Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating.

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Hippocampal AMPA receptor assemblies and mechanism of allosteric inhibition.

AMPA-selective glutamate receptors mediate the transduction of signals between the neuronal circuits of the hippocampus1. The trafficking, localization, kinetics and pharmacology of AMPA receptors are tuned by an ensemble of auxiliary protein subunits, which are integral membrane proteins that associate with the receptor to yield bona fide receptor signalling complexes2. Thus far, extensive studies of recombinant AMPA receptor-auxiliary subunit complexes using engineered protein constructs have not been able to faithfully elucidate the molecular architecture of hippocampal AMPA receptor complexes. Here we obtain mouse hippocampal, calcium-impermeable AMPA receptor complexes using immunoaffinity purification and use single-molecule fluorescence and cryo-electron microscopy experiments to elucidate three major AMPA receptor-auxiliary subunit complexes. The GluA1-GluA2, GluA1-GluA2-GluA3 and GluA2-GluA3 receptors are the predominant assemblies, with the auxiliary subunits TARP-γ8 and CNIH2-SynDIG4 non-stochastically positioned at the B'/D' and A'/C' positions, respectively. We further demonstrate how the receptor-TARP-γ8 stoichiometry explains the mechanism of and submaximal inhibition by a clinically relevant, brain-region-specific allosteric inhibitor.

Plasma Proteomic Kinetics in Response to Acute Exercise.

Regular exercise has many favorable effects on human health, which may be mediated in part by the release of circulating bioactive factors during each bout of exercise. Limited data exist regarding the kinetic responses of plasma proteins during and after acute exercise. Proteomic profiling of 4163 proteins was performed using a large-scale, affinity-based platform in 75 middle-aged adults who were referred for treadmill exercise stress testing. Plasma proteins were quantified at baseline, peak exercise, and 1-h postexercise, and those with significant changes at both exercise timepoints were further examined for their associations with cardiometabolic traits and change with aerobic exercise training in the Health, Risk Factors, Exercise Training and Genetics Family Study, a 20-week exercise intervention study. A total of 765 proteins changed (false discovery rate < 0.05) at peak exercise compared to baseline, and 128 proteins changed (false discovery rate < 0.05) at 1-h postexercise. The 56 proteins that changed at both timepoints included midkine, brain-derived neurotrophic factor, metalloproteinase inhibitor 4, and coiled-coil domain-containing protein 126 and were enriched for secreted proteins. The majority had concordant direction of change at both timepoints. Across all proteins assayed, gene set enrichment analysis showed increased abundance of coagulation-related proteins at 1-h postexercise. Forty-five proteins were associated with at least one measure of adiposity, lipids, glucose homeostasis, or cardiorespiratory fitness in Health, Risk Factors, Exercise Training and Genetics Family Study, and 20 proteins changed with aerobic exercise training. We identified hundreds of novel proteins that change during acute exercise, most of which resolved by 1 h into recovery. Proteins with sustained changes during exercise and recovery may be of particular interest as circulating biomarkers and pathways for further investigation in cardiometabolic diseases. These data will contribute to a biochemical roadmap of acute exercise that will be publicly available for the entire scientific community.

Omics-driven investigation of the biology underlying intrinsic submaximal working capacity and its trainability.

Submaximal exercise capacity is an indicator of cardiorespiratory fitness with clinical and public health implications. Submaximal exercise capacity and its response to exercise programs are characterized by heritability levels of about 40%. Using physical working capacity (power output) at a heart rate of 150 beats/min (PWC150) as an indicator of submaximal exercise capacity in subjects of the HERITAGE Family Study, we have undertaken multi-omics and in silico explorations of the underlying biology of PWC150 and its response to 20 wk of endurance training. Our goal was to illuminate the biological processes and identify panels of genes associated with human variability in intrinsic PWC150 (iPWC150) and its trainability (dPWC150). Our bioinformatics approach was based on a combination of genome-wide association, skeletal muscle gene expression, and plasma proteomics and metabolomics experiments. Genes, proteins, and metabolites showing significant associations with iPWC150 or dPWC150 were further queried for the enrichment of biological pathways. We compared genotype-phenotype associations of emerging candidate genes with reported functional consequences of gene knockouts in mouse models. We investigated the associations between DNA variants and multiple muscle and cardiovascular phenotypes measured in HERITAGE subjects. Two panels of prioritized genes of biological relevance to iPWC150 (13 genes) and dPWC150 (6 genes) were identified, supporting the hypothesis that genes and pathways associated with iPWC150 are different from those underlying dPWC150. Finally, the functions of these genes and pathways suggested that human variation in submaximal exercise capacity is mainly driven by skeletal muscle morphology and metabolism and red blood cell oxygen-carrying capacity.NEW & NOTEWORTHY Multi-omics and in silico explorations of the genes and underlying biology of submaximal exercise capacity and its response to 20 wk of endurance training were undertaken. Prioritized genes were identified: 13 genes for variation in submaximal exercise capacity in the sedentary state and 5 genes for the response level to endurance training, with no overlap between them. Genes and pathways associated with submaximal exercise capacity in the sedentary state are different from those underlying trainability.

Comparative review of airway anesthesia and sedation methods for awake intubation.

PURPOSE OF REVIEW: Successful awake intubation hinges upon adequate airway anesthesia and sedation for patient comfort. This review will summarize relevant anatomy and regional anesthesia techniques to achieve airway anesthesia, and compare various airway anesthesia and sedation regimens. RECENT FINDINGS: Overall, nerve blocks consistently provided superior airway anesthesia, shorter time to intubation, higher patient comfort, and higher postintubation patient satisfaction. Additionally, ultrasound guidance can further provide benefit by reducing the amount of local anesthetic administered, leading to denser blockade, and proving invaluable in challenging clinical situations. Regarding sedation methods, numerous studies supported the use of dexmedetomidine, with or without the addition of supplemental sedation, such as midazolam, ketamine, or opioids. SUMMARY: Emerging evidence has indicated that nerve blocks for airway anesthesia may be superior to other methods of topicalization. Additionally, dexmedetomidine can be useful, both as monotherapy and with supplemental sedatives, to safely provide anxiolysis for the patient and increase success. However, it is crucial to note that the method of airway anesthesia and sedation regimen should be adapted to each patient and clinical situation, and knowledge of multiple techniques and sedation regimens can best equip anesthesiologists to do so.

Structural basis of kainate subtype glutamate receptor desensitization.

Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this 'desensitization ring' is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.

Risk Prediction in Cardiogenic Shock: Current State of Knowledge, Challenges and Opportunities.

Cardiogenic shock (CS) is a condition associated with high mortality rates in which prognostication is uncertain for a variety of reasons, including its myriad causes, its rapidly evolving clinical course and the plethora of established and emerging therapies for the condition. A number of validated risk scores are available for CS prognostication; however, many of these are tedious to use, are designed for application in a variety of populations and fail to incorporate contemporary hemodynamic parameters and contemporary mechanical circulatory support interventions that can affect outcomes. It is important to separate patients with CS who may recover with conservative pharmacological therapies from those in who may require advanced therapies to survive; it is equally important to identify quickly those who will succumb despite any therapy. An ideal risk-prediction model would balance incorporation of key hemodynamic parameters while still allowing dynamic use in multiple scenarios, from aiding with early decision making to device weaning. Herein, we discuss currently available CS risk scores, perform a detailed analysis of the variables in each of these scores that are most predictive of CS outcomes and explore a framework for the development of novel risk scores that consider emerging therapies and paradigms for this challenging clinical entity.

Flavonoids in the Treatment of Neuropathic Pain.

PURPOSE OF REVIEW: Chronic pain continues to present a large burden to the US healthcare system. Neuropathic pain, a common class of chronic pain, remains particularly difficult to treat despite extensive research efforts. Current pharmacologic regimens exert limited efficacy and wide, potentially dangerous side effect profiles. This review provides a comprehensive, preclinical evaluation of the literature regarding the role of flavonoids in the treatment of neuropathic pain. RECENT FINDINGS: Flavonoids are naturally occurring compounds, found in plants and various dietary sources, which may have potential benefit in neuropathic pain. Numerous animal-model studies have demonstrated this benefit, including reversal of hyperalgesia and allodynia. Flavonoids have also exhibited an anti-inflammatory effect relevant to neuropathic pain, as evidenced by the reduction in multiple pro-inflammatory mediators, such as TNF-α, NF-κB, IL-1ß, and IL-6. Flavonoids represent a potentially new treatment modality for neuropathic pain in preclinical models, though human clinical evidence is yet to be explored at this time.

Structural mechanism of glutamate receptor activation and desensitization.

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

Physician adherence to 'Seattle' and 'International' ECG criteria in adolescent athletes: An analysis of compliance by specialty, experience, and practice environment.

