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Ca2+/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that is linked to learning and memory. In this study, we focused on understanding how interactions between CaMKIIα and the actin crosslinking protein α-actinin-2 underlie long-lasting changes in dendritic spine architecture. We found that association of the two proteins was unexpectedly elevated following stimulation of NMDA receptors to trigger structural LTP in primary hippocampal neurons. Furthermore, disruption of interactions between the two proteins prevented the accumulation of enlarged mushroom-type dendritic spines following NMDA receptor activation. α-actinin-2 binds to the regulatory segment of CaMKII. Calorimetry experiments, and a crystal structure of α-actinin-2 EF hands 3 and 4 in complex with the CaMKII regulatory segment, indicate that the regulatory segment of autoinhibited CaMKII is not fully accessible to α-actinin-2. Pull-down experiments show that occupation of the CaMKII substrate binding groove by GluN2B markedly increases α-actinin-2 access to the CaMKII regulatory segment. Overall, our study provides new mechanistic insight into the molecular basis of structural LTP and reveals an added layer of sophistication to the function of CaMKII.

Opioid use has much increased in several countries during the last two decades, accompanied by a rise in associated morbidity and mortality, especially in the United States. Data on a possible opioid crisis are scarcer in Europe. We performed a study aiming to assess the frequency of adverse drug reactions (ADR) related to opioids in patients presenting to the emergency unit (EU) of a geriatric tertiary Swiss University Hospital. This particular setting is intended for patients aged 75 and older. Our retrospective, monocentric survey of opioid use and related ADR was conducted over two months in 2018. The main and secondary outcomes were the frequency of EU visits considered due to an opioid ADR and insufficient pain relief, respectively. Current opioid use was identified in 20.3% (n=99) of the 487 included EU visits (mean age 86). An ADR was the suspected cause of the EU visit in 22 opioid users, mainly fall-related injury and gastrointestinal disorders. All these patients had at least one comorbid condition. In 19/22 cases (86%) of ADR, a drug-drug interaction might have been involved. In 12 opioid users (12%), insufficient pain relief was suspected as the cause of the EU visit. In conclusion, one-third of opioid users visiting a geriatric EU consulted for a problem related to its use mainly adverse drug-related reaction (22%) followed by insufficient pain relief (12%).

AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP(+). In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35–55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes towards LTD. This non- canonical mode of PKA regulation may underlie many other cellular processes.

Background Improving clinical reasoning education has been identified as an important strategy to reduce diagnostic error—an important cause of adverse patient outcomes. Clinical reasoning is fundamental to each specialty, yet the extent to which explicit instruction in clinical reasoning occurs across specialties in the clerkship years remains unclear. Method The Alliance for Clinical Education (ACE) Clinical Reasoning Workgroup and the Directors of Clinical Skills Courses (DOCS) Clinical Reasoning Workgroup collaborated to develop a clinical reasoning needs assessment survey. The survey questionnaire covered seven common clinical reasoning topics including illness scripts, semantic qualifiers, cognitive biases and dual process theory. Questionnaires were delivered electronically through ACE member organizations, which are primarily composed of clerkship leaders across multiple specialties. Data was collected between March of 2019 and May of 2020. Results Questionnaires were completed by 305 respondents across the six organizations. For each of the seven clinical reasoning topics, the majority of clerkship leaders (range 77.4% to 96.8%) rated them as either moderately important or extremely important to cover during the clerkship curriculum. Despite this perceived importance, these topics were not consistently covered in respondents’ clerkships (range 29.4% to 76.4%) and sometimes not covered anywhere in the clinical curriculum (range 5.1% to 22.9%). Conclusions Clerkship educators across a range of clinical specialties view clinical reasoning instruction as important, however little curricular time is allocated to formally teach the various strategies. Faculty development and restructuring of curricular time may help address this potential gap.

AMPA-type glutamate receptors, which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization, causing a progressive current decline in the continued presence of agonist. Here authors show that homomeric Q/R edited AMPARs still allow ions to flow when the receptors are desensitized.

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

Cultured hippocampal slices from rodents, in which the architecture and functional properties of the hippocampal network are largely preserved, have proved to be a powerful substrate for studying healthy and pathological neuronal mechanisms. Here, we delineate the membrane-interface method for maintaining organotypic slices in culture for several weeks. The protocol includes procedures for dissecting hippocampus from rat brain, and collecting slices using a vibratome. This method provides the experimenter with easy access to both the brain tissue and culture medium, which facilitates genetic and pharmacological manipulations and enables experiments that incorporate imaging and electrophysiology. The method is generally applicable to rats of different ages, and to different brain regions, and can be modified for culture of slices from other species including mice.

