Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca(2+)] ([Ca(2+)](o)) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca(2+)](o) sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca(2+)](o) than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.Our experiments identify CaSR as the first component in the [Ca(2+)](o) sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca(2+)](o) will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca(2+)](o) following its entry via voltage-activated Ca(2+) channels. CaSR may detects such falls in [Ca(2+)](o) and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca(2+)](o) on release probability. CaSR is positioned to detect the dynamic changes of [Ca(2+)](o) and provide presynaptic feedback that will alter brain excitability

Genetic Causes of Cardiomyopathy in Children: First Results From the Pediatric Cardiomyopathy Genes Study

Pediatric cardiomyopathy is a genetically heterogeneous disease with substantial morbidity and mortality. Current guidelines recommend genetic testing in children with hypertrophic, dilated, or restrictive cardiomyopathy, but practice variations exist. Robust data on clinical testing practices and diagnostic yield in children are lacking. This study aimed to identify the genetic causes of cardiomyopathy in children and to investigate clinical genetic testing practices. Methods and Results Children with familial or idiopathic cardiomyopathy were enrolled from 14 institutions in North America. Probands underwent exome sequencing. Rare sequence variants in 37 known cardiomyopathy genes were assessed for pathogenicity using consensus clinical interpretation guidelines. Of the 152 enrolled probands, 41% had a family history of cardiomyopathy. Of 81 (53%) who had undergone clinical genetic testing for cardiomyopathy before enrollment, 39 (48%) had a positive result. Genetic testing rates varied from 0% to 97% between sites. A positive family history and hypertrophic cardiomyopathy subtype were associated with increased likelihood of genetic testing (P=0.005 and P=0.03, respectively). A molecular cause was identified in an additional 21% of the 63 children who did not undergo clinical testing, with positive results identified in both familial and idiopathic cases and across all phenotypic subtypes. Conclusions A definitive molecular genetic diagnosis can be made in a substantial proportion of children for whom the cause and heritable nature of their cardiomyopathy was previously unknown. Practice variations in genetic testing are great and should be reduced. Improvements can be made in comprehensive cardiac screening and predictive genetic testing in first-degree relatives. Overall, our results support use of routine genetic testing in cases of both familial and idiopathic cardiomyopathy

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Probing the mechanism of the dedicated NO sensor [4Fe-4S] NsrR: the effect of cluster ligand environment

NsrR from Streptomyces coelicolor is a bacterial nitric oxide (NO) sensor/nitrosative stress regulator as its primary function, and has been shown to have differential response at low, mid, and high levels of NO. These must correspond to discrete structural changes at the protein-bound [4Fe-4S] cluster in response to stepwise nitrosylation of the cluster. We have investigated the effect of the monohapto carboxylate ligand in the site differentiated [4Fe-4S] cluster cofactor of the protein NsrR on modulating its reactivity to NO with a focus on indentifying mechanistic intermediates. We have prepared a synthetic model [4Fesingle bond4S] cluster complex with tripodal ligand and one single site differentiated site occupied by either thiolate or carboxylate ligand. We report here the mechanistic details of sequential steps of nitrosylation as observed by ESI MS and IR spectroscopy. Parallel non-denaturing mass spectrometry analyses were performed using site-differentiated variants of NsrR with the native aspartic acid, cysteine, or alanine in the position of the forth ligand to the cluster. A mono-nitrosylated synthetic [4Fe-4S] cluster was observed for the first time in a biologically-relevant thiolate-based coordination environment. Combined synthetic and protein data give unprecedented clarity in the modulation of nitrosylation of a [4Fesingle bond4S] cluster

Solution and Solid State Correlations of Antimalarial Drug Actions: NMR and Crystallographic Studies of Drug Interactions with a Heme Model

Solution NMR has been used in tandem with a diamagnetic non-iron heme model compound as a simple and effective tool to rapidly probe the structures of the bound complexes formed between the metalloporphyrin and antimalarial drugs from the 4-aminoquinoline, 4-methylenehydroxylquinoline, and 8-aminoquinoline subfamilies. The ability of gallium­(III) protoporphyrin IX to mimic heme chemistry is exploited. The 4-aminoquinolines quinacrine and amodiaquine and two novel 3-halo chloroquine analogues are found to bind to the metalloporphyrin through hydrogen-bonding and stacking interactions, while halofantrine and the 4-methylenehydroxylquinolines, quinine and mefloquine bind through the alcohol group of the drug. In each case, detailed structural information is available from the NMR assessment. The mefloquine model is confirmed crystallographically. The 8-aminoquinoline primaquine does not interact strongly. These tools show promise for future applications in assessing antimalarials in preclinical development for heme-binding drug targets

Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster

Iron–sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress. Both iron–sulfur proteins and synthetic model clusters are extremely sensitive to nitrosylation, tending towards rapid multi-step reaction and cluster degradation. Reaction of protein-bound iron–sulfur clusters with nitric oxide can be stopped at partial nitrosylation in vivo, and repair of protein-bound nitrosylated clusters is possible in the cellular environment. We have used a combination of infrared, EPR, and UV-visible spectroscopies to show that a model [4Fe4S] cluster-containing protein, A. ferroxidans high potential iron–sulfur protein (HiPIP), reacts with NO to give a product mixture dominated by Roussin's