Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Greenhouse gas mitigation potential in smallholder agroecosystem of southern Ethiopia.

In developing countries, it is critical that novel and swift strategies are devised to help direct and prioritize potential greenhouse gas (GHG) mitigation activities. The Carbon Benefit Project (CBP) analysis tool is a modular, web-based system that allows a consistent comparison of various projects by providing a standardized GHG benefits protocol. In this study, we used the CBP tool to estimate the GHG mitigation potential of the agriculture, forestry, and other land uses (AFOLU) sector and prioritize components for their GHG benefits in three districts of Wolaita Zone, southern Ethiopia. The study area is 90,731 ha of which about 2% was covered by forest, 7% by grassland, 78% by annual crops, 12% by home garden and 1% by settlements. The livestock population in the study area was 512,622 heads. Using the CBP's Detailed Assessment, we estimated mitigation potential in the AFOLU consisting of different managements strategies for a period between 2016 and 2030 in the smallholder agricultural landscape. The results showed an overall GHG benefit of 1,725,052 (±5%) Mg CO2e from the projected scenario in the study area. The GHG benefit was in the order of biomass C (683,757 Mg CO2e) > soil C (619,210 Mg CO2e) > livestock (408,981 Mg CO2e) illustrating the greater mitigation potential of trees in different systems. The soil C plus biomass C was high in agroforestry systems, and this component had the highest priority for GHG mitigation. This was followed by high enteric methane emission reduction in the livestock category. The GHG emission from manure increased by 71,633 Mg CO2e in the project because manure was not managed. The surprisingly low GHG benefit of the forest was primarily because of its low land cover (i.e., about 2%) in the agroecosystem. Despite the low GHG benefit in the cropland from best management practices, the improved soil quality in it can affect GHG benefits from other land uses by contributing to their conservation through food security. Thus, a comprehensive project may be a viable strategy in a mitigation effort at the agroecosystem level because of the interactions amongst the components. The CBP analysis tool is useful in prioritizing mitigation activities and may be an option to quantify GHG benefits if studies collate Teir 2 factors in data scarce areas.

Components of tart cherry juice inhibit NFκB activation and inflammation in acute gout.

OBJECTIVES: To identify the anthocyanin content in tart cherry juice concentrate (TCJC) and establish the anti-inflammatory effect of in a murine acute gout model. METHODS: The main anthocyanins in the TCJC were identified by liquid chromatography mass spectroscopy (LCMS). TCJC or phosphate-buffered saline (PBS) as control were administered daily by oral gavage to BALB/C-Tg(NFκB-RE-luc)-Xen mice that harbour a firefly luciferase cDNA reporter under the regulation of 3 Nuclear factor-κB (NF-κB) response elements. After 14 days, gouty inflammation was induced by intra-articular injection of monosodium urate (MSU) crystals into the tibio-tarsal joint (ankle). NF-κB activity was measured locally in the injected ankle using the Xenogen in vivo imaging system (IVIS), and decalcified feet/ankles were paraffin-embedded and analysed histopathologically. RESULTS: The major anthocyanin compound present in TCJC was cyanidin 3-glucosylrutinoside followed by cyanidin 3-rutinoside. In the murine acute gout model, MSU injection increased NF-κB activity and oral administration of TCJC significantly reduced NF-κB activity in mouse foot, and ankle joints as assessed by IVIS analysis. Bioluminescent imaging detection of NF-κB activation was inhibited approximately 2-fold relative to control mice receiving PBS. Histopathologic examination showed suppression of infiltrates into the tibio-tarsal joint space of the mice receiving TCJC compared to PBS-treated control counterparts. CONCLUSIONS: The major anthocyanin in TCJC was cyanidin 3-glucosylrutinoside. Clinically relevant doses of TCJC significantly inhibit inflammation and NF-κB activation induced by MSU crystals.

Pathological manifestation of autoimmune myocarditis is detected prior to glomerulonephritis in a murine model of lupus nephritis.

