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Background: Patellar tendinopathy is a common chronic condition caused by mechanical loading that leads to patellar tendon (PT) degeneration, with an estimated incidence of 13% across all sports in adolescents. Musculoskeletal ultrasound (MSK-US) evaluation is becoming more common in the diagnostic process, but normative data are extremely rare for the adolescent population. These normative data are needed to aid in proper interpretation of the MSK-US image. Purpose: The purpose of this study was to determine standard reference values for thickness, width, and cross-sectional area (CSA) of the PT using MSK-US in asymptomatic adolescent athletes. Methods: IRB approval, parental consent, and athlete assent were obtained prior to data collection. Bilateral PT were scanned using a portable US unit with a 12-4MHz linear array transducer. Long axis images were captured at the apex and 1cm distal to the patella, 1 cm proximal to the insertion and at the PT insertion (Figure 1a & 1b). Short axis images were captured 1 cm proximal and distal to the attachments. PT thickness, width, and CSA were measured using ImageJ by a single evaluator. Additional exploratory analyses were also conducted. Results: Seventy-one adolescent athletes (27 males, 44 females) between 11-18 years old (13.8 ±1.8 years) volunteered. Mean values for PT thickness, width, and CSA are found in Table 1. Significant differences in PT thickness and CSA between sex were identified at each measurement location (P(52)=.348, P=.014, right PT: r(49)=.369, P=.007) and weight (left PT: r(50 )=.290, P=.041). There was no correlation with age. Conclusion: These data represent one of the largest healthy cohorts of adolescent athletes from which normative data have been collected. This preliminary analysis reveals some interesting trends, such as the thickness of the PT is greatest at the insertion and origin, while thinner at mid-tendon, with the insertion the thicker of the two. Age was not correlated with PT size, but distal PT CSA was larger with taller and heavier individuals. These data will contribute to the paucity of adolescent PT MSK-US data, which should aid clinicians in interpretation, diagnostic accuracy, and management of active adolescent patients with patellar tendinopathy. [Figure: see text][Table: see text]

Changing with the timesPandemic spread of a virus in naïe populations can select for mutations that alter pathogenesis, virulence, and/or transmissibility. The ancestral form of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged from China has now been largely replaced by strains containing the mutation D614G (Asp614-to-Gly) in the viral spike protein. Houet al.compared the characteristics of the new variant against the ancestral form in a series of experiments in human cells and animal models. The variant is better at infecting upper-airway epithelial cells and replicates in greater numbers than the ancestral virus. Evidence indicates modest, if any, significant changes to virulence in animal models. Therefore, the virus appears to have evolved for greater transmissibility in humans rather than for greater pathogenicity. The mutation renders the new virus variant more susceptible to neutralizing antisera without altering the efficacy of vaccine candidates currently under development.Science, this issue p.1464

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS-CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibitingACE2expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission.

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.

Objectives: In this paper, we examined diet quality and associations between changes in skin carotenoids and body composition among selected NCAA Division I athletes. Methods: Athletes from women's (rowing, swimming, gymnastics) and men's (swimming, wrestling) teams at a large Midwest university (N = 129) completed one online food frequency questionnaire and 2 in-person visits, once in-season and once out-of-season, to assess skin carotenoids and body composition. Diet quality was assessed via Healthy Eating Index-2015 (HEI). Carotenoids were measured via resonance Raman spectroscopy and body composition via dual-energy x-ray absorptiometry. ANOVA and Pearson correlations were used to test differences between teams and determine association between changes from in-season to out-of-season. Results: Mean HEI score for all athletes was 71.0. Women's rowing reported the highest diet quality (73.5), men's wrestling lowest (56.5). Skin carotenoids decreased for all teams, except men's wrestling, from in-season to out-of-season. Body fat percentage increased for women and decreased for men. There was a moderate inverse association between changes in skin carotenoids and body fat percentage (r = -.334, p = .001). Conclusions: Suboptimal diet quality coupled with decreases in skin carotenoids and increases in body fat percentage from in-season to out-of-season may justify dietitian-led interventions year-round to improve dietary patterns in collegiate athletes.

