Congenital anemia reveals distinct targeting mechanisms for master transcription factor GATA1.

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Development of an Artificial Intelligence-Based Image Recognition System for Time-Sequence Analysis of Tracheal Intubation.

BACKGROUND: Total intubation time (TIT) is an objective indicator of tracheal intubation (TI) difficulties. However, large variations in TIT because of diverse initial and end targets make it difficult to compare studies. A video laryngoscope (VLS) can capture images during the TI process. By using artificial intelligence (AI) to detect airway structures, the start and end points can be freely selected, thus eliminating the inconsistencies. Further deconstructing the process and establishing time-sequence analysis may aid in gaining further understanding of the TI process. METHODS: We developed a time-sequencing system for analyzing TI performed using a #3 Macintosh VLS. This system was established and validated on 30 easy TIs performed by specialists and validated using TI videos performed by a postgraduate-year (PGY) physician. Thirty easy intubation videos were selected from a cohort approved by our institutional review board (B-ER-107-088), and 6 targets were labeled: the lip, epiglottis, laryngopharynx, glottic opening, tube tip, and a black line on the endotracheal tube. We used 887 captured images to develop an AI model trained using You Only Look Once, Version 3 (YOLOv3). Seven cut points were selected for phase division. Seven experts selected the cut points. The expert cut points were used to validate the AI-identified cut points and time-sequence data. After the removal of the tube tip and laryngopharynx images, the durations between 5 identical cut points and sequentially identified the durations of 4 intubation phases, as well as TIT. RESULTS: The average and total losses approached 0 within 150 cycles of model training for target identification. The identification rate for all cut points was 92.4% (194 of 210), which increased to 99.4% (179 of 180) after the removal of the tube tip target. The 4 phase durations and TIT calculated by the AI model and those from the expert exhibited strong Pearson correlation (phase I, r = 0.914; phase II, r = 0.868; phase III, r = 0.964; and phase IV, r = 0.949; TIT, r = 0.99; all P < .001). Similar findings were obtained for the PGY's observations (r > 0.95; P < .01). CONCLUSIONS: YOLOv3 is a powerful tool for analyzing images recorded by VLS. By using AI to detect the airway structures, the start and end points can be freely selected, resolving the heterogeneity resulting from the inconsistencies in the TIT cut points across studies. Time-sequence analysis involving the deconstruction of VLS-recorded TI images into several phases should be conducted in further TI research.

Transcription factor expression defines subclasses of developing projection neurons highly similar to single-cell RNA-seq subtypes.

We are only just beginning to catalog the vast diversity of cell types in the cerebral cortex. Such categorization is a first step toward understanding how diversification relates to function. All cortical projection neurons arise from a uniform pool of progenitor cells that lines the ventricles of the forebrain. It is still unclear how these progenitor cells generate the more than 50 unique types of mature cortical projection neurons defined by their distinct gene-expression profiles. Moreover, exactly how and when neurons diversify their function during development is unknown. Here we relate gene expression and chromatin accessibility of two subclasses of projection neurons with divergent morphological and functional features as they develop in the mouse brain between embryonic day 13 and postnatal day 5 in order to identify transcriptional networks that diversify neuron cell fate. We compare these gene-expression profiles with published profiles of single cells isolated from similar populations and establish that layer-defined cell classes encompass cell subtypes and developmental trajectories identified using single-cell sequencing. Given the depth of our sequencing, we identify groups of transcription factors with particularly dense subclass-specific regulation and subclass-enriched transcription factor binding motifs. We also describe transcription factor-adjacent long noncoding RNAs that define each subclass and validate the function of Myt1l in balancing the ratio of the two subclasses in vitro. Our multidimensional approach supports an evolving model of progressive restriction of cell fate competence through inherited transcriptional identities.

Optic Perineuritis Distinguishing Arteritic Ischaemic from Amiodarone-Associated Optic Neuropathy.

Giant cell arteritis (GCA) is a common medium to large vessel vasculitis of the elderly that can lead to permanent vision loss. Neuroimaging in GCA may reveal optic nerve sheath enhancement, which is a cardinal finding of optic perineuritis (OPN). The clinical manifestations of GCA can mimic that of other ocular disorders including amiodarone-associated optic neuropathy (AAON). We report a case of biopsy-proven GCA in a patient initially suspected to have AAON. This patient presented with bilateral optic disc oedema in conjunction with subacute predominately monocular vision loss and enhancement of the corresponding optic nerve sheath on neuroimaging. Clinicians should recognise that clinical and neuroimaging findings of GCA can mimic a variety of ocular and orbital pathologies including idiopathic OPN and AAON.

Biocompatible and Na+-sensitive thin-film transistor for biological fluid sensing.