OBJECTIVE: Since enhanced cardiac magnetic resonance imaging (cMRI) signals have been associated with lupus disease activity in humans prior to renal failure and novel, cardiac-focused therapeutic strategies could be investigated with an associated animal model, autoimmune myocarditis was characterized in murine lupus nephritis (NZM2410). METHODS: Weekly blood urea nitrogen (BUN) levels and weights were recorded. Cardiac function was assessed by echocardiogram. Myocardial edema was measured with quantitative T2 cMRI mapping. Endpoint serum and cardiac tissue were collected for histopathological analysis and cytokine measurements. RESULTS: Despite showing no signs of significant renal disease, mice displayed evidence of myocarditis and fibrosis histologically at 30-35 weeks. Moreover, T2 cMRI mapping displayed robust signals and analysis of sagittal heart sections showed significant myocardium thickening. Cytokine expression levels of IL-2, IL-10, TNF-α, CXCL1, and IL-6 were significantly enhanced in serum. Echocardiograms demonstrated significantly increased ventricular diameters and reduced ejection fractions, while immunohistochemical staining identified CD4+ and CD8+ T cells, and IL-17 in cardiac infiltrates. Human lupus cardiac tissue showed similar histopathology with enhanced infiltrates by H&E, fibrosis, and CD4+ detection. CONCLUSIONS: Histopathology, functional abnormalities, and enhanced cMRI signals indicative of myocarditis are detected in NZM2410 mice without glomerulonephritis, which supports the primary pathological role of autoimmune-mediated, cardiac-targeted inflammation in lupus.

Site-Dependent Spin Delocalization and Evidence of Ferrimagnetism in Atomically Precise Au25(SR)180 Clusters as Seen by Solution 13C NMR Spectroscopy.

We report a simple but detailed solution 13C nuclear magnetic resonance spectroscopic study of atomically precise neutral Au25(SR)180 (SR = alkyl thiolate) clusters. The paramagnetic 13C Knight shift of alkyl chain carbons, which is proportional to the local electron spin density, exhibits an electron spin delocalization that exponentially decays along the alkyl chain. The magnitude and decay constant of the observed electron spin delocalization, although largely independent of alkyl chain length, depend on where, that is, "in" versus "out" (vide infra) position, the alkyl chain is bound, in agreement with density functional theory calculations. Notably, the determined position-dependent decay constants, 1.70/Å and 0.41/Å for "in" and "out" ligands, respectively, not only could have important ramifications in molecular spintronics but are also comparable to measured decay constants in molecular electrical conductance of alkyl chains, potentially offering an alternative, simple method for estimating the latter. Moreover, the negative intercept temperatures of linear fits of reciprocal 13C (as well its bound 1H) Knight shift versus temperature strongly suggest the existence of local ferrimagnetism in individual Au25(SR)180 clusters.

An observational study of high- and low-abundance anti-retroviral resistance mutations among treatment-naïve people living with HIV in New Zealand between 2012 and 2017.

HIV resistance genotyping detects drug resistance mutations (DRMs) in ≥20% of circulating virus within an infected individual (high-abundance DRMs). Deep sequencing also detects DRMs in smaller viral subpopulations (low-abundance DRMs), although these are of uncertain importance. In this retrospective analysis of 292 treatment-naïve patients, high-abundance DRMs were present in 30/292 (10%) patients, but only one (0.3%) had resistance to first-line anti-retrovirals. Low-abundance DRMs were present in 36/247 (15%) patients, but none who received anti-retrovirals for which these were present had virologic failure. These findings demonstrate that starting first-line therapy in treatment-naïve patients need not be delayed while awaiting resistance testing.

A preliminary survey of anthropogenic gadolinium in water and sediment of a constructed wetland.

Gadolinium (Gd) is a rare earth element used in magnetic resonance imaging (MRI) contrast agents that has recently been identified as an emerging contaminant of concern due to its possible toxic effects and accumulation in the environment. The objectives of this preliminary study were to determine the occurrence and fate of Gd in surface water and sediment of a constructed wetland that receives effluent from a wastewater treatment plant. The rate of anthropogenic Gd entering the wetland was determined to be approximately 25 g Gd day-1, with surface water concentrations in the parts per trillion. Anthropogenic Gd concentrations in surface waters decreased as a function of distance from the inlet site to near the outfall, and were three orders of magnitude higher in sediment than in surface water suggesting that the wetland was providing a sink for Gd possibly through plant uptake and incorporation in organic biomass. An anthropogenic Gd anomaly was observed with an average GdAnt/GdGeo ratio of 5.34. Sediment with higher total organic carbon (TOC) tended to be higher in anthropogenic Gd, suggesting that Gd sequestration may occur through uptake by plants and/or through flocculation and deposition of natural organic matter.

The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.

Evaluating Potential Distribution of High-Risk Aquatic Invasive Species in the Water Garden and Aquarium Trade at a Global Scale Based on Current Established Populations.