High altitude temperate mountains have long been considered devoid of life owing to high extinction or low speciation rates during the Pleistocene. We performed a phylogenetic and population genomic investigation of an emblematic high-altitude plant clade (Androsacesect.Aretia, 31 currently recognized species), based on plant surveys conducted during alpinism expeditions. Surprisingly, we inferred that this clade originated in the Miocene and continued diversifying through Pleistocene glaciations, and discovered three novel species ofAndrosacedwelling on different bedrock types on the rooftops of the Alps. This suggests that temperate high mountains have been cradles of plant diversity even during the Pleistocene, with in-situ speciation driven by the combined action of geography and geology. Our findings have an unexpected historical relevance: H.-B. de Saussure likely observed one of these species during his 1788 expedition to the Mont Blanc and we describe it here, over two hundred years after its first sighting.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expres-sion in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-in-fected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings high-light the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host in-teractions in protective immunity, host susceptibility, and virus pathogenesis.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1. Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

14 pages; International audience; Savanna is a species-rich biome, that includes many modern mammal lineages and C4 grass (Poaceae) species. The greater productivity and grazing pressure associated with savannas is likely attributable to the foliar traits of the grasses they support. Thus, it is important to understand the complex relationships between the abiotic environment, foliar attributes and the floristic composition of savanna grasses, and the supported grazer densities. We sampled 37 grass communities in the Kruger National Park (South Africa) across three soil types and along a rainfall gradient and found that these communities lack strong phylogenetic structure. We then measured specific leaf area and leaf tensile strength for 384 individuals representing 66 species and found that both traits were strongly phylogenetically structured and associated with both rainfall and soil type. Finally, we found that grazer densities in the Park are correlated with the foliar traits of the associated grass communities, but the resolution of our data do not allow for a thorough analysis of this association. Our results demonstrate the complex interactions between climate, soils and grazer densities relative to C4 grass functional traits.

It is widely accepted that sea level changes intermittently inun- dated the Sunda Shelf throughout the Pleistocene, separating Java, Sumatra and Borneo from the Malay Peninsula and from each other. On this basis, the dynamics of the biodiversity hotspot of Sundaland is consistently regarded as solely contingent on glacial sea level os- cillations, with interglacial highstands creating intermittent dispersal barriers between disjunct landmasses. However, recent findings on the geomorphology of the currently submerged Sunda shelf sug- gest that it subsided during the Pleistocene and that, over the Late Pliocene and Quaternary, is was never submerged prior to Marine Isotope Stage 11 (MIS 11, 400 ka). This would have enabled the dispersal of terrestrial organisms regardless of sea level variations until 400 ka and hampered movements thereafter, at least during interglacial periods. Existing phylogeographic data for terrestrial organisms conform to this scenario: available divergence time esti- mates reveal an 8‐ to 9‐fold increase in the rate of vicariance be- tween landmasses of Sundaland after 400 ka, corresponding to the onset of episodic flooding of the Sunda shelf. These results highlight how reconsidering the paleogeographic setting of Sundaland chal- lenges understanding the mechanisms generating Southeast Asian biodiversity.

Rationale: Non–cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized. Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity. Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured. Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV(1) and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non–cystic fibrosis bronchiectasis mucus concentration by 5%. Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

We hypothesized that dynamic perfluorinated gas MRI would sensitively detect mild cystic fibrosis (CF) lung disease. This cross-sectional study enrolled 20 healthy volunteers and 24 stable subjects with CF, including a subgroup of subjects with normal forced expiratory volume in the first second (FEV(1); >80% predicted, n = 9). Dynamic fluorine-19–enhanced MRI ((19)F MRI) were acquired during sequential breath holds while breathing perfluoropropane (PFP) and during gas wash-out. Outcomes included the fraction of lung without significant ventilation (ventilation defect percent, VDP) and time constants that described PFP wash-in and wash-out kinetics. VDP values (mean ± SD) of healthy controls (3.87% ± 2.7%) were statistically different from moderate CF subjects (19.5% ± 15.5%, P = 0.001) but not from mild CF subjects (10.4% ± 9.9%, P = 0.24). In contrast, the fractional lung volume with slow gas wash-out was elevated both in subjects with mild (9.61% ± 4.87%; P = 0.0066) and moderate CF (16.01% ± 5.01%; P = 0.0002) when compared with healthy controls (3.84% ± 2.16%) and distinguished mild from moderate CF (P = 0.006). (19)F MRI detected significant ventilation abnormalities in subjects with CF. The ability of gas wash-out kinetics to distinguish between healthy and mild CF lung disease subjects makes (19)F MRI a potentially valuable method for the characterization of early lung disease in CF. This study has been registered at ClinicalTrials.gov (NCT03489590).

