Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Mechanosignalling via integrins directs fate decisions of pancreatic progenitors.

The pancreas originates from two epithelial evaginations of the foregut, which consist of multipotent epithelial progenitors that organize into a complex tubular epithelial network. The trunk domain of each epithelial branch consists of bipotent pancreatic progenitors (bi-PPs) that give rise to both duct and endocrine lineages, whereas the tips give rise to acinar cells1. Here we identify the extrinsic and intrinsic signalling mechanisms that coordinate the fate-determining transcriptional events underlying these lineage decisions1,2. Single-cell analysis of pancreatic bipotent pancreatic progenitors derived from human embryonic stem cells reveal that cell confinement is a prerequisite for endocrine specification, whereas spreading drives the progenitors towards a ductal fate. Mechanistic studies identify the interaction of extracellular matrix (ECM) with integrin α5 as the extracellular cue that cell-autonomously, via the F-actin-YAP1-Notch mechanosignalling axis, controls the fate of bipotent pancreatic progenitors. Whereas ECM-integrin α5 signalling promotes differentiation towards the duct lineage, endocrinogenesis is stimulated when this signalling cascade is disrupted. This cascade can be disrupted pharmacologically or genetically to convert bipotent pancreatic progenitors derived from human embryonic stem cells to hormone-producing islet cells. Our findings identify the cell-extrinsic and intrinsic mechanotransduction pathway that acts as gatekeeper in the fate decisions of bipotent pancreatic progenitors in the developing pancreas.

Liquid Thyroxine Improves Outcomes in Hypothyroid Patients With Small Intestinal Bacterial Overgrowth and Irritable Bowel Syndrome.

OBJECTIVE: Malabsorption of levothyroxine (LT4) is often seen in patients with hypothyroidism and gastrointestinal (GI) conditions. Our study was designed to establish the prevalence of small intestinal bacterial overgrowth (SIBO) in patients with hypothyroidism and irritable bowel syndrome (IBS), and to demonstrate that liquid LT4 is more consistently absorbed vs tablet, leading to improvement in thyroid and GI symptoms. METHODS: This was a single-center, open label, prospective cohort study of liquid LT4 in 75 adult patients with hypothyroidism and IBS. Patients were transitioned from LT4 tablets to solution at equivalent dosing. Patients returned at 6 and 12 weeks for repeat thyroid levels and completion of validated questionnaires. A standard 2-hour SIBO breath test was administered at Week 6. Patients recorded daily stool appearance and frequency. RESULTS: Prevalence of SIBO was 65.3%. Liquid LT4 normalized thyroid stimulating hormone (TSH) in a higher percentage of patients vs tablet (77.55% vs 57.14%); significantly decreased TSH in subjects with SIBO; improved hypothyroid symptoms, IBS symptoms, stool appearance in all groups, and significantly altered bowel frequency among those with SIBO. CONCLUSION: Small intestinal bacterial overgrowth (SIBO) is common in patients with hypothyroidism and IBS. Among SIBO patients, LT4 tablets were inefficiently absorbed, leading to suboptimal thyroid control; however, transitioning from LT4 tablets to solution normalized TSH and improved hypothyroid symptoms. Liquid LT4 also significantly improved GI symptoms in all patients with hypothyroidism and IBS, regardless of SIBO status. Additionally, 1 in 5 patients had complete resolution of IBS symptoms after switching from LT4 tablets to solution, independent of changes in TSH.

Effects of electronic cigarette e-liquid flavouring on cigarette craving.