Aquatic non-native invasive species are commonly traded in the worldwide water garden and aquarium markets, and some of these species pose major threats to the economy, the environment, and human health. Understanding the potential suitable habitat for these species at a global scale and at regional scales can inform risk assessments and predict future potential establishment. Typically, global habitat suitability models are fit for freshwater species with only climate variables, which provides little information about suitable terrestrial conditions for aquatic species. Remotely sensed data including topography and land cover data have the potential to improve our understanding of suitable habitat for aquatic species. In this study, we fit species distribution models using five different model algorithms for three non-native aquatic invasive species with bioclimatic, topographic, and remotely sensed covariates to evaluate potential suitable habitat beyond simple climate matches. The species examined included a frog (Xenopus laevis), toad (Bombina orientalis), and snail (Pomacea spp.). Using a unique modeling approach for each species including background point selection based on known established populations resulted in robust ensemble habitat suitability models. All models for all species had test area under the receiver operating characteristic curve values greater than 0.70 and percent correctly classified values greater than 0.65. Importantly, we employed multivariate environmental similarity surface maps to evaluate potential extrapolation beyond observed conditions when applying models globally. These global models provide necessary forecasts of where these aquatic invasive species have the potential for establishment outside their native range, a key component in risk analyses.

Meningitis in adults: diagnosis and management.

Bacterial meningitis is a medical emergency. All clinicians who provide acute medical care require a sound understanding of the priorities of managing a patient with suspected meningitis during the first hour. These include obtaining blood cultures, performing lumbar puncture and initiating appropriate therapy, while avoiding harmful delays such as those that result from not administering treatment until neuroimaging has been performed. Despite the increasing availability of newer diagnostic techniques, the interpretation of cerebrospinal fluid parameters remains a vital skill for clinicians. International and local guidelines differ with regard to initial empirical therapy of bacterial meningitis in adults; the North American guideline recommends ceftriaxone and vancomycin for all patients, while the Australian, UK and European guidelines recommend that vancomycin only be added for patients who are more likely to have pneumococcal meningitis or who have a higher likelihood of being infected with a strain of Streptococcus pneumoniae with reduced susceptibility to ceftriaxone. Patients with risk factors for Listeria meningitis also require an anti-Listeria agent, such as benzylpenicillin, to be added to this treatment regimen. Dexamethasone should be a routine component of empirical therapy due to its proven role in reducing morbidity and mortality from pneumococcal meningitis.

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

A survival guide to Landsat preprocessing.

Landsat data are increasingly used for ecological monitoring and research. These data often require preprocessing prior to analysis to account for sensor, solar, atmospheric, and topographic effects. However, ecologists using these data are faced with a literature containing inconsistent terminology, outdated methods, and a vast number of approaches with contradictory recommendations. These issues can, at best, make determining the correct preprocessing workflow a difficult and time-consuming task and, at worst, lead to erroneous results. We address these problems by providing a concise overview of the Landsat missions and sensors and by clarifying frequently conflated terms and methods. Preprocessing steps commonly applied to Landsat data are differentiated and explained, including georeferencing and co-registration, conversion to radiance, solar correction, atmospheric correction, topographic correction, and relative correction. We then synthesize this information by presenting workflows and a decision tree for determining the appropriate level of imagery preprocessing given an ecological research question, while emphasizing the need to tailor each workflow to the study site and question at hand. We recommend a parsimonious approach to Landsat preprocessing that avoids unnecessary steps and recommend approaches and data products that are well tested, easily available, and sufficiently documented. Our focus is specific to ecological applications of Landsat data, yet many of the concepts and recommendations discussed are also appropriate for other disciplines and remote sensing platforms.

A chimeric human-mouse model of Sjögren's syndrome.

Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.

Estrogen modulation of endosome-associated toll-like receptor 8: an IFNα-independent mechanism of sex-bias in systemic lupus erythematosus.

Females of child-bearing age are more resistant to infectious disease and have an increased risk of systemic lupus erythematosus (SLE). We hypothesized that estrogen-induced gene expression could establish an immunoactivated state which would render enhanced defense against infection, but may be deleterious in autoimmune development. Using peripheral blood mononuclear cells (PBMCs), we demonstrate enhanced responses with immunogen stimulation in the presence of 17ß-estradiol (E2) and gene array analyses reveal toll-like receptor 8 (TLR8) as an E2-responsive candidate gene. TLR8 expression levels are up-regulated in SLE and PBMCs stimulated with TLR8 agonist display a female sex-biased, E2-sensitive response. Moreover, we identify a putative ERα-binding region near the TLR8 locus and blocking ERα expression significantly decreases E2-mediated TLR8 induction. Our findings characterize TLR8 as a novel estrogen target gene that can lower the inflammatory threshold and implicate an IFNα-independent inflammatory mechanism that could contribute to higher SLE incidence in women.

Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu.

OBJECTIVE: Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell-deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. METHODS: FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)-null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1-null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. RESULTS: FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. CONCLUSION: These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity.

Lung cancer survival and stage at diagnosis in Australia, Canada, Denmark, Norway, Sweden and the UK: a population-based study, 2004-2007.

BACKGROUND: The authors consider whether differences in stage at diagnosis could explain the variation in lung cancer survival between six developed countries in 2004-2007. METHODS: Routinely collected population-based data were obtained on all adults (15-99 years) diagnosed with lung cancer in 2004-2007 and registered in regional and national cancer registries in Australia, Canada, Denmark, Norway, Sweden and the UK. Stage data for 57 352 patients were consolidated from various classification systems. Flexible parametric hazard models on the log cumulative scale were used to estimate net survival at 1 year and the excess hazard up to 18 months after diagnosis. RESULTS: Age-standardised 1-year net survival from non-small cell lung cancer ranged from 30% (UK) to 46% (Sweden). Patients in the UK and Denmark had lower survival than elsewhere, partly because of a more adverse stage distribution. However, there were also wide international differences in stage-specific survival. Net survival from TNM stage I non-small cell lung cancer was 16% lower in the UK than in Sweden, and for TNM stage IV disease survival was 10% lower. Similar patterns were found for small cell lung cancer. CONCLUSIONS: There are comparability issues when using population-based data but, even given these constraints, this study shows that, while differences in stage at diagnosis explain some of the international variation in overall lung cancer survival, wide disparities in stage-specific survival exist, suggesting that other factors are also important such as differences in treatment. Stage should be included in international cancer survival studies and the comparability of population-based data should be improved.

How researchers calculate students' grade point average in other courses has minimal impact.

Grade point average in "other" courses (GPAO) is an increasingly common measure used to control for prior academic performance and to predict future academic performance. In previous work, there are two distinct approaches to calculating GPAO, one based on only courses taken concurrently (term GPAO) and one based on all previous courses taken (cumulative GPAO). To our knowledge, no one has studied whether these methods for calculating the GPAO result in equivalent analyses and conclusions. As researchers often use one definition or the other without comment on why that choice was made, if the two calculations of GPAO are different, researchers might be inducing systematic error into their results and publishing potentially inaccurate conclusions. We looked at more than 3,700 courses at a public, research-intensive university over a decade and found limited evidence that the choice of GPAO calculation affects the conclusions. At most, one in seven courses could be affected. Further analysis suggests that there may be situations where one form of GPAO may be preferred over the other when it comes to examining inequity in courses or predicting student grades. However, we did not find sufficient evidence to universally recommend one form of GPAO over the other.

Retinal Blood Vessel Analysis Using Optical Coherence Tomography (OCT) in Multiple Sclerosis.

Background: Both greater retinal neurodegenerative pathology and greater cardiovascular burden are seen in people with multiple sclerosis (pwMS). Studies also describe multiple extracranial and intracranial vascular changes in pwMS. However, there have been few studies examining the neuroretinal vasculature in MS. Our aim is to determine differences in retinal vasculature between pwMS and healthy controls (HCs) and to determine the relationship between retinal nerve fiber layer (RNFL) thickness and retinal vasculature characteristics. Methods: A total of 167 pwMS and 48 HCs were scanned using optical coherence tomography (OCT). Earlier OCT scans were available for 101 pwMS and 35 HCs for an additional longitudinal analysis. Segmentation of retinal vasculature was performed in a blinded manner in MATLAB's optical coherence tomography segmentation and evaluation GUI (OCTSEG) software. Results: PwMS has fewer retinal blood vessels when compared to HCs (35.1 vs. 36.8, p = 0.017). Over the 5.4 year follow up, and when compared to HCs, pwMS has a significant decrease in number of retinal vessels (average loss of -3.7 p = 0.007). Moreover, the total vessel diameter in pwMS does not change when compared to the increase in vessel diameter in the HCs (0.06 vs. 0.3, p = 0.017). Only in pwMS is there an association between lower RNFL thickness and fewer retinal vessel number and smaller diameter (r = 0.191, p = 0.018 and r = 0.216, p = 0.007). Conclusions: Over 5 years, pwMS exhibit significant retinal vascular changes that are related to greater atrophy of the retinal layers.

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis.

Introduction: Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Methods: Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. Results: RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Discussion: Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.

Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets.

In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies.