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BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ∼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

Graphene liquid cell electron microscopy has the necessary temporal and spatial resolution to enable the in situ observation of nanoscale dynamics in solution. However, the chemistry of the solution in the liquid cell during imaging is as yet poorly understood due to the generation of a complex mixture of radiolysis products by the electron beam. In this work, the etching trajectories of nanocrystals were used as a probe to determine the effect of the electron beam dose rate and preloaded etchant, FeCl3, on the chemistry of the liquid cell. Initially, illuminating the sample at a low electron beam dose rate generates hydrogen bubbles, providing a reservoir of sacrificial reductant. Increasing the electron beam dose rate leads to a constant etching rate that varies linearly with the electron beam dose rate. Comparing these results with the oxidation potentials of the species in solution, the electron beam likely controls the total concentration of oxidative species in solution and FeCl3 likely controls the relative ratio of oxidative species, independently determining the etching rate and chemical potential of the reaction, respectively. Correlating these liquid cell etching results with the ex situ oxidative etching of gold nanocrystals using FeCl3 provides further insight into the liquid cell chemistry while corroborating the liquid cell dynamics with ex situ synthetic behavior. This understanding of the chemistry in the liquid cell will allow researchers to better control the liquid cell electron microscopy environment, allowing new nanoscale materials science experiments to be conducted systematically in a reproducible manner.

There is a strong link between integrins and interleukin-1β (IL-1β), but the specifics of the role of integrins in IL-1β signaling are unclear. We describe that IL-1β specifically bound to integrins αvβ3 and α5β1. The E128K mutation in the IL1R-binding site enhanced integrin binding. We studied whether direct integrin binding is involved in IL-1β signaling. We compared sequences of IL-1β and IL-1 receptor antagonist (IL1RN), which is an IL-1β homologue but has no agonistic activity. Several surface-exposed Lys residues are present in IL-1β, but not in IL1RN. A disulfide linkage is present in IL1RN, but is not in IL-1β because of natural C117F mutation. Substitution of the Lys residues to Glu markedly reduced integrin binding of E128K IL-1β, suggesting that the Lys residues mediate integrin binding. The Lys mutations reduced, but did not completely abrogate, agonistic action of IL-1β. We studied whether the disulfide linkage plays a role in agonistic action of IL-1β. Reintroduction of the disulfide linkage by the F117C mutation did not affect agonistic activity of WT IL-1β, but effectively reduced the remaining agonistic activity of the Lys mutants. Also, deletion of the disulfide linkage in IL1RN by the C116F mutation did not make it agonistic. We propose that the direct binding to IL-1β to integrins is primarily important for agonistic IL-1β signaling, and that the disulfide linkage indirectly affects signaling by blocking conformational changes induced by weak integrin binding to the Lys mutants. The integrin-IL-1β interaction is a potential target for drug discovery.

Objective. To characterize educational attainment and experiences of current US pharmacy school deans and chairs. Methods. A cross-sectional study using a publicly available listing of accredited schools and information. Results. Among 134 deans and 301 chairs, 79.9% and 65.5% held a professional degree (BSPharm and/or PharmD), 33.6% and 26.2% completed PGY-1 residencies, 12.7% and 15.6% completed post-PharmD fellowships, 23.1% and 33.9% completed post-doctoral fellowships, and 13.4% and 18.3% held BPS certification, respectively. Fewer than 1 in 5 were employed at an alma mater. Ninety (20.7%) deans and chairs completed AACP's Academic Leadership Fellows Program. Average current tenure was 5.7 and 5.1 years for deans and chairs, respectively. Conclusion. The majority of deans and chairs held a pharmacy professional degree and the prevalence of post-graduate educational and leadership training is increasing. Future research should apply mixed methods to investigate educational attainment and employment experience of deans and chairs, institutional hiring trends, and how these characteristics compare between newer and established programs.

