Juvenile social play predicts adult reproductive success in male bottlenose dolphins.

For over a century, the evolution of animal play has sparked scientific curiosity. The prevalence of social play in juvenile mammals suggests that play is a beneficial behavior, potentially contributing to individual fitness. Yet evidence from wild animals supporting the long-hypothesized link between juvenile social play, adult behavior, and fitness remains limited. In Western Australia, adult male bottlenose dolphins (Tursiops aduncus) form multilevel alliances that are crucial for their reproductive success. A key adult mating behavior involves allied males using joint action to herd individual females. Juveniles of both sexes invest significant time in play that resembles adult herding-taking turns in mature male (actor) and female (receiver) roles. Using a 32-y dataset of individual-level association patterns, paternity success, and behavioral observations, we show that juvenile males with stronger social bonds are significantly more likely to engage in joint action when play-herding in actor roles. Juvenile males also monopolized the actor role and produced an adult male herding vocalization ("pops") when playing with females. Notably, males who spent more time playing in the actor role as juveniles achieved more paternities as adults. These findings not only reveal that play behavior provides male dolphins with mating skill practice years before they sexually mature but also demonstrate in a wild animal population that juvenile social play predicts adult reproductive success.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Neonatal Chylothorax and Early Fluid Overload After Cardiac Surgery: Retrospective Analysis of the Neonatal and Pediatric Heart and Renal Outcomes Network Registry (2015-2018).

OBJECTIVES: To evaluate the association between postoperative cumulative fluid balance (FB) and development of chylothorax in neonates after cardiac surgery. DESIGN: Multicenter, retrospective cohort identified within the Neonatal and Pediatric Heart and Renal Outcomes Network (NEPHRON) Registry. SETTING: Twenty-two hospitals were involved with NEPHRON, from September 2015 to January 2018. PATIENTS: Neonates (< 30 d old) undergoing index cardiac operation with or without cardiopulmonary bypass (CPB) entered into the NEPHRON Registry. Postoperative chylothorax was defined in the Pediatric Cardiac Critical Care Consortium as lymphatic fluid in the pleural space secondary to a leak from the thoracic duct or its branches. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of the 2240 NEPHRON patients, 4% ( n = 89) were treated for chylothorax during postoperative day (POD) 2-21. Median (interquartile range [IQR]) time to diagnosis was 8 (IQR 6, 12) days. Of patients treated for chylothorax, 81 of 89 (91%) had CPB and 68 of 89 (76%) had Society of Thoracic Surgeons-European Association for Cardiothoracic Surgery Congenital Heart Surgery 4-5 operations. On bivariate analysis, chylothorax patients had higher POD 1 FB (3.2 vs. 1.1%, p = 0.014), higher cumulative POD 2 FB (1.5 vs. -1.5%, p < 0.001), achieved negative daily FB by POD 1 less often (69% vs. 79%, p = 0.039), and had lower POD 1 urine output (1.9 vs. 3. 2 mL/kg/day, p ≤ 0.001) than those without chylothorax. We failed to identify an association between presence or absence of chylothorax and peak FB (5.2 vs. 4.9%, p = 0.9). Multivariable analysis shows that higher cumulative FB on POD 2 was associated with greater odds (odds ratio [OR], 95% CI) of chylothorax development (OR 1.5 [95% CI, 1.1-2.2]). Further multivariable analysis shows that chylothorax was independently associated with greater odds of longer durations of mechanical ventilation (OR 5.5 [95% CI, 3.7-8.0]), respiratory support (OR 4.3 [95% CI, 2.9-6.2]), use of inotropic support (OR 2.9 [95% CI, 2.0-4.3]), and longer hospital length of stay (OR 3.7 [95% CI, 2.5-5.4]). CONCLUSIONS: Chylothorax after neonatal cardiac surgery for congenital heart disease (CHD) is independently associated with greater odds of longer duration of cardiorespiratory support and hospitalization. Higher early (POD 2) cumulative FB is associated with greater odds of chylothorax. Contemporary, prospective studies are needed to assess whether early fluid mitigation strategies decrease postoperative chylothorax development.

Competitive Sports Participation is Associated with Decreased Risk of Obesity in Children with Bicuspid Aortic Valve.

