in traumatic injuries that require timely, high-level care. However, no study to date has specifically examined timely access to trauma centers from national parks. This study aimed to examine the accessibility of trauma care from national parks by calculating the travel time by ground and air from each park to its nearest trauma center. Using these calculations, the percentage of parks by census region with timely access to a trauma center was determined. METHODS: This was a cross-sectional study analyzing travel times by ground and air transport between national parks and their closest adult advanced trauma center (ATC) in 2018. A list of parks was compiled from the National Parks Service (NPS) website, and the location of trauma centers from the 2018 National Emergency Department Inventory (NEDI)-USA database. Ground and air transport times were calculated using Google Maps and ArcGIS, with medians and interquartile ranges reported by US census region. Percentage of parks by region with timely trauma center access-defined as access within 60 minutes of travel time-were determined based on these calculated travel times. RESULTS: In 2018, 83% of national parks had access to an adult ATC within 60 minutes of air travel, while only 26% had timely access by ground. Trauma center access varied by region, with median travel times highest in the West for both air and ground transport. At a national level, national parks were unequally distributed, with the West housing the most parks of all regions. CONCLUSION: While most national parks had timely access to a trauma center by air travel, significant gaps in access remain for ground, the extent of which varies greatly by region. To improve the accessibility of trauma center expertise from national parks, the study highlights the potential that increased implementation of trauma telehealth in emergency departments (EDs) may have in bridging these gaps.

Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

BACKGROUND: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA). RESULTS: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1ß, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo. CONCLUSIONS: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

De Novo Assembly of the Northern Cardinal (Cardinalis cardinalis) Genome Reveals Candidate Regulatory Regions for Sexually Dichromatic Red Plumage Coloration.

Northern cardinals (Cardinalis cardinalis) are common, mid-sized passerines widely distributed in North America. As an iconic species with strong sexual dichromatism, it has been the focus of extensive ecological and evolutionary research, yet genomic studies investigating the evolution of genotype-phenotype association of plumage coloration and dichromatism are lacking. Here we present a new, highly-contiguous assembly for C. cardinalis We generated a 1.1 Gb assembly comprised of 4,762 scaffolds, with a scaffold N50 of 3.6 Mb, a contig N50 of 114.4 kb and a longest scaffold of 19.7 Mb. We identified 93.5% complete and single-copy orthologs from an Aves dataset using BUSCO, demonstrating high completeness of the genome assembly. We annotated the genomic region comprising the CYP2J19 gene, which plays a pivotal role in the red coloration in birds. Comparative analyses demonstrated non-exonic regions unique to the CYP2J19 gene in passerines and a long insertion upstream of the gene in C. cardinalis Transcription factor binding motifs discovered in the unique insertion region in C. cardinalis suggest potential androgen-regulated mechanisms underlying sexual dichromatism. Pairwise Sequential Markovian Coalescent (PSMC) analysis of the genome reveals fluctuations in historic effective population size between 100,000-250,000 in the last 2 millions years, with declines concordant with the beginning of the Pleistocene epoch and Last Glacial Period. This draft genome of C. cardinalis provides an important resource for future studies of ecological, evolutionary, and functional genomics in cardinals and other birds.