Cultured hippocampal slices from rodents, in which the architecture and functional properties of the hippocampal network are largely preserved, have proved to be a powerful substrate for studying healthy and pathological neuronal mechanisms. Here, we delineate the membrane-interface method for maintaining organotypic slices in culture for several weeks. The protocol includes procedures for dissecting hippocampus from rat brain, and collecting slices using a vibratome. This method provides the experimenter with easy access to both the brain tissue and culture medium, which facilitates genetic and pharmacological manipulations and enables experiments that incorporate imaging and electrophysiology. The method is generally applicable to rats of different ages, and to different brain regions, and can be modified for culture of slices from other species including mice.

This study analyzed 27 surface sediment samples from the Tamaulipas Continental shelf to determine the spatial-temporal distribution of V and Cd (spring-summer 2016; summer 2017). Average V concentrations (99 ± 18 mg g

We investigated the spatial and temporal distribution of V and Cd in surface sediments of the Yucatan Shelf to establish current average values for the area. V and Cd concentrations are similar to those reported internationally for limestone rocks and surface marine sediments. The observed variability of V concentrations between cruises may be the result of changes in ocean current direction in summer (SW-S) and strong prevailing winds in autumn (N-NE). In addition to the hydrodynamics described above, Cd variations may also be associated with inputs of particulate material by upwelling events and subsequent transport and distribution to the shelf by the Yucatan Channel current (autumn). Considering that both metals showed significant spatial and temporal differences, a range of values for V (0.4-1.5 µg g

Surface water samples from the Perdido study area presented Cd and V concentrations similar to those reported internationally for waters with: (1) fossil fuel extraction, processing and burning, and (2) sites polluted by anthropogenic wastewater. Results showed an order of magnitude increase in time for Cd, therefore, no general average value was established. For V, however, results of this study suggest a general average value of 1.4 µg L

Purpose Periocular inverted papilloma (IP) is a rare, locally aggressive tumor with a propensity for recurrence and malignant transformation. Historically treated via wide excision, this study examines the characteristics and management of periocular IP, comparing those confined to the nasolacrimal system with those invading the orbit. Methods An Institutional Review Board-approved, Health Insurance Portability and Accountability Act-compliant retrospective, comparative case series was conducted in patients with IP of the orbit or nasolacrimal system across 15 clinical sites. Results Of 25 patients, 22 met inclusion criteria with 9 limited to the nasolacrimal system and 13 invading the orbit. Mean age was 60.4 years, 55% were women, all were unilateral. Mean follow-up was 48 months. Rates of smoking, dust and/or aerosol exposure, human papillomavirus (HPV) status, and inflammatory polyps were elevated compared to rates in the general population (45%, 18%, 18%, and 14%, respectively). Bony erosion on computed tomography scans was statistically significantly associated with orbit-invading IP (p = 0.002). Treatment involved all confined IP undergoing surgery alone while 39% of orbit-invading IP also received radiation therapy and/or chemotherapy (p = 0.054). Orbit-invading IP was more likely to be excised with wide margins than IP confined to the nasolacrimal system (85% vs. 22%, p = 0.007). Overall rates of malignancy, recurrence, and patient mortality from IP were found to be 27%, 23%, and 9%, respectively. Conclusions IP invading the orbit typically requires aggressive therapy, while IP confined to the nasolacrimal system may be treated more conservatively. Using risk factors, characteristics, and outcomes, a treatment algorithm was created to guide management.

INTRODUCTION AND OBJECTIVE:Urologists' use of minimally invasive surgery (MIS) has increased rapidly in the last decade. In reaction this, the Urology RRC set required minimums of MIS cases for res...