[Aim] Intraspecific trait variation (ITV) within natural plant communities can be large, influencing local ecological processes and dynamics. Here, we shed light on how ITV in vegetative and floral traits responds to large‐scale abiotic and biotic gradients (i.e., climate and species richness). Specifically, we tested whether associations of ITV with temperature, precipitation and species richness were consistent with any of four hypotheses relating to stress tolerance and competition. Furthermore, we estimated the degree of correlation between ITV in vegetative and floral traits and how they vary along the gradients., [Location] Global., [Time period] 1975–2016., [Major taxa studied] Herbaceous and woody plants., [Methods] We compiled a dataset of 18,401 measurements of the absolute extent of ITV (measured as the coefficient of variation) in nine vegetative and seven floral traits from 2,822 herbaceous and woody species at 2,372 locations., [Results] Large‐scale associations between ITV and climate were trait specific and more prominent for vegetative traits, especially leaf morphology, than for floral traits. The ITV showed pronounced associations with climate, with lower ITV values in colder areas and higher values in drier areas. The associations of ITV with species richness were inconsistent across traits. Species‐specific associations across gradients were often idiosyncratic, and covariation in ITV was weaker between vegetative and floral traits than within the two trait groups. [Main conclusions] Our results show that, depending on the traits considered, ITV either increased or decreased with climate stress and species richness, suggesting that both factors can constrain or enhance ITV, which might foster plant‐population persistence in stressful conditions. Given the species‐specific responses and covariation in ITV, associations can be hard to predict for traits and species not yet studied. We conclude that consideration of ITV can improve our understanding of how plants cope with stressful conditions and environmental change across spatial and biological scales., Open Access funding was provided by Ulm University under the DEAL‐agreement.

Engineered T cells expressing chimeric antigen receptors (CARs) on their cell surface can redirect antigen specificity. This ability makes CARs one of the most promising cancer therapeutic agents. CAR-T cells for treating patients with B cell hematological malignancies have shown impressive results. Clinical manifestation has yielded several trials, so far five CAR-T cell therapies have received US Food and Drug Administration (FDA) approval. However, emerging clinical data and recent findings have identified some immune-related toxicities due to CAR-T cell therapy. Given the outcome and utilization of the same proof of concept, further investigation in other hematological malignancies, such as leukemias, is warranted. This review discusses the previous findings from the pre-clinical and human experience with CAR-T cell therapy. Additionally, we describe recent developments of novel targets for adoptive immunotherapy. Here we present some of the early findings from the pre-clinical studies of CAR-T cell modification through advances in genetic engineering, gene editing, cellular programming, and formats of synthetic biology, along with the ongoing efforts to restore the function of exhausted CAR-T cells through epigenetic remodeling. We aim to shed light on the new targets focusing on acute myeloid leukemia (AML).

OBJECTIVES Our purpose was to determine whether ultrasound (US)-aided instruction and practice on musculoskeletal anatomy would improve first-year medical students' ability to locate and identify specific soft tissue structures by unaided palpation in the upper and lower extremities of healthy human models. METHODS This study was a randomized crossover design with 49 first-year medical students randomly assigned to 1 of 2 groups. Each group was provided expert instruction and hands-on practice using US to scan and study soft tissue structures. During session 1, group A learned the anatomy of the upper extremities, whereas group B learned the lower. Students were then tested on their proficiency in locating 4 soft tissue structures (2 upper and 2 lower extremities) through palpation of a human model. During session 2, group A learned lower extremities, and group B learned upper. At the end of session 2, students repeated the assessment. RESULTS After the first instructional session, neither group performed significantly better on identifying and locating the soft tissue landmarks they learned aided by US. After the second instructional session, however, scores for both groups increased approximately 20 percentage points, indicating that both groups performed significantly better on palpating and identifying both the upper and lower extremity soft tissue landmarks (Cohen d = 0.89 and 0.82, respectively). CONCLUSIONS Time and practice viewing soft tissue structures with US assistance seems to have a "palpation-with-eyes" effect that improves students' abilities to correctly locate, palpate, and identify limb-specific soft tissue structures once the US assistance is removed.