BACKGROUND: E-liquid flavour restrictions may discourage electronic cigarette (e-cigarette) uptake among youth. However, possible unintended consequences may include reduced appeal and effectiveness of e-cigarettes for smoking cessation. Non-tobacco flavours appear to be important for smoking cessation, but how and why are currently unclear. METHODS: We conducted an experimental study in a UK sample of adult daily smokers using an independent groups design (N=84). Participants were randomised to use an e-cigarette with nicotine-containing fruit/sweet-flavoured e-liquid (blackcurrant, strawberry, vanilla, caramel) or unflavoured e-liquid for 1 week. The primary outcomes were average, peak and cue-elicited cigarette craving (the latter was assessed using a cue exposure task). The secondary outcomes were smoking lapse occurrence, enjoyment of the e-cigarette, ease of transitioning from smoking to using an e-cigarette, intentions to continue using an e-cigarette, intentions and motivation to quit smoking, return to smoking, and continuation of e-cigarette use. RESULTS: E-liquid flavouring did not appear to have an effect on average cigarette craving (b 0.18, 95% CI -0.44 to 0.79, p=0.57), peak cigarette craving (b -0.12, 95% CI -0.59 to 0.35, p=0.62) or cue-elicited cigarette craving (b -0.21, 95% CI -3.86 to 3.43, p=0.91). We did not find evidence of a difference in secondary outcomes. CONCLUSIONS: We did not find evidence to suggest that nicotine-containing fruit/sweet-flavoured and unflavoured e-liquids have different effects on cigarette cravings after 1 week of use. Further research is needed to establish if differences emerge over longer periods of exposure and extend to smoking cessation outcomes.

Aggression Results in the Phosphorylation of ERK1/2 in the Nucleus Accumbens and the Dephosphorylation of mTOR in the Medial Prefrontal Cortex in Female Syrian Hamsters.

Like many social behaviors, aggression can be rewarding, leading to behavioral plasticity. One outcome of reward-induced aggression is the long-term increase in the speed in which future aggression-based encounters is initiated. This form of aggression impacts dendritic structure and excitatory synaptic neurotransmission in the nucleus accumbens, a brain region well known to regulate motivated behaviors. Yet, little is known about the intracellular signaling mechanisms that drive these structural/functional changes and long-term changes in aggressive behavior. This study set out to further elucidate the intracellular signaling mechanisms regulating the plasticity in neurophysiology and behavior that underlie the rewarding consequences of aggressive interactions. Female Syrian hamsters experienced zero, two or five aggressive interactions and the phosphorylation of proteins in reward-associated regions was analyzed. We report that aggressive interactions result in a transient increase in the phosphorylation of extracellular-signal related kinase 1/2 (ERK1/2) in the nucleus accumbens. We also report that aggressive interactions result in a transient decrease in the phosphorylation of mammalian target of rapamycin (mTOR) in the medial prefrontal cortex, a major input structure to the nucleus accumbens. Thus, this study identifies ERK1/2 and mTOR as potential signaling pathways for regulating the long-term rewarding consequences of aggressive interactions. Furthermore, the recruitment profile of the ERK1/2 and the mTOR pathways are distinct in different brain regions.

Examining the bidirectional association between emotion recognition and social autistic traits using observational and genetic analyses.

BACKGROUND: There is mixed evidence for an association between autism spectrum disorder (ASD) and emotion recognition deficits. We sought to assess the bidirectionality of this association using phenotypic and genetic data in a large community sample. METHODS: Analyses were conducted in three stages. First, we examined the bidirectional association between social autistic traits at age 8 years and emotion recognition task (ERT) responses at age 24 years (Study 1; N = 3,562); and between Diagnostic Analysis of Non-Verbal Accuracy (DANVA) emotion recognition responses at age 8 years and social autistic traits at age 10 years (Study 2; N = 9,071). Next, we used genetic analyses (Study 3) to examine the association between polygenic risk scores for ASD and outcomes for the ERT and DANVA. The genetic correlation between ASD and ERT responses at age 24 was also estimated. Analyses were conducted in the Avon Longitudinal Study of Parents and Children. RESULTS: Social autistic traits at age 8 years were negatively associated with later total correct responses on ERT in Study 1 (b = -0.18; 95% CI: -0.27 to -0.09). We also found evidence of an association in Study 2 (b = -0.04; 95% CI: -0.05 to -0.03). We found the opposite association, that is positive, between the ASD polygenic risk score and ERT (b = 0.40; 95% CI: 0.10 to 0.70); however, this association varied across different p-value thresholds and would not survive multiple testing, so should be interpreted with caution. We did not find evidence of a genetic correlation between ASD and ERT. CONCLUSION: We found an observational association between poorer emotion recognition and increased social autistic traits. Our genetic analyses may suggest a shared genetic aetiology between these or a potential causal pathway; however, future research would benefit from using better powered GWAS to examine this further. Our results may inform interventions targeting emotion recognition.

Wood ash and water: Cause of superficial alkaline burns in a toddler.