BACKGROUND: Screening electrocardiography (ECG) for athletes is both controversial and rapidly evolving. While identifying an abnormal ECG could detect a serious cardiovascular disease, falsely interpreting a benign ECG pattern as abnormal can lead to unnecessary testing, cost, and anxiety. Though recent refinements of athlete screening ECG criteria have significantly improved its accuracy, in clinical practice physician adherence to these criteria may vary. METHODS: We analyzed physician adherence to contemporary athlete ECG criteria in a large, national athlete screening registry. 1577 consecutive screening ECGs were independently re- interpreted to assess for physician adherence to Seattle Criteria or International Criteria, respective to the criteria in place when the screening was performed. We further determined the most common ECG interpretations that deviated from these criteria, and analyzed physician characteristics for independent predictors of adherence to published ECG criteria. RESULTS: Though overall adherence to contemporary criteria was high, 60.4% of ECGs interpreted as abnormal did not meet athlete ECG criteria for pathology when independently re- read. The most common ECG patterns misinterpreted as abnormal were isolated left ventricular hypertrophy (LVH) and non-pathologic T-wave inversions (TWI). Multivariate regression identified three independent predictors of adherence to athlete ECG criteria: participating in a screening overseen by the organization's medical leadership, electrophysiology specialists and adult cardiologists. CONCLUSION: This study highlights the need for quality control measures and continued clinician education in a controversial and rapidly evolving field. Clinician education for athlete screening ECG criteria should emphasize the recent changes in how TWI and LVH voltage criteria should be interpreted in this patient population.

How to Manage Temporary Mechanical Circulatory Support Devices in the Critical Care Setting: Translating Physiology to the Bedside.

The incidence of cardiogenic shock and the utilization of mechanical circulatory support devices are increasing in the US. In this review we discuss the pathophysiology of cardiogenic shock through basic hemodynamic and myocardial energetic principles. We also explore the commonly used platforms for temporary mechanical circulatory support, their advantages, disadvantages and practical considerations relating to implementation and management. It is through the translation of underlying physiological principles that we can attempt to maximize the clinical utility of circulatory support devices and improve outcomes in cardiogenic shock.

Ambulatory central VA-ECMO with biventricular decompression for acute cardiogenic shock.

We describe the off-pump insertion of a biventricular assist device with extra-corporeal membrane oxygenation (ECMO): a novel technique that allows for ambulatory central veno-arterial (VA) ECMO with direct biventricular decompression.

Peripheral VA-ECMO with direct biventricular decompression for refractory cardiogenic shock.

Cardiogenic shock and cardiac arrest are life-threatening emergencies that result in high mortality rates. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) via peripheral cannulation is an option for patients who do not respond to conventional therapies. Left ventricular (LV) distention is a major limitation with peripheral VA-ECMO and is thought to contribute to poor recovery and the inability to wean off VA-ECMO. We report on a novel technique that combines peripheral VA-ECMO with off-pump insertion of a trans-apical LV venting cannula and a right ventricular decompression cannula.

Intraoperative thermographic imaging to assess myocardial distribution of Del Nido cardioplegia.

We describe the intraoperative non-invasive use of an infrared (IR) camera to monitor Del Nido cardioplegia delivery in patients undergoing cardiac surgery. Thermal pictures were taken pre- and post-cardioplegia and at timed points after arrest, and compared to readings from a transseptal temperature probe. There was good concordance between the transseptal probe and the IR camera temperature readings. This non-invasive technique, which assesses cardioplegic distribution, may help to determine when additional doses of Del Nido cardioplegia are required during periods of cardioplegic arrest.

Assessment of Proliferative Potential of Odontogenic Keratocyst and Dentigerous Cyst using Podoplanin: An Immunohistochemical Study.

INTRODUCTION: Odontogenic cysts are commonly encountered lesions among head and neck pathologies. Odontogenic keratocyst (OKC) has unique features of recurrence and local aggressiveness. Podoplanin (PDP) is a lymphatic endothelial marker and is shown to be expressed in a variety of tissues. Hence, we planned to assess the significance of PDP in OKC and dentigerous cyst (DC). MATERIALS AND METHODS: The present study included assessment of immunoexpression of PDP in OKC and DC. Twenty specimens each of OKC and DC were included in the present study and were stained with D2-40 antibody. All the sections were analyzed and were categorized as negative staining, weakly positive staining, and strongly positive staining. All the results were analyzed by Statistical Package for the Social Sciences (SPSS) software. RESULTS: We detected PDP-positive staining in the cell membrane and cytoplasm of the cells of basal cell layer and supra-basal cell layers. In DC cases, we observed positive staining only in cases associated with inflammation. CONCLUSION: Podoplanin does play a significant role in enhancing the local invasive and neoplastic properties of OKC. CLINICAL SIGNIFICANCE: Podoplanin expression in OKC is potentially associated with moderate invasive nature of the neighboring structures.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.