Individuals with bicuspid aortic valve (BAV) have historically been advised to avoid contact sports and isometric exercise for risk of increasing aortic dilation and valve disease. There is mounting evidence that current sports participation guidelines qualify children for a high rate of sports exclusion, and that this population is at increased risk of obesity. The primary aim of this study was to evaluate relationship between sports participation and aortic dilation in children with bicuspid aortic valve and secondarily the relationship between competitive sports participation and obesity. We performed a review of children between the ages of 8 and 17 years with isolated BAV followed at Oregon Health & Science University. We excluded those with other congenital heart diseases, genetic conditions, prior cardiac intervention, and inability to ambulate independently. Parents completed a phone survey detailing their child's level of daily activity and participation in competitive sports. Demographic information and most recent echo findings were collected from the electronic medical record. We found no difference between the aortic diameters of athletes vs non-athletes. We also found that sports participation and daily activity were both associated with a decreased likelihood of obesity (OR 0.24, 95% CI 0.078-0.73 and OR 0.24, 95% CI 0.081-0.71 respectively). In summary, in our sample population, competitive sports participation was associated with a decreased risk of obesity. Competitive sports participation does not appear to be associated with an increased risk of aortic dilation in our population.

Sports participation, activity, and obesity in children who have undergone the Fontan procedure.

BACKGROUND: Children with CHD are at risk for obesity and low levels of activity. These factors are associated with an increased risk of poor outcome. Participation in organised sports is an important avenue for children to maintain physical activity, though the relationship between sports participation and obesity has not been examined in the Fontan population. METHODS: We performed a cross-sectional study of children aged 8-18 who had been evaluated between January 1, 2015 and October 1, 2019 at the Doernbecher Children's Hospital outpatient paediatric cardiology clinic and had previously undergone a Fontan. Patients were excluded if they were unable to ambulate independently or if they had undergone a heart transplant. Patient characteristics were recorded from the electronic medical record. Parents were interviewed via a telephone survey and asked to describe their child's activity levels and sports participation. RESULTS: Our final cohort included 40 individuals, 74% were male. The overall prevalence of obesity (CDC BMI >95% for sex/age) in the cohort (23%) was significantly higher in non-athletes (33%) than athletes (0) (p = 0.02). There was no difference in cardiac complications or comorbidities between athletes and non-athletes. Athletes were more likely to meet daily activity recommendations (p = 0.05). CONCLUSION: Fontan patients who do not participate in sports are significantly more likely to be obese and less likely to be active than those who do. This is the first study to demonstrate the association between competitive sports participation and decreased likelihood of obesity in the Fontan population.

Development of consensus recommendations for the management of post-operative chylothorax in paediatric CHD.

OBJECTIVE: A standardised multi-site approach to manage paediatric post-operative chylothorax does not exist and leads to unnecessary practice variation. The Chylothorax Work Group utilised the Pediatric Critical Care Consortium infrastructure to address this gap. METHODS: Over 60 multi-disciplinary providers representing 22 centres convened virtually as a quality initiative to develop an algorithm to manage paediatric post-operative chylothorax. Agreement was objectively quantified for each recommendation in the algorithm by utilising an anonymous survey. "Consensus" was defined as ≥ 80% of responses as "agree" or "strongly agree" to a recommendation. In order to determine if the algorithm recommendations would be correctly interpreted in the clinical environment, we developed ex vivo simulations and surveyed patients who developed the algorithm and patients who did not. RESULTS: The algorithm is intended for all children (<18 years of age) within 30 days of cardiac surgery. It contains rationale for 11 central chylothorax management recommendations; diagnostic criteria and evaluation, trial of fat-modified diet, stratification by volume of daily output, timing of first-line medical therapy for "low" and "high" volume patients, and timing and duration of fat-modified diet. All recommendations achieved "consensus" (agreement >80%) by the workgroup (range 81-100%). Ex vivo simulations demonstrated good understanding by developers (range 94-100%) and non-developers (73%-100%). CONCLUSIONS: The quality improvement effort represents the first multi-site algorithm for the management of paediatric post-operative chylothorax. The algorithm includes transparent and objective measures of agreement and understanding. Agreement to the algorithm recommendations was >80%, and overall understanding was 94%.

The short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction.

Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.

Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH.

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696-10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered.IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the ß1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

The Utility of Echocardiography in Pediatric Patients with Structurally Normal Hearts and Suspected Endocarditis.

The objective of this study was to evaluate the utility of transthoracic echocardiography (TTE) in children with structurally normal hearts suspected of having infective endocarditis (IE). We hypothesized that the diagnostic yield of TTE is minimal in low-risk patients with normal hearts. We performed a retrospective chart review of TTEs performed for concern for endocarditis at a pediatric tertiary care referral center in Portland, Oregon. Three hundred patients met inclusion criteria (< 21 years old, completed TTE for IE from 2005 to 2015, no history of congenital heart disease or endocarditis). We recorded findings that met the modified Duke criteria (MDC) including fever, positive blood culture, and vascular/immunologic findings; presence of a central line; whether or not patients were diagnosed with IE clinically; and if any changes to antibiotic regimens were made based on TTE. Ten patients (3%) had echocardiograms consistent with IE. When compared to the clinical diagnosis of IE, the positive predictive value (PPV) of one positive blood culture without other major/minor MDC was 0. Similarly, the PPV of two positive blood cultures without other major/minor criteria was 0.071. Patients should be evaluated using the MDC to assess the clinical probability of IE prior to performing a TTE. Patients with a low probability for IE should not undergo TTE as it has a low diagnostic yield and patients are unlikely to be diagnosed with disease.

Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59.

The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

Determination of right ventricular function with adjusted systolic to diastolic duration ratio after transannular patch repair of tetralogy of Fallot.

BACKGROUND: Early detection of right ventricular dysfunction after transannular patch for tetralogy of Fallot (TOF-TAP) is essential for management. OBJECTIVES: To evaluate echocardiographic metrics of ventricular function correlate with functional MRI measurements, in patients with TOF-TAP. METHODS: A retrospective review of patients with TOF-TAP between 2007 and 2017 who had an echocardiogram and MRI within six months were analyzed. Systolic to diastolic ratio (SD ratio) was measured from the tricuspid regurgitation and adjusted for heart rate. Tricuspid Annular Plane Systolic Excursion (TAPSE), Fractional Area Change (FAC), and shortening fraction (SF) were additionally measured. Echocardiographic measurements were correlated with MRI assessment of right ventricular ejection fraction (RVEF), left ventricular ejection fraction (LVEF), right ventricular end-diastolic volume index (RVEDi), and right to left ventricle volume ratio (RV/LV). RESULTS: Of the 53 patients (mean age 12.8 years) that met inclusion criteria, 45 (85%) had available TR jets for SD ratio analysis. The HR adjusted SD ratio negatively correlated with RVEF (r = -.359, P = .016), LVEF (r = -.317, P = .038) and positively with RV/LV EDV ratio (r = .347, P = .024). TAPSE, FAC, and SF measurements did not show significant correlation. CONCLUSION: In patients with TOF-TAP, there is a moderate negative correlation between heart rate adjusted SD ratio and MRI metrics of ventricular function, suggesting that decreased filling time is a marker for reduced right ventricular function. The SD ratio may be a useful echocardiographic tool for serial evaluation of in this population.

Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor.

Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the Ceacam1a gene, called Ceacam1b, producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including ß-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein.

Clinical history and management recommendations of the smooth muscle dysfunction syndrome due to ACTA2 arginine 179 alterations.

PURPOSE: Smooth muscle dysfunction syndrome (SMDS) due to heterozygous ACTA2 arginine 179 alterations is characterized by patent ductus arteriosus, vasculopathy (aneurysm and occlusive lesions), pulmonary arterial hypertension, and other complications in smooth muscle-dependent organs. We sought to define the clinical history of SMDS to develop recommendations for evaluation and management. METHODS: Medical records of 33 patients with SMDS (median age 12 years) were abstracted and analyzed. RESULTS: All patients had congenital mydriasis and related pupillary abnormalities at birth and presented in infancy with a patent ductus arteriosus or aortopulmonary window. Patients had cerebrovascular disease characterized by small vessel disease (hyperintense periventricular white matter lesions; 95%), intracranial artery stenosis (77%), ischemic strokes (27%), and seizures (18%). Twelve (36%) patients had thoracic aortic aneurysm repair or dissection at median age of 14 years and aortic disease was fully penetrant by the age of 25 years. Three (9%) patients had axillary artery aneurysms complicated by thromboembolic episodes. Nine patients died between the ages of 0.5 and 32 years due to aortic, pulmonary, or stroke complications, or unknown causes. CONCLUSION: Based on these data, recommendations are provided for the surveillance and management of SMDS to help prevent early-onset life-threatening complications.

Widening participation in medicine? New insights from school students' aspirations.

OBJECTIVES: Students from lower socio-economic status backgrounds continue to be under-represented in medical education. Although various initiatives have been implemented by universities to widen participation, their effectiveness and their timing remain contentious. Prior studies have primarily focused on students already on a medical pathway, with little analytical attention given to the aspirations of primary and secondary school-aged students. The aim of this study was to identify the characteristics of students who express early interest in medicine and ascertain the degree to which diversification of the future medical student cohort is indicated. METHODS: As part of a longitudinal study of educational and occupational aspirations (2012-2015), students in Years 3-12 (n = 6492) from government schools in New South Wales, Australia, completed an annual online survey. Their individual responses were linked with prior academic achievement and demographic data. Logistic regression models were used to examine the significance of student- and school-related variables as predictors of interest in medicine. RESULTS: Significant predictors were: being in the early years of secondary school, possessing high cultural capital, coming from a language background other than English, being female, and perceiving oneself as 'well above average' relative to peers. Socio-economic status was a significant predictor when examined independently, but not when all variables were considered in the full regression model. CONCLUSIONS: For medical schools seeking to widen participation, this study underscores the importance of recognising the intersection of other factors with socio-economic status and how they contribute to students' aspirational biographies. If medical schools are to select from a more diverse range of applicants, recruitment strategies must take into account the discursive positioning of the discipline. Sustained outreach into primary and secondary schools may be critical to interrupting the current social reproduction of medical schooling.

Echocardiographic Screening of Rheumatic Heart Disease in American Samoa.

While rheumatic heart disease (RHD) is a treatable disease nearly eradicated in the United States, it remains the most common form of acquired heart disease in the developing world. This study used echocardiographic screening to determine the prevalence of RHD in children in American Samoa. Screening took place at a subset of local schools. Private schools were recruited and public schools underwent cluster randomization based on population density. We collected survey information and performed a limited physical examination and echocardiogram using the World Heart Federation protocol for consented school children aged 5-18 years old. Of 2200 students from two private high schools and two public primary schools, 1058 subjects consented and were screened. Overall, 133 (12.9%) children were identified as having either definite (3.5%) or borderline (9.4%) RHD. Of the patients with definitive RHD, 28 subjects had abnormal mitral valves with pathologic regurgitation, three mitral stenosis, three abnormal aortic valves with pathologic regurgitation, and seven borderline mitral and aortic valve disease. Of the subjects with borderline disease, 77 had pathologic mitral regurgitation, 12 pathologic aortic regurgitation, and 7 at least two features of mitral valve disease without pathologic regurgitation or stenosis. Rheumatic heart disease remains a major cause of morbidity and mortality worldwide. The prevalence of RHD in American Samoa (12.9%) is to date the highest reported in the world literature. Echocardiographic screening of school children is feasible, while reliance on murmur and Jones criteria is not helpful in identifying children with RHD.

Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins.

UNLABELLED: The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other ß-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of ß-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution. IMPORTANCE: Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 ß-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of ß-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of ß-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1.

UNLABELLED: Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (ß-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the ß-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human ß-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and ß-CoVs in groups B and C bind to their specific receptor proteins. Thus, two ß-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE: Mouse hepatitis virus, a ß-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another ß-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory ß-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two ß-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

Platform technology to generate broadly cross-reactive antibodies to α-helical epitopes in hemagglutinin proteins from influenza A viruses.

We have utilized a de novo designed two-stranded α-helical coiled-coil template to display conserved α-helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface-exposed residues of the native α-helix in the native HA protein of interest displayed on the surface of the two-stranded α-helical coiled-coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α-helix in the native protein. We investigated the highly conserved sequence region 421-476 of HA by inserting 21 or 28 residue sequences from this region into our template. The cross-reactivity of the resulting rabbit polyclonal antibodies prepared to these immunogens was determined using a series of HA proteins from H1N1, H2N2, H3N2, H5N1, H7N7, and H7N9 virus strains which are representative of Group 1 and Group 2 virus subtypes of influenza A. Antibodies from region 449-476 were Group 1 specific. Antibodies to region 421-448 showed the greatest degree of cross-reactivity to Group 1 and Group 2 and suggested that this region has a great potential as a "universal" synthetic peptide vaccine for influenza A. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 144-159, 2016.

Elevated uric acid and obesity-related cardiovascular disease risk factors among hypertensive youth.

BACKGROUND: Uric acid (UA) is associated with high blood pressure in adolescents and with left ventricular hypertrophy (LVH) and cardiovascular disease (CVD) in adults. We sought to determine if UA is independently associated with CVD risk factors and left ventricular mass (LVM) over time in hypertensive youth. METHODS: This was a 1-year prospective observational study of hypertensive children aged 3-19 years. Cross-sectional and longitudinal associations of serum UA with CVD risk factors and LVM were explored. RESULTS: Of the 49 children who completed both the baseline and 12-month assessments, at baseline the mean age was 13.8 years and mean UA was 5.5 mg/dL; 24% had elevated UA, 51% were overweight/obese and 39% had LVH. Measures of adiposity, low high-density lipoprotein cholesterol, high-sensitivity C-reactive protein, LVM and LVH were all significantly associated with elevated UA at baseline, but not with change over time. Each 1 mg/dL increase in baseline UA was associated with a 2.5 g/m(2.7) increase in the LVM index at follow-up (95% confidence interval 0.64, 4.39; p = 0.01); after adjustment for age, sex, race, body mass index z-score, change in UA, time, blood pressure and medication use, this association was no longer significant. CONCLUSIONS: Hypertensive children with elevated UA have a higher prevalence of obesity-related CVD risk factors. Among hypertensive children, UA may be a marker of adiposity and not an independent CVD risk factor.

Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses.

From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Children's Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.