It is widely acknowledged that metaphysical assumptions, commitments and presuppositions play an important role in science. Yet according to the empiricist there is no place for metaphysics as traditionally understood in the scientific enterprise. In this paper I aim to take a first step towards reconciling these seemingly irreconcilable claims. In the first part of the paper I outline a conception of metaphysics and its relation to science that should be congenial to empiricists, motivated by van Fraassen’s work on ‘stances’. There has been a considerable about of recent work devoted to van Fraassen’s ‘stance’ view, but it has not on the whole been noticed that the view has the potential to motivate a general empiricist conception of the relation between science and metaphysics. In the second and third sections I discuss two examples from biology to illustrate this conception: metaphysical punctuationism, and its relation to and influence on the thesis of punctuated equilibrium; and dialectical biology as defended by Levins and Lewontin.

Understanding how expression of airway secretory mucins MUC5B and MUC5AC is regulated in health and disease is important to elucidating the pathogenesis of mucoobstructive respiratory diseases. The transcription factor SPDEF (sterile α-motif pointed domain epithelial specific transcription factor) is a key regulator of MUC5AC, but its role in regulating MUC5B in health and in mucoobstructive lung diseases is unknown. Characterization of Spdef-deficient mice upper and lower airways demonstrated region-specific, Spdef-dependent regulation of basal Muc5b expression. Neonatal Spdef-deficient mice exhibited reductions in BAL Muc5ac and Muc5b. Adult Spdef-deficient mice partially phenocopied Muc5b-deficient mice as they exhibited reduced Muc5b in nasopharyngeal and airway epithelia but not in olfactory Bowman glands, 75% incidence of nasopharyngeal hair/mucus plugs, and mild bacterial otitis media, without defective mucociliary clearance in the nasopharynx. In contrast, tracheal mucociliary clearance was reduced in Spdef-deficient mice in the absence of lung disease. To evaluate the role of Spdef in the development and persistence of Muc5b-predominant mucoobstructive lung disease, Spdef-deficient mice were crossed with Scnn1b-transgenic (Scnn1b-Tg) mice, which exhibit airway surface dehydration-induced airway mucus obstruction and inflammation. Spdef-deficient Scnn1b-Tg mice exhibited reduced Muc5ac, but not Muc5b, expression and BAL content. Airway mucus obstruction was not decreased in Spdef-deficient Scnn1b-Tg mice, consistent with Muc5b-dominant Scnn1b disease, but increased airway neutrophilia was observed compared with Spdef-sufficient Scnn1b-Tg mice. Collectively, these results indicate that Spdef regulates baseline Muc5b expression in respiratory epithelia but does not contribute to Muc5b regulation in a mouse model of Muc5b-predominant mucus obstruction caused by airway dehydration.

Rationale: Rural residence is associated with poor outcomes in several chronic diseases. The association between rural residence and chronic obstructive pulmonary disease (COPD) exacerbations remains unclear.Objectives: In this work, we sought to determine the independent association between rural residence and COPD-related outcomes, including COPD exacerbations, airflow obstruction, and symptom burden.Methods: A total of 1,684 SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study) participants with forced expiratory volume in 1 second/forced vital capacity < 0.70 had geocoding-defined rural-urban residence status determined (N = 204 rural and N = 1,480 urban). Univariate and multivariate logistic and negative binomial regressions were performed to assess the independent association between rurality and COPD outcomes, including exacerbations, lung function, and symptom burden. The primary exposure of interest was rural residence, determined by geocoding of the home address to the block level at the time of study enrollment. Additional covariates of interest included demographic and clinical characteristics, occupation, and occupational exposures. The primary outcome measures were exacerbations determined over a 1-year course after enrollment by quarterly telephone calls and at an annual research clinic visit. The odds ratio (OR) and incidence rate ratio (IRR) of exacerbations that required treatment with medications, including steroids or antibiotics (total exacerbations), and exacerbations leading to hospitalization (severe exacerbations) were determined after adjusting for relevant covariates.Results: Rural residence was independently associated with a 70% increase in the odds of total exacerbations (OR, 1.70 [95% confidence interval (CI), 1.13-2.56]; P = 0.012) and a 46% higher incidence rate of total exacerbations (IRR 1.46 [95% CI, 1.02-2.10]; P = 0.039). There was no association between rural residence and severe exacerbations. Agricultural occupation was independently associated with increased odds and incidence of total and severe exacerbations. Inclusion of agricultural occupation in the analysis attenuated the association between rural residence and the odds and incidence rate of total exacerbations (OR, 1.52 [95% CI, 1.00-2.32]; P = 0.05 and IRR 1.39 [95% CI, 0.97-1.99]; P = 0.07). There was no difference in symptoms or airflow obstruction between rural and urban participants.Conclusions: Rural residence is independently associated with increased odds and incidence of total, but not severe, COPD exacerbations. These associations are not fully explained by agriculture-related exposures, highlighting the need for future research into potential mechanisms of the increased risk of COPD exacerbations in the rural population.

