Regulation of Chromatin Modifications through Coordination of Nucleus Size and Epithelial Cell Morphology Heterogeneity.

Cell morphology heterogeneity within epithelial collectives is a pervasive phenomenon intertwined with tissue mechanical properties. Despite its widespread occurrence, the underlying mechanisms driving cell morphology heterogeneity and its consequential biological ramifications remain elusive. Here, we investigate the dynamic evolution of epithelial cell morphology and nucleus morphology during crowding, unveiling a consistent correlation between the two. Our investigation reveals a persistent log-normal probability distribution characterizing both cell and nucleus areas across diverse crowding stages and epithelial model systems. We showed that this morphological diversity arises from asymmetric partitioning during cell division and is perpetuated through actomyosin-mediated regulation of cell-nucleus size coordination. Moreover, we provide insights into the impact of nucleus morphology on chromatin dynamics, demonstrating that constraining nucleus area leads to downregulation of the euchromatic mark H3K9ac and upregulation of the heterochromatic mark H3K27me3 through modulation of histone demethylase UTX expression. These findings under-score the significance of cell morphology heterogeneity as a driver of chromatin state diversity, shaping functional variability within epithelial tissues.

Memorability shapes perceived time (and vice versa).

Visual stimuli are known to vary in their perceived duration. Some visual stimuli are also known to linger for longer in memory. Yet, whether these two features of visual processing are linked is unknown. Despite early assumptions that time is an extracted or higher-order feature of perception, more recent work over the past two decades has demonstrated that timing may be instantiated within sensory modality circuits. A primary location for many of these studies is the visual system, where duration-sensitive responses have been demonstrated. Furthermore, visual stimulus features have been observed to shift perceived duration. These findings suggest that visual circuits mediate or construct perceived time. Here we present evidence across a series of experiments that perceived time is affected by the image properties of scene size, clutter and memorability. More specifically, we observe that scene size and memorability dilate time, whereas clutter contracts it. Furthermore, the durations of more memorable images are also perceived more precisely. Conversely, the longer the perceived duration of an image, the more memorable it is. To explain these findings, we applied a recurrent convolutional neural network model of the ventral visual system, in which images are progressively processed over time. We find that more memorable images are processed faster, and that this increase in processing speed predicts both the lengthening and the increased precision of perceived durations. These findings provide evidence for a link between image features, time perception and memory that can be further explored with models of visual processing.

Control of neurogenic competence in mammalian hypothalamic tanycytes.

Hypothalamic tanycytes, radial glial cells that share many features with neuronal progenitors, can generate small numbers of neurons in the postnatal hypothalamus, but the identity of these neurons and the molecular mechanisms that control tanycyte-derived neurogenesis are unknown. In this study, we show that tanycyte-specific disruption of the NFI family of transcription factors (Nfia/b/x) robustly stimulates tanycyte proliferation and tanycyte-derived neurogenesis. Single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analysis reveals that NFI (nuclear factor I) factors repress Sonic hedgehog (Shh) and Wnt signaling in tanycytes and modulation of these pathways blocks proliferation and tanycyte-derived neurogenesis in Nfia/b/x-deficient mice. Nfia/b/x-deficient tanycytes give rise to multiple mediobasal hypothalamic neuronal subtypes that can mature, fire action potentials, receive synaptic inputs, and selectively respond to changes in internal states. These findings identify molecular mechanisms that control tanycyte-derived neurogenesis, which can potentially be targeted to selectively remodel the hypothalamic neural circuitry that controls homeostatic physiological processes.

Early decrease in basophil sensitivity to Ara h 2 precedes sustained unresponsiveness after peanut oral immunotherapy.