Though seemingly innocuous, the combination of wood ash and water can result in an alkaline solution with a pH up to 12, making it a burn risk. A 2-year-old boy developed a significant irritant reaction to his arms and legs while playing outdoors in wood ashes with a water gun. This case is novel because an unintentional, trivial exposure resulted in ulcerative irritant dermatitis. It is necessary to raise awareness of the corrosive risk from wood ash and water in the medical community, especially in primary and emergency care settings, to improve recognition of ash-water burns and irritant contact dermatitis and to ensure appropriate treatment.

Question order bias in retrospective evaluations of item and associative recognition.

Previous research has demonstrated a retrospective memory bias in metacognitive judgments regarding performance on general knowledge questions: Test-takers rate their own performance more optimistically when tests begin with easy questions than when tests begin with hard questions. An anchoring heuristic has been proposed to explain this finding, in which experience with the early questions constrains global performance evaluations of the test. In the current study we report on two experiments using tasks of item recognition and associative recognition to investigate the generality of question order bias. As predicted by an anchoring explanation, participants' estimates of performance were higher for item recognition tests beginning with easy items. However, the effect was reversed in the associative recognition task: Participants' estimates of performance were higher for tests beginning with hard items. Specific recollections, if present, may have a greater impact on test performance perception than more general global impressions.

Localised inhibition of FGF signalling in the third pharyngeal pouch is required for normal thymus and parathyroid organogenesis.

The thymus and parathyroid glands are derived from the third pharyngeal pouch endoderm. The mechanisms that establish distinct molecular domains in the third pouch and control the subsequent separation of these organ primordia from the pharynx are poorly understood. Here, we report that mouse embryos that lack two FGF feedback antagonists, Spry1 and Spry2, display parathyroid and thymus hypoplasia and a failure of these organ primordia to completely separate from the pharynx. We show that FGF ligands and downstream reporter genes are expressed in highly regionalised patterns in the third pouch and that sprouty gene deletion results in upregulated FGF signalling throughout the pouch endoderm. As a consequence, the initiation of markers of parathyroid and thymus fate is altered. In addition, a normal apoptotic programme that is associated with the separation of the primordia from the pharynx is disrupted, resulting in the maintenance of a thymus-pharynx attachment and a subsequent inability of the thymus to migrate to its appropriate position above the heart. We demonstrate that the sprouty genes function in the pharyngeal endoderm itself to control these processes and that the defects in sprouty-deficient mutants are, at least in part, due to hyper-responsiveness to Fgf8. Finally, we provide evidence to suggest that parathyroid hypoplasia in these mutants is due to early gene expression defects in the third pouch, whereas thymus hypoplasia is caused by reduced proliferation of thymic epithelial cells in the thymus primordium.

Preparation of Dental Resins Resistant to Enzymatic and Hydrolytic Degradation in Oral Environments.

The short average service life of traditional dental composite restorative materials and increasing occurrence of secondary caries adjacent to composite restorations and sealants are necessitating the development of new, longer lasting compositions. Novel monomers and their polymers, reinforcing fillers, and adhesive components are needed. The goal of this research is to develop resin systems for use in restorations, sealants, and other dental services that are superior in properties and endurance to currently used bisphenol A glycidyl dimethacrylate/triethylene glycol dimethacrylate (Bis-GMA/TEGDMA) and urethane-dimethacrylate products. Ether-based monomers and their polymers that were not susceptible to enzymatic or hydrolytic degradation were prepared and characterized. They showed no degradation under hydrolytic and enzymatic challenges, whereas the hydrolysis of ester links weakened contemporary resins within 16 days under these challenges. The success of the ether-based materials is promising in making durable systems that are subjected to long-term biochemical and hydrolytic challenges in oral environments.

Endoderm-specific deletion of Tbx1 reveals an FGF-independent role for Tbx1 in pharyngeal apparatus morphogenesis.

BACKGROUND: The T-box transcription factor Tbx1, is essential for the normal development of multiple organ systems in the embryo. One of the most striking phenotypes in Tbx1-/- embryos is the failure of the caudal pharyngeal pouches to evaginate from the foregut endoderm. Despite considerable interest in the role of Tbx1 in development, the mechanisms whereby Tbx1 controls caudal pouch formation have remained elusive. In particular, the question as to how Tbx1 expression in the pharyngeal endoderm regulates pharyngeal pouch morphogenesis in the mouse embryo is not known. RESULTS: To address this question, we produced mouse embryos in which Tbx1 was specifically deleted from the pharyngeal endoderm and, as expected, embryos failed to form caudal pharyngeal pouches. To determine the molecular mechanism, we examined expression of Fgf3 and Fgf8 ligands and downstream effectors. Although Fgf8 expression is greatly reduced in Tbx1-deficient endoderm, FGF signaling levels are unaffected. Furthermore, pouch morphogenesis is only partially perturbed by the loss of both Fgf3 and Fgf8 from the endoderm, indicating that neither are required for pouch formation. CONCLUSIONS: Tbx1 deletion from the pharyngeal endoderm is sufficient to cause caudal pharyngeal arch segmentation defects by FGF-independent effectors that remain to be identified.

Quadriceps strength and endurance in fibrotic idiopathic interstitial pneumonia.

BACKGROUND AND OBJECTIVE: Quadriceps muscle dysfunction is an important contributor to exercise limitation in chronic obstructive pulmonary disease, but little is known about skeletal muscle function and its impact on exercise capacity in patients with fibrotic idiopathic interstitial pneumonia (IIP). The aim of the study was to compare quadriceps strength and endurance in patients with fibrotic IIP and healthy controls, and relate it to exercise capacity. METHODS: Quadriceps strength and endurance, as well as respiratory muscle strength, and 6-min walk distance were compared among 25 patients with fibrotic IIP, forced vital capacity mean (standard deviation) 78.7 (14.0) %predicted, carbon monoxide transfer factor 40.3 (10.9) %predicted and 33 age-matched healthy controls using non-volitional measures. Quadriceps strength was assessed using magnetic femoral nerve stimulation (quadriceps twitch force), and endurance using the decay in force in response to repetitive magnetic stimulation of the quadriceps over 5 min. RESULTS: Both groups had comparable anthropometrics, gender proportion and respiratory muscle strength. Patients were significantly weaker than controls; quadriceps twitch force 10.1 (3.0) kg versus 8.0 (2.4) kg (P = 0.013). Quadriceps force in response to repetitive magnetic stimulation declined significantly more rapidly in patients during the endurance protocol (P < 0.001). In controls, there was a significant relationship between 6-min walk distance and quadriceps twitch force (r 0.40, P = 0.038), and quadriceps endurance (r 0.59, P = 0.016). In patients, only PaO2 and inspiratory muscle strength were retained as independent correlates of 6-min walk distance (r(2) = 0.3 P = 0.022). CONCLUSIONS: Quadriceps strength and endurance are reduced in patients with fibrotic IIP compared with healthy controls, but are less tightly correlated with exercise performance.

Impression formation of tests: retrospective judgments of performance are higher when easier questions come first.

Four experiments are reported on the importance of retrospective judgments of performance (postdictions) on tests. Participants answered general knowledge questions and estimated how many questions they answered correctly. They gave higher postdictions when easy questions preceded difficult questions. This was true when time to answer each question was equalized and constrained, when participants were instructed not to write answers, and when questions were presented in a multiple-choice format. Results are consistent with the notion that first impressions predominate in overall perception of test difficulty.

QuickFit: A High-Throughput RT-qPCR-Based Assay to Quantify Viral Growth and Fitness In Vitro.

Quantifying viral growth rates is key to understanding evolutionary dynamics and the potential for mutants to escape antiviral drugs. Defining evolutionary escape paths and their impact on viral fitness allows for the development of drugs that are resistant to escape. In the case of HIV, combination antiretroviral therapy can successfully prevent or treat infection, but it relies on strict adherence to prevent escape. Here, we present a method termed QuickFit that enables the quantification of viral fitness by employing large numbers of parallel viral cultures to measure growth rates accurately. QuickFit consistently recapitulated HIV growth measurements obtained by traditional approaches, but with significantly higher throughput and lower rates of error. This method represents a promising tool for rapid and consistent evaluation of viral fitness.

Age related trends in the utilization of neoadjuvant chemotherapy for muscle invasive bladder cancer.

PURPOSE: The current standard of care for muscle invasive bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Previous research has shown under-utilization of NAC for treatment of MIBC, especially among the elderly. Our aim was to stratify NAC use by decade of life and analyze trends in use over time along with recording pathologic downstaging and perioperative outcomes. MATERIALS AND METHODS: The National Cancer Database was queried for patients with cT2-4NanyM0 MIBC treated with RC from 2010 to 2016 with urothelial carcinoma. Nineteen thousand nine hundred fifty seven patients met criteria for analysis. We retrospectively analyzed trends in use of NAC, readmission rate, mortality rate, and pathologic downstaging with NAC all stratified by decade of life. RESULTS: Of the 19,957 patients treated with RC for MIBC, only 30.9% underwent NAC. There was a statistically significant increase in NAC use across all age groups from 2010 to 2016. Receipt of NAC was associated with decreased age on univariate analysis (P < 0.001) and on logistic regression (OR: 0.617 for age 70-79, OR: 0.221 for age ≥80 vs. age <60; P < 0.001). Patients receiving NAC were more likely to exhibit pathologic downstaging at time of RC (OR: 3.907; P < 0.001), and this trend held for each age group examined. Among patients receiving NAC, the risk of 30 and 90-day mortality was associated with increasing age; however, age was not associated with 30-day readmission for those receiving NAC. CONCLUSION: Rates of NAC use prior to RC have increased among all age groups with the lowest utilization rate among the elderly. NAC use was associated with greater pathologic downstaging in all age groups. These data show a promising trend in the uptake of the gold standard for treatment of MIBC; however, the underlying etiology of differing rates of NAC utilization remains to be determined.

Quality and Readability of Online Health Information on Common Urologic Cancers: Assessing Barriers to Health Literacy in Urologic Oncology.

INTRODUCTION: A growing number of Americans search online for health information related to urologic oncologic care each year. The American Medical Association recommends that medical information be written at a maximum sixth-grade level in order to be comprehensible by the majority of patients. As such, it is important to assess the quality and readability of online patient education material that patients are being exposed to. METHODS: A Google search was performed using the terms "testicular cancer," "prostate cancer," "kidney cancer," and "bladder cancer," and the top 30 results for each were reviewed. Websites were categorized based on their source. Readability was assessed using the Flesch-Kincaid Grade Level, the Gunning Frequency of Gobbledygook, and the Simple Measure of Gobbledygook indices. Quality was assessed using the DISCERN Quality Index (1-5 scale). RESULTS: A total of 91 websites were included in our analysis. On average, online health information pertaining to urologic cancers is written at a 10th- to 11th-grade reading level, which is significantly higher than that of an average American adult and that recommended by the American Medical Association (P < .01). The overall quality of websites was 3.4 ± 0.7, representing moderate to high quality. There was no significant difference in readability based on cancer type or information source. CONCLUSIONS: Despite being of moderate to high quality, online patient education materials related to common urologic cancers are often written at a grade level that exceeds the reading level of an average American adult. This presents as a barrier to online health literacy and calls into question the utility of these resources.

Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

An engineered prodrug selectively suppresses ß-lactam resistant bacteria in a mixed microbial setting.

The rise of ß-lactam resistance necessitates new strategies to combat bacterial infections. We purposefully engineered the ß-lactam prodrug AcephPT to exploit ß-lactamase activity to selectively suppress resistant bacteria producing extended-spectrum-ß-lactamases (ESBLs). Selective targeting of resistant bacteria requires avoiding interaction with penicillin-binding proteins, the conventional targets of ß-lactam antibiotics, while maintaining recognition by ESBLs to activate AcephPT only in resistant cells. Computational approaches provide a rationale for structural modifications to the prodrug to achieve this biased activity. We show AcephPT selectively suppresses gram-negative ESBL-producing bacteria in clonal populations and in mixed microbial cultures, with effective selectivity for both lab strains and clinical isolates expressing ESBLs. Time-course NMR experiments confirm hydrolytic activation of AcephPT exclusively by ESBL-producing bacteria. In mixed microbial cultures, AcephPT suppresses proliferation of ESBL-producing strains while sustaining growth of ß-lactamase-non-producing bacteria, highlighting its potential to combat ß-lactam resistance while promoting antimicrobial stewardship.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.