A single-nucleotide polymorphism (rs35705950) in the mucin 5B (MUC5B) gene promoter is associated with pulmonary fibrosis and interstitial features on chest CT but may also have beneficial effects. In non-Hispanic whites in the COPDGene cohort with interstitial features (n=454), the MUC5B promoter polymorphism was associated with a 61% lower odds of a prospectively reported acute respiratory disease event (P=0.001), a longer time-to-first event (HR=0.57; P=0.006) and 40% fewer events (P=0.016). The MUC5B promoter polymorphism may have a beneficial effect on the risk of acute respiratory disease events in smokers with interstitial CT features.

International audience; Organisms have evolved a diversity of life-history strategies to cope with variation in their environment. Persistence as adults and/or seeds across recruitment events allows species to dampen the effects of environmental fluctuations. The evolution of life cycles with overlapping generations should thus permit the colonization of environments with uncertain recruitment. We tested this hypothesis in Leucadendron (Proteaceae), a genus with high functional diversity native to fire-prone habitats in the South African fynbos. We analyzed the joint evolution of life-history traits (adult survival and seed-bank strategies) and ecological niches (climate and fire regime), using comparative methods and accounting for various sources of uncertainty. In the fynbos, species with canopy seed banks that are unable to survive fire as adults display nonoverlapping generations. In contrast, resprouters with an underground seed bank may be less threatened by extreme climatic events and fire intervals, given their iteroparity and long-lasting seed bank. Life cycles with nonoverlapping generations indeed jointly evolved with niches with less exposure to frost but not with those with less exposure to drought. Canopy seed banks jointly evolved with niches with more predictable fire return, compared to underground seed banks. The evolution of extraordinary functional diversity among fynbos plants thus reflects, at least in part, the diversity of both climates and fire regimes in this region.

A natural splice deletion of rat α-ENaC was previously reported to produce reduced ENaC current that was not attributable to reduced surface expression. Kota et al. show that this ENaC variant resists furin cleavage, implicating α-ENaC residues 34–82 in ENaC posttranslational processing., Epithelial Na+ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (INa) that results from expression of a previously identified, naturally occurring splice variant of the α subunit (α-ENaC), in which residues 34–82 are deleted (αΔ34–82). αΔ34–82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal INa, which can largely be recovered by exogenous trypsin. With this αΔ34–82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced INa of αΔ34–82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, INa in oocytes expressing αΔ34–82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing αΔ34–82-ENaC. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to nuclear factor kappa B (NF-κB) signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed lymphocyte-specific protein tyrosine kinase (LCK) recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NOD scid gamma (NSG) mice. We also engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased central memory phenotype, expansion, and LCK recruitment to the CAR. This enhanced function was dependent on the positioning of the two co-stimulatory domains. A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. Bi-specific CAR T cells exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion.ConclusionThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono-specific and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