BACKGROUND: Only some patients with peanut allergy undergoing oral immunotherapy (OIT) achieve sustained clinical response. Basophil activation could provide a functional surrogate of efficacy. OBJECTIVE: We hypothesized that changes in basophil sensitivity and area under the curve (AUC) to the immunodominant allergen Ara h 2 correlate with clinical responses to OIT. METHODS: Children with peanut allergy aged 7 to 13 years were enrolled in a single-center, open-label peanut OIT trial. Levels of specific immunoglobulins were measured throughout OIT. Peripheral blood from multiple time points was stimulated in vitro with peanut allergens for flow cytometric assessment of the percentage of CD63hi activated basophils. RESULTS: Twenty-two of 30 subjects were successfully treated with OIT; after avoidance, 9 achieved sustained unresponsiveness (SU), and 13 had transient desensitization (TD). Basophil sensitivity, measured by using the dose that induces 50% of the maximal basophil response, to Ara h 2 stimulation decreased from baseline in subjects with SU (after OIT, P = .0041; after avoidance, P = .0011). At 3 months of OIT, basophil sensitivity in subjects with SU decreased from baseline compared with that in subjects with TD (median, 18-fold vs 3-fold; P = .01), with a receiver operating characteristic of 0.84 and optimal fold change of 4.9. Basophil AUC to Ara h 2 was suppressed after OIT equally in subjects with SU and those with TD (P = .4). After avoidance, basophil AUC rebounded in subjects with TD but not those with SU (P < .001). Passively sensitized basophils suppressed with postavoidance SU plasma had a lower AUC than TD plasma (6.4% vs 38.9%, P = .03). CONCLUSIONS: Early decreases in basophil sensitivity to Ara h 2 correlate with SU. Basophil AUC rebounds after avoidance in subjects with TD. Therefore, different aspects of basophil activation might be useful for monitoring of OIT efficacy.

Data-driven programmatic approach to analysis of basophil activation tests.

BACKGROUND: Conventional data analysis of flow cytometry-based basophil activation testing requires repetitive, labor-intensive analysis that hampers efforts to standardize testing for clinical applications. Using an open-source platform, we developed and implemented a programmatic approach to the analysis of the basophil activation test (BAT) by flow cytometry. METHODS: Using the BÜHLMANN FlowCAST® assay, peripheral blood from peanut allergic patients undergoing oral immunotherapy was incubated with peanut allergens (Arah1, Arah2, Arah6, whole peanut extract) and stained with fluorescent antibodies to CCR3 and CD63 for the development of a data-driven programmatic analysis using Bioconductor and R. Basophil identification using clustering and classification was validated using manually gated comparisons in an experimental subset. Reproducibility of CD63 upregulation set on unstimulated or anti-FcERI stimulated basophils was compared. RESULTS: BAT analysis of 294 experiments was successful in 91.5% using the above approach, with a total of 7,166 individual basophil activation tests from 269 experiments. We estimate this represents a net saving of 1340 min of labor by a skilled operator. Medium-based gating correlated to respective manual gating more closely than anti-FcERI based gating (R = 0.96 vs. R = 0.84, P < 0.001). Only 2% of the basophil activation results were significantly different from manual gating. Quality measures of the experiments and other measures of basophil activation were also provided by the analysis. CONCLUSIONS: We present a novel data-driven flow cytometric platform for the analysis of clinical basophil activation testing, providing a high throughput objective approach to basophil activation analysis. © 2017 International Clinical Cytometry Society.

Peanut oral immunotherapy transiently expands circulating Ara h 2-specific B cells with a homologous repertoire in unrelated subjects.