BACKGROUND AND PURPOSE: To date, normal values for optic nerve diameter (OND) and optic nerve sheath diameter (ONSD) for transorbital sonography (TOS) have only been reported by individual small-scale studies, exposing a great variability in the measurement of the OND and ONSD. METHODS: We performed a systematic review and metanalysis of available to date studies on TOS evaluation of adults without elevated intracranial pressure to provide an overview of the published literature, measuring methods and further specify normal values for OND and ONSD. RESULTS: In total, we included 39 studies with 2,927 healthy volunteers (mean age 36.1 years, 44.4% female), so that a total of 5,854 eyes were examined. All pooled analyses were based on random effect models. Mean values for OND were provided in 13 studies. Calculated mean pooled OND value was 3.08 mm (95% confidence interval [CI], 2.9-3.25), with low heterogeneity across studies (I2 = 12.7%). Thirty-four studies provided mean values for ONSD measurement. The pool of mean ONSD measurements was 4.78 mm (95% CI, 4.63-4.94), with evidence of substantial heterogeneity between estimates ONSD (I2 = 50.6%). There were no significant differences (P =.139) in the subsequent subgroup analysis for the different geographic continents. Also, no significant differences could be recorded for the effect of age (P =.824) or gender (P =.093). CONCLUSIONS: TOS is a frequently described and widely used method. We provide reference values of OND and ONSD that are based on metanalytical analysis. Different measuring methods of ONSD result in higher heterogeneity. Subgroup analysis revealed no significant correlation between ONSD and age, gender, or geographic origin. © 2020 by the American Society of Neuroimaging

Purpose To evaluate the effectiveness of orbital radiotherapy (ORT) in the treatment of thyroid eye disease (TED)-compressive optic neuropathy. Methods A retrospective review of patients with corticosteroid-responsive compressive optic neuropathy due to TED treated with ORT. Study was conducted in compliance with Health Insurance Portability and Accountability Act. One hundred four patients (163 orbits) with a mean age of 61.7 years met inclusion criteria. Seventy-four percent (77/104) were female, and 32.7% (34/104) were current or previous smokers. A total absorbed dose of 2000 cGy fractionated in 10 treatment doses over the course of 2 weeks was administered to the retroocular tissues according to a standard protocol. The primary end point was failure of ORT, defined as persistent optic neuropathy following completion of radiotherapy that mandated urgent orbital decompression surgery. Results Ninety-eight of 104 (94%) patients or 152 of 163 (93.3%) orbits did not require orbital decompression surgery during the acute phase. Patients who responded successfully to ORT had similar improvements in visual acuity, color vision, Humphrey threshold visual field testing, and afferent pupillary defects compared with patients who failed ORT and underwent urgent decompression surgery. Only 36.7% of successfully treated patients ultimately underwent elective surgery, including orbital decompression, strabismus, or eyelid surgery, during the inactive phase of TED. Conclusions The data from this study, the largest retrospective review reported to date, supports the use of ORT in eyes with corticosteroid-responsive TED-compressive optic neuropathy. ORT may favorably alter the natural history of active-phase TED by preventing recurrent compressive optic neuropathy after withdrawal of corticosteroids.

γ-aminobutyric acid receptors (GABA(A)Rs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result following receptor dysfunction. A critical component of most native GABA(A)Rs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact on anxiety, stress and depression, and for therapeutic drugs such as general anaesthetics. To understand the basis for modulating GABA(A)R function, high-resolution structures are required. Here we present the first atomic structures of a GABA(A)R chimera at 2.8Å resolution, including those bound with potentiating and inhibitory neurosteroids. These define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable neurosteroids to be exploited for therapeutic drug design to regulate GABA(A)Rs in neurological disorders.

Every year since 2000, five to ten ecological restoration projects have been implemented on public and private lands in the Puget Sound region of Washington State by the University of Washington Restoration Ecology Network (UW-REN) capstone program. Students, faculty, and community partners have collaborated to implement these restoration projects, improve ecological function at the sites, and build site stewardship. Approximately twenty-nine, 2,000 m2 projects from the first ten years of the capstone projects were retrospectively evaluated using a variety of response variables that could reflect ecological “success”, including native species cover, richness, and diversity in the restoration plant community. We hypothesized that certain elements of restoration design and implementation, such as selected techniques for invasive vegetation management and the resulting degree of site stewardship, would strongly contribute to the success of these restoration projects. Stewardship was found to contribute to native species cover, richness, and diversity. Native plant species richness and diversity responded to initial invasive control techniques; stem-only removal did not work as well as root system removal for native species richness or diversity. The use of wood chip and cardboard-with-wood chip sheet mulches had less clear effects on species richness. An interaction between mulch and control technique was found to contribute to species diversity evenness. Overall, this study of restoration projects led by university students in cooperation with community partners highlights the importance of specific elements of restoration design and implementation and due diligence in the form of long-term stewardship.