Summary Background Increased concentrations of eosinophils in blood and sputum in chronic obstructive pulmonary disease (COPD) have been associated with increased frequency of exacerbations, reduced lung function, and corticosteroid responsiveness. We aimed to assess whether high eosinophil concentrations in either sputum or blood are associated with a severe COPD phenotype, including greater exacerbation frequency, and whether blood eosinophils are predictive of sputum eosinophils. Methods We did a multicentre observational study analysing comprehensive baseline data from SPIROMICS in patients with COPD aged 40–80 years who had a smoking history of at least 20 pack-years, recruited from six clinical sites and additional subsites in the USA between Nov 12, 2010, and April 21, 2015. Inclusion criteria for this analysis were SPIROMICS baseline visit data with complete blood cell counts and, in a subset, acceptable sputum counts. We stratified patients on the basis of blood and sputum eosinophil concentrations and compared their demographic characteristics, as well as results from questionnaires, clinical assessments, and quantitative CT (QCT). We also analysed whether blood eosinophil concentrations reliably predicted sputum eosinophil concentrations. This study is registered with ClinicalTrials.gov (NCT01969344). Findings Of the 2737 patients recruited to SPIROMICS, 2499 patients were smokers and had available blood counts, and so were stratified by mean blood eosinophil count: 1262 patients with low ( 1 percentage predicted than the low sputum eosinophil group both before (65·7% [IQR 51·8–81·3] vs 75·7% [59·3–90·2], p vs 82·9% [67·8–95·9], p=0·001) bronchodilation. QCT density measures for emphysema and air trapping were significantly higher in the high sputum eosinophil group than the low sputum eosinophil group. Exacerbations requiring corticosteroids treatment were more common in the high versus low sputum eosinophil group (p=0·002). FEV 1 percentage predicted was significantly different between low and high blood eosinophil groups, but differences were less than those observed between the sputum groups. The high blood eosinophil group had slightly increased airway wall thickness (0·02 mm difference, p=0·032), higher St George Respiratory Questionnaire symptom scores (p=0·037), and increased wheezing (p=0·018), but no evidence of an association with COPD exacerbations (p=0·35) or the other indices of COPD severity, such as emphysema measured by CT density, COPD assessment test scores, Body-mass index, airflow Obstruction, Dyspnea, and Exercise index, or Global Initiative for Chronic Obstructive Lung Disease stage. Blood eosinophil counts showed a weak but significant association with sputum eosinophil counts (receiver operating characteristic area under the curve of 0·64, p Interpretation In a large, well characterised cohort of former and current smoking patients with a broad range of COPD severity, high concentrations of sputum eosinophils were a better biomarker than high concentrations of blood eosinophils to identify a patient subgroup with more severe disease, more frequent exacerbations, and increased emphysema by QCT. Blood eosinophils alone were not a reliable biomarker for COPD severity or exacerbations, or for sputum eosinophils. Clinical trials targeting eosinophilic inflammation in COPD should consider assessing sputum eosinophils. Funding National Institutes of Health, and National Heart, Lung, and Blood Institute.

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitic and emphysematous components. In one biophysical model, the concentration of mucin on the airway surfaces is hypothesized to be a key variable that controls mucus transport in healthy persons versus cessation of transport in persons with muco-obstructive lung diseases. Under this model, it is postulated that a high mucin concentration produces the sputum and disease progression that are characteristic of chronic bronchitis.MethodsWe characterized the COPD status of 917 participants from the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) using questionnaires administered to participants, chest tomography, spirometry, and examination of induced sputum. Total mucin concentrations in sputum were measured with the use of size-exclusion chromatography and refractometry. In 148 of these participants, the respiratory secreted mucins MUC5AC and MUC5B were quantitated by means of mass spectrometry. Data from chronic-bronchitis questionnaires and data on total mucin concentrations in sputum were also analyzed in an independent 94-participant cohort.ResultsMean (±SE) total mucin concentrations were higher in current or former smokers with severe COPD than in controls who had never smoked (3166±402 vs. 1515±152 μg per milliliter) and were higher in participants with two or more respiratory exacerbations per year than in those with zero exacerbations (4194±878 vs. 2458±113 μg per milliliter). The absolute concentrations of MUC5B and MUC5AC in current or former smokers with severe COPD were approximately 3 times as high and 10 times as high, respectively, as in controls who had never smoked. Receiver-operating-characteristic curve analysis of the association between total mucin concentration and a diagnosis of chronic bronchitis yielded areas under the curve of 0.72 (95% confidence interval [CI], 0.65 to 0.79) for the SPIROMICS cohort and 0.82 (95% CI, 0.73 to 0.92) for the independent cohort.ConclusionsAirway mucin concentrations may quantitate a key component of the chronic bronchitis pathophysiologic cascade that produces sputum and mediates disease severity. Studies designed to explore total mucin concentrations in sputum as a diagnostic biomarker and therapeutic target for chronic bronchitis appear to be warranted. (Funded by the National Heart, Lung, and Blood Institute and others.).