BACKGROUND: Peanut oral immunotherapy (PNOIT) induces persistent tolerance to peanut in a subset of patients and induces specific antibodies that might play a role in clinical protection. However, the contribution of induced antibody clones to clinical tolerance in PNOIT is unknown. OBJECTIVE: We hypothesized that PNOIT induces a clonal, allergen-specific B-cell response that could serve as a surrogate for clinical outcomes. METHODS: We used a fluorescent Ara h 2 multimer for affinity selection of Ara h 2-specific B cells and subsequent single-cell immunoglobulin amplification. The diversity of related clones was evaluated by means of next-generation sequencing of immunoglobulin heavy chains from circulating memory B cells with 2x250 paired-end sequencing on the Illumina MiSeq platform. RESULTS: Expression of class-switched antibodies from Ara h 2-positive cells confirms enrichment for Ara h 2 specificity. PNOIT induces an early and transient expansion of circulating Ara h 2-specific memory B cells that peaks at week 7. Ara h 2-specific sequences from memory cells have rates of nonsilent mutations consistent with affinity maturation. The repertoire of Ara h 2-specific antibodies is oligoclonal. Next-generation sequencing-based repertoire analysis of circulating memory B cells reveals evidence for convergent selection of related sequences in 3 unrelated subjects, suggesting the presence of similar Ara h 2-specific B-cell clones. CONCLUSIONS: Using a novel affinity selection approach to identify antigen-specific B cells, we demonstrate that the early PNOIT-induced Ara h 2-specific B-cell receptor repertoire is oligoclonal and somatically hypermutated and shares similar clonal groups among unrelated subjects consistent with convergent selection.

Schottky barrier thin film transistors using solution-processed n-ZnO.

Solution-processed ZnO thin films are attractive as active materials in thin film transistors (TFTs) for low-cost electronic device applications. However, the lack of true enhancement mode operation, low mobility, and unreliability in transistor characteristics due to the high density of traps and other defects present challenges in using such TFTs in circuits. We demonstrate in this report that the electrical characteristics of such TFTs can be improved by source injection barriers. Asymmetrical Schottky source metal-oxide-semiconductor field-effect transistors (MOSFETs) have been fabricated by utilizing heavily doped solution-processed ZnO as the active layer. n(+)-ZnO was obtained by using triethylamine as the stabilizer in the solution process instead of the more commonly used monoethanolamine. Au was chosen for source metallization to create a Schottky contact to the ZnO and an Al ohmic contact was chosen as the drain. Voltage applied to the gate induced field emission through the Schottky barrier and allowed modulation of the drain current by varying the width of the barrier. By operating the asymmetrical MOSFET when the Schottky contact is reverse biased, effective control over the transistor characteristics was obtained.

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface.

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

Characterization of vascular injury responses to stent insertion in an ex-vivo arterial perfusion model.

PURPOSE: To develop an ex-vivo arterial perfusion model to evaluate vascular responses to bare metal stents (BMS) and drug-eluting stents (DES) in porcine carotid arteries. MATERIALS AND METHODS: Porcine carotid arteries with BMS or DES were cultured under hemodynamic stimuli for 24 hours and 72 hours. Vascular responses of arteries with stents were assessed by cellular functionality and gene expression and compared with a noninjured (NI) control group at each time point. Cellular functionality was confirmed with sequential dosing of norepinephrine (NE), acetylcholine (ACH), and sodium nitroprusside (SNP). QuantiGene (Panomics, Fremont, California) branched DNA (bDNA) assay was used to evaluate gene expression of endothelial cell (EC) and smooth muscle cell (SMC) biomarkers and compare it with responses of in-vivo arteries with stents. Bromodeoxyuridine (BrDU) stain was also used to detect cellular proliferation in the ex-vivo arteries with stents. RESULTS: EC relaxation and SMC contraction in response to vasoactivators indicated the arteries remained viable and functional for at least 72 hours in culture. SMC-dependent contractility and EC-dependent relaxation were lower in arteries with stents compared with NI arteries. Greater SMC proliferation was observed in BMS arteries compared with DES arteries. Cellular proliferation, EC function, and SMC marker expression at acute time points were similar between both models suggesting that the ex-vivo arterial model can provide comparative predictions of stent injury in vivo. CONCLUSIONS: The ex-vivo arterial perfusion model can be used as a quick and less costly (than current in-vivo and some in-vitro perfusion testing models) approach for evaluating the vascular responses to various stent design parameters (eg, strut thickness, strut width).