cAMP-dependent protein kinase (PKA) plays a central role in important biological processes including synaptic plasticity and sympathetic stimulation of the heart. Elevations of cAMP trigger release of PKA catalytic (C) subunits from PKA holoenzymes, thereby coupling cAMP to protein phosphorylation. Uncontrolled C subunit activity, such as occurs in genetic disorders in which regulatory subunits are depleted, is pathological. Anchoring proteins that associate with PKA regulatory subunits are important for localising PKA activity in cells. However, anchoring does not directly explain how unrestrained ‘free swimming' of C subunits is avoided following C subunit release. In this review, I discuss new mechanisms that have been posited to account for this old problem. One straightforward explanation is that cAMP does not trigger C subunit dissociation but instead activates intact PKA holoenzymes whose activity is restrained through anchoring. A comprehensive comparison of observations for and against cAMP-activation of intact PKA holoenzymes does not lend credence to this mechanism. Recent measurements have revealed that PKA regulatory subunits are expressed at very high concentrations, and in large molar excess relative to C subunits. I discuss the implications of these skewed PKA subunit concentrations, before considering how phosphorylation of type II regulatory subunits and myristylation of C subunits are likely to contribute to controlling C subunit diffusion and recapture in cells. Finally, I speculate on future research directions that may be pursued on the basis of these emerging mechanisms.

Let R be a finite commutative ring with identity. The idempotent graph of R is the simple undirected graph I(R) with vertex set, the set of all nontrivial idempotents of R and two distinct vertices x and y are adjacent if and only if xy = 0. In this paper, we have determined all isomorphism classes of finite commutative rings with identity whose I(R) has genus one or two. Also we have determined all isomorphism classes of finite commutative rings with identity whose I(R) has crosscap one. Also we study the the book embedding of toroidal idempotent graphs and classify finite commutative rings whose I(R) is a ring graph.

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53., Oncoprotein E6 facilitates the E6AP-catalyzed ubiquitination of p53. Here, the authors study the structural basis of this process by qualitative and quantitative cross-linking mass spectrometry, providing insights into E6AP-E6-p53 complex assembly and the conformational dynamics that enable p53 ubiquitination.

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca2+ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca2+ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca2+ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behaviour. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca2+ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes.

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.

Brain trauma is known to result in heterogeneous patterns of tissue damage and altered neuronal and glial metabolism that evolve over time following injury; however, little is known on the longitudinal evolution of these changes. In this study, magnetic resonance spectroscopic imaging (MRSI) was used to map the distributions of altered metabolism in a single subject at five time points over a period of 28 months following injury.Magnetic resonance imaging and volumetric MRSI data were acquired in a subject that had experienced a moderate traumatic brain injury (Glasgow Coma Scale 13) at five time points, from 7 weeks to 28 months after injury. Maps of N-acetylaspartate (NAA), total choline (Cho), and total creatine signal were generated and differences from normal control values identified using a z-score image analysis method.The z-score metabolite maps revealed areas of significantly reduced NAA and increased Cho, predominately located in frontal and parietal white matter, which evolved over the complete course of the study. A map of the ratio of Cho/NAA showed the greatest sensitivity to change, which indicated additional metabolic changes throughout white matter. The metabolic changes reduced over time following injury, though with abnormal values remaining in periventricular regions.The use of z-score image analysis for MRSI provides a method for visualizing diffuse changes of tissue metabolism in the brain. This image visualization method is of particularly effective for visualizing widespread and diffuse metabolic changes, such as that due to traumatic injury.

AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression. Ca2+ directly regulates AKAP79 through its effector calmodulin (CaM), but the molecular basis of this regulation was previously unknown. Here, we report that CaM recognizes a ‘1-4-7-8’ pattern of hydrophobic amino acids starting at Trp79 in AKAP79. Cross-linking coupled to mass spectrometry assisted mapping of the interaction site. Removal of the CaM-binding sequence in AKAP79 prevents formation of a Ca2+-sensitive interface between AKAP79 and calcineurin, and increases resting cellular PKA phosphorylation. We determined a crystal structure of CaM bound to a peptide encompassing its binding site in AKAP79. CaM adopts a highly compact conformation in which its open Ca2+-activated C-lobe and closed N-lobe cooperate to recognize a mixed α/310 helix in AKAP79. The structure guided a bioinformatic screen to identify potential sites in other proteins that may employ similar motifs for interaction with CaM., Nature Communications, 8 (1), ISSN:2041-1723