Down syndrome (DS), trisomy 21, is caused by increased dose of genes present on human chromosome 21 (HSA21). The gene-dose hypothesis argues that a change in the dose of individual genes or regulatory sequences on HSA21 is necessary for creating DS-related phenotypes, including cognitive impairment. We focused on a possible role for Kcnj6, the gene encoding Kir3.2 (Girk2) subunits of a G-protein-coupled inwardly-rectifying potassium channel. This gene resides on a segment of mouse Chromosome 16 that is present in one extra copy in the genome of the Ts65Dn mouse, a well-studied genetic model of DS. Kir3.2 subunit-containing potassium channels serve as effectors for a number of postsynaptic metabotropic receptors including GABAB receptors. Several studies raise the possibility that increased Kcnj6 dose contributes to synaptic and cognitive abnormalities in DS. To assess directly a role for Kcnj6 gene dose in cognitive deficits in DS, we produced Ts65Dn mice that harbor only 2 copies of Kcnj6 (Ts65Dn:Kcnj6++- mice). The reduction in Kcnj6 gene dose restored to normal the hippocampal level of Kir3.2. Long-term memory, examined in the novel object recognition test with the retention period of 24h, was improved to the level observed in the normosomic littermate control mice (2N:Kcnj6++). Significantly, both short-term and long-term potentiation (STP and LTP) was improved to control levels in the dentate gyrus (DG) of the Ts65Dn:Kcnj6++- mouse. In view of the ability of fluoxetine to suppress Kir3.2 channels, we asked if fluoxetine-treated DG slices of Ts65Dn:Kcnj6+++ mice would rescue synaptic plasticity. Fluoxetine increased STP and LTP to control levels. These results are evidence that increased Kcnj6 gene dose is necessary for synaptic and cognitive dysfunction in the Ts65Dn mouse model of DS. Strategies aimed at pharmacologically reducing channel function should be explored for enhancing cognition in DS.

We have reported that integrins crosstalk with growth factors through direct binding to growth factors (e.g., fibroblast growth factor-1, insulin-like growth factor 1 (IGF1), neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor [e.g., integrin/IGF1/IGF1 receptor (IGF1R)]. IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανβ3 and α6β4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανβ3 and α6β4, and induced proliferation of CHO cells (IGF1R+) that express ανβ3 or α6β4 (β3- or α6β4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling.

Background: There is little consensus regarding the indications and utility of urinary tract imaging and type of imaging to perform in patients presenting with acute pyelonephritis (APN). Aims: The aims of this systematic review were to, among patients with APN, (i) identify the proportion of patients investigated with ultrasound (US), (ii) identify the proportion of abnormal US and (iii) identify the proportion of patients with a change in management resulting from abnormal US. Methods: A comprehensive search covered two electronic databases (Medline and EMBASE), with selection of studies performed independently by two investigators. Inclusion criteria were English language APN diagnosis and quantification of patients assessed with US or abnormal US results. Quality appraisal used the Newcastle–Ottawa instrument. Results: There were 35 studies included. The proportion of patients assessed with US was reported in 16 manuscripts and ranged from 20% to 94%, with significant heterogeneity and publication bias. The proportion of abnormal US was reported in 31 manuscripts and ranged from 7% to 79%. The proportion of abnormal US leading to change in management was reported in five studies and ranged from 7% to 59%. There was marked heterogeneity among studies included in all three subgroups. Conclusions: Patients with APN are commonly investigated with US, but only a small proportion have abnormalities and appear to be associated with changes in clinical management. The use of routine US for APN is therefore questioned. The identification of clinical variables for appropriate selection of patients to investigate with US requires further research. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Gastric sleeve stenosis (GSS) is an increasingly common adverse event following sleeve gastrectomy for which objective diagnostic criteria are lacking. Impedance planimetry measurements show promise in characterizing GSS, though normal and abnormal benchmark values have never been established. METHODS: This was a retrospective analysis of upper endoscopies performed with impedance planimetry for suspected GSS. A bariatric endoscopist, blind to impedance planimetry measurements, assessed gastric sleeve anatomy and graded GSS severity. Impedance planimetry of diameter and distensibility index (DI) were obtained using 3 different balloon volumes (30, 40, and 50 mL). RESULTS: A total of 110 upper endoscopies were included. Distribution of GSS was graded as none, mild, moderate, and severe in 19 (17%), 27 (25%), 34 (31%), and 30 (27%) procedures, respectively. In normal gastric sleeve anatomy, mean (±SD) diameter and DI measurements using consecutive balloon volumes ranged from 19.1 (±5.5) to 23.2 (±1.7) and 16.8 (±4.9) to 23.1 (±10.9), respectively. In severe GSS, mean diameter and DI measurements ranged from 10.3 (±3.0) to 16.6 (±2.1) and 7.5 (±2.4) to 7.7 (±2.4), respectively. When stratified by severity, impedance planimetry measurements of diameter and DI were significantly lower with each subsequent increase in GSS grade regardless of balloon fill volumes (P ≤ 0.001). DISCUSSION: Impedance planimetry measurements provide objective assessment in the diagnosis of GSS and correlate with luminal narrowing. A diameter ≥20 mm and a DI ≥15 mm 2/mm Hg, as measured by impedance planimetry, are predictive of normal gastric sleeve anatomy. This study provides new benchmark values for the diagnosis and severity of GSS. [ABSTRACT FROM AUTHOR]

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.