In this study, we develop a Na+-sensitive thin-film transistor (TFT) for a biocompatible ion sensor and investigate its cytotoxicity. A transparent amorphous oxide semiconductor composed of amorphous In-Ga-Zn-oxide (a-InGaZnO) is utilized as a channel of the Na+-sensitive TFT, which includes an indium tin oxide (ITO) film as the source and drain electrodes and a Ta2O5 thin-film gate, onto which a Na+-sensitive membrane is coated. As one of the Na+-sensitive membranes, the polyvinyl chloride (PVC) membrane with bis(12-crown-4) as the ionophore used on the TFT sensors shows good sensitivity and selectivity to changes in Na+ concentration but has high cytotoxicity owing to the leaching of its plasticizer to the solution; the plasticizer is added to solve and entrap the ionophore in the PVC membrane. On the other hand, a plasticizer-free Na+-sensitive membrane, the fluoropolysilicone (FPS) membrane with the bis(12-crown-4) ionophore, also reduces cell viability owing to the leaching of the ionophore. However, the FPS membrane with calix[4]arene as the ionophore on the gate of TFT sensors exhibits not only favorable electrical properties but also the lack of cytotoxicity. Thus, considering structural flexibility of TFTs, a platform based on TFT sensors coated with the Na+-sensitive FPS membrane containing calix[4]arene is suitable as a biocompatible Na+ sensing system for the continuous monitoring of ionic components in biological fluids such as sweat and tears.

Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion.

Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance.IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.

Sensing Responses Based on Transfer Characteristics of InAs Nanowire Field-Effect Transistors.

Nanowire-based field-effect transistors (FETs) have demonstrated considerable promise for a new generation of chemical and biological sensors. Indium arsenide (InAs), by virtue of its high electron mobility and intrinsic surface accumulation layer of electrons, holds properties beneficial for creating high performance sensors that can be used in applications such as point-of-care testing for patients diagnosed with chronic diseases. Here, we propose devices based on a parallel configuration of InAs nanowires and investigate sensor responses from measurements of conductance over time and FET characteristics. The devices were tested in controlled concentrations of vapour containing acetic acid, 2-butanone and methanol. After adsorption of analyte molecules, trends in the transient current and transfer curves are correlated with the nature of the surface interaction. Specifically, we observed proportionality between acetic acid concentration and relative conductance change, off current and surface charge density extracted from subthreshold behaviour. We suggest the origin of the sensing response to acetic acid as a two-part, reversible acid-base and redox reaction between acetic acid, InAs and its native oxide that forms slow, donor-like states at the nanowire surface. We further describe a simple model that is able to distinguish the occurrence of physical versus chemical adsorption by comparing the values of the extracted surface charge density. These studies demonstrate that InAs nanowires can produce a multitude of sensor responses for the purpose of developing next generation, multi-dimensional sensor applications.

Direct Electrochemical Signaling in Organic Electrochemical Transistors Comprising High-Conductivity Double-Network Hydrogels.

In composite hydrogels, the high electrical performance of poly(3,4-ethylenedioxythiophene) complexed with poly(styrenesulfonate) (PEDOT:PSS) is integrated with complementary structural and electrochemical functions via a rationally designed poly(acrylamide) second network incorporating phenylboronic acid (PBA). Free-standing double-network hydrogels prepared by a simple one-pot radical polymerization exhibit state-of-the-art electrical conductivity (∼20 S cm-1 in phosphate buffered saline) while retaining a degree of hydration similar to that of biological soft tissues. Low resistance contacts to Au electrodes are formed via facile thermo-mechanical annealing and demonstrate stability over a month of continuous immersion, thus enabling hydrogels to serve as channels of organic electrochemical transistors (OECTs). Despite thicknesses of ∼100 µm, gating of hydrogel OECTs is efficient with transconductances gm ∼ 40 mS and on/off ratios of 103 in saturation mode operation, whereas sufficiently high conductivity enables linear mode operation (gm ∼ 1 mS at -10 mV drain bias). This drives a shift of sensing strategy toward detection of electrochemical signals originating within the bulky channel. A kinetic basis for glucose detection via diol esterification on PBA is identified as the coupling of PBA equilibrium to electrocatalyzed O2 reduction occurring on PEDOT in cathodic potentials. Hydrogel OECTs inherently amplify this direct electrochemical signal, demonstrating the viability of a new class of soft, structural biosensors.

Nonlinear Chemical Sensitivity Enhancement of Nanowires in the Ultralow Concentration Regime.

Much recent attention has been focused on the development of field-effect transistors based on low-dimensional nanostructures for the detection and manipulation of molecules. Because of their extraordinarily high charge sensitivity, InAs nanowires present an excellent material system in which to probe and study the behavior of molecules on their surfaces and elucidate the underlying mechanisms dictating the sensor response. So far, chemical sensors have relied on slow, activated processes restricting their applicability to high temperatures and macroscopic adsorbate coverages. Here, we identify the transition into a highly sensitive regime of chemical sensing at ultralow concentrations (<1 ppm) via physisorption at room temperature using field-effect transistors with channels composed of several thousand InAs nanowires and ethanol as a simple analyte molecule. In this regime, the nanowire conductivity is dictated by a local gating effect from individual dipoles, leading to a nonlinear enhancement of the sensitivity. At higher concentrations (>1 ppm), the nanowire channel is globally gated by a uniform dipole layer at the nanowire surface. The former leads to a dramatic increase in sensitivity due to weakened screening and the one-dimensional geometry of the nanowire. In this regime, we detect concentrations of ethanol vapor as low as 10 ppb, 100 times below the lowest concentrations previously reported. Furthermore, we demonstrate electrostatic control of the sensitivity and dynamic range of the InAs nanowire-based sensor and construct a unified model that accurately describes and predicts the sensor response over the tested concentration range (10 ppb to 10 ppm).

Structural and functional characterization of G protein-coupled receptors with deep mutational scanning.

The >800 human G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.