Mucus hyper-secretion is a hallmark feature of asthma and other muco-obstructive airway diseases. The mucin proteins MUC5AC and MUC5B are the major glycoprotein components of mucus and have critical roles in airway defense. Despite the biomedical importance of these two proteins, the loci that regulate them in the context of natural genetic variation have not been studied. To identify genes that underlie variation in airway mucin levels, we performed genetic analyses in founder strains and incipient lines of the Collaborative Cross (CC) in a house dust mite mouse model of asthma. CC founder strains exhibited significant differences in MUC5AC and MUC5B, providing evidence of heritability. Analysis of gene and protein expression of Muc5ac and Muc5b in incipient CC lines (n = 154) suggested that post-transcriptional events were important regulators of mucin protein content in the airways. Quantitative trait locus (QTL) mapping identified distinct, trans protein QTL for MUC5AC (chromosome 13) and MUC5B (chromosome 2). These two QTL explained 18 and 20% of phenotypic variance, respectively. Examination of the MUC5B QTL allele effects and subsequent phylogenetic analysis allowed us to narrow the MUC5B QTL and identify Bpifb1 as a candidate gene. Bpifb1 mRNA and protein expression were upregulated in parallel to MUC5B after allergen challenge, and Bpifb1 knockout mice exhibited higher MUC5B expression. Thus, BPIFB1 is a novel regulator of MUC5B.

Objectives Ultrasound imaging is commonly used to teach basic anatomy to medical students. The purpose of this study was to determine whether learning musculoskeletal anatomy with ultrasound improved performance on medical students' musculoskeletal physical examination skills. Methods Twenty-seven first-year medical students were randomly assigned to 1 of 2 instructional groups: either shoulder or knee. Both groups received a lecture followed by hands-on ultrasound scanning on live human models of the assigned joint. After instruction, students were assessed on their ability to accurately palpate 4 anatomic landmarks: the acromioclavicular joint, the proximal long-head biceps tendon, and the medial and lateral joint lines of the knee. Performance scores were based on both accuracy and time. A total physical examination performance score was derived for each joint. Scores for instructional groups were compared by a 2-way analysis of variance with 1 repeated measure. Significant findings were further analyzed with post hoc tests. Results All students performed significantly better on the knee examination, irrespective of instructional group (F = 14.9; df = 1.25; P = .001). Moreover, the shoulder instruction group performed significantly better than the knee group on the overall assessment (t = –3.0; df = 25; P

Efficient delivery of aerosols to the lungs via the nasal route has been difficult to achieve, but it may offer benefits over the traditional oral route for a range of patient populations. Because slow, continuous delivery of short-acting agents could improve safety, tolerability, compliance, and efficacy when compared with the rapid, intermittent aerosol treatments delivered by mouthpiece or mask, a novel trans-nasal pulmonary aerosol delivery (tPAD) device was developed. The tPAD incorporates an aerosol particle-size selection chamber and a custom nasal cannula that are specifically optimized for aerosol delivery to the lung via the nasal route. The tPAD device produced a steady aerosol output (∼2 mL/h) from an optimized nasal cannula with negligible rainout in the cannula for up to 8 hours. The generated aerosol particles were small enough to minimize nasal deposition [volume median diameter (VMD) = 1.4 μm].In this proof-of-concept study, gamma scintigraphy was used to quantitate deposition efficiency ofThe tPAD device achieved high pulmonary deposition (39% ± 8%), based on emitted dose, and matched that of the oral jet nebulizer (36% ± 9%). Low fractions of aerosol deposition in the head and nose region were observed for tPAD (6% ± 6%) and jet nebulizer deliver (1% ± 1%) as well.A profile of high pulmonary deposition efficiency and low nasal dose may enable the sustained use of the tPAD platform with a variety of therapeutic agents for a range of pulmonary disorders.

Explaining the variation in diversification rate across groups of plants has long been an important goal of botanists. In plants, complex scenarios involving a combination of extrinsic opportunities and intrinsic traits have been used to explain rapid diversification in certain groups. However, we feel that a very simple trait has been neglected from theories of plant diversification, namely plant height. Here, we argue that decreasing plant size should generally lead to an increase in speciation rate and a decrease in extinction rate. Theory suggests that all population genetic processes involved in speciation are influenced by plant size and its correlates, including seed dispersal distance, population size, generation time and the spatial scale at which plants perceive environmental heterogeneity. In addition, several of these variables, notably population size, also influence rates of extinction. We support our arguments with an empirical analysis showing that plant height is indeed negatively correlated with net diversification rate across families of angiosperms. Finally, we outline how the finer aspects of our hypothesis could be tested, at both micro- and macroevolutionary scales. In addition to strengthening our understanding of the effect of plant size on evolutionary processes, such a research agenda should contribute novel insights to speciation theory in general.

Stephen Jay Gould's views on the ontology of species were an important plank of his revisionist program in evolutionary theory. In this paper I cast a critical eye over those views. I focus on three central aspects of Gould's views on species: the relation between the Darwinian and the metaphysical notions of individuality, the relation between the ontology of species and macroevolution, and the issue of contextualism and conventionalism about the metaphysics of species.

Airway diseases, including cigarette smoke-induced chronic bronchitis, cystic fibrosis, and primary ciliary dyskinesia are associated with decreased mucociliary clearance (MCC). However, it is not known whether a simple reduction in MCC or concentration-dependent mucus adhesion to airway surfaces dominates disease pathogenesis or whether decreasing the concentration of secreted mucins may be therapeutic. To address these questions, Scnn1b-Tg mice, which exhibit airway mucus dehydration/adhesion, were compared to and crossed with Muc5b- and Muc5ac-deficient mice. Absence of Muc5b caused a 90% reduction in MCC, whereas Scnn1b-Tg mice exhibited an ~50% reduction. However, the degree of MCC reduction did not correlate with bronchitic airways pathology, which was observed only in Scnn1b-Tg mice. Ablation of Muc5b significantly reduced the extent of mucus plugging in Scnn1b-Tg mice. However, complete absence of Muc5b in Scnn1b-Tg mice was associated with increased airway inflammation, suggesting that Muc5b is required to maintain immune homeostasis. Loss of Muc5ac had few phenotypic consequences in Scnn1b-Tg mice. These data suggest that: (1) mucus hyperconcentration dominates over MCC reduction alone to produce bronchitic airways pathology; (2) Muc5b is the dominant contributor to the Scnn1b-Tg phenotype; and (3) therapies that limit mucin secretion may reduce plugging, but complete Muc5b removal from airway surfaces may be detrimental.

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

Introduction: The Sharp-Purser Test (SPT) is used to assess for atlantoaxial instability (AI) in patients with rheumatoid arthritis (RA). The test is commonly used by clinicians; however, many experts argue it lacks reliability and validity along with concerns of safety. The primary purpose of this review is to determine the diagnostic accuracy of the SPT to detect AI. Methods: A search of five databases was performed from inception to 19 December 2018 using search terms related to the SPT. Studies were eligible for inclusion if the SPT was used on a patient/participant. Methodological quality assessment of diagnostic studies was performed with the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) for studies that reported data to calculate sensitivity (SN), specificity (SP), positive likelihood ratio (+LR), and negative likelihood ratio (-LR). Results: The search yielded 1009 articles, and 32 studies met the inclusion criteria for analysis. Meta-analysis on diagnostic accuracy studies assessing the SPT was not possible due to statistical heterogeneity. Six diagnostic accuracy studies assessed the SN of the SPT ranging from 0.19 to 1.00. Four of the studies assessed SP of the SPT ranging from 0.71 to 0.98. The +LR was identified in 4 studies was 0.655, 1.73, 22, and 17.25. The -LR was 1.14, 0.799, 0.571, and 0.323. Seven RCTs utilized the SPT to screen for AI, and the SPT was used in 18 case reports. Conclusion: The SPT may be inappropriate to use due to inconsistent validity, poor inter-rater reliability, and potential to cause harm. Level of evidence: 1

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

While biodiversity hotspots are typically identified on the basis of species number per unit area, their exceptional richness is often attributed, either implicitly or explicitly, to high diversification rates. High species concentrations, however, need not reflect rapid diversification, with the diversity of some hotspots accumulating at modest rates over long timespans. Here we explore the relationship between diversification in time vs. diversification in space and develop the concept of diversification density to describe the spatial scale of species accumulation in a clade. We investigate how plant height is associated with both aspects of diversification in Alooideae, a large plant subfamily with its center of diversity in the Greater Cape Floristic Region. We first reconstruct a time-calibrated phylogeny for Alooideae and demonstrate an evolutionary tendency towards reduced plant height. While plant height does not correlate with diversification rate across Alooideae it does so with diversification per unit space: clades of small plants tend to have the highest diversification densities. Furthermore, we find that diversification in time vs. space are uncorrelated. Our results show that diversification rate and density can be decoupled, and suggest that while some biodiversity hotspots might have been generated by high diversification rates, others are the product of high diversification density.

Muco-Obstructive Lung Diseases This review covers the normal mechanisms of mucus formation and how they are abnormal in common conditions — such as COPD, cystic fibrosis, and non–cystic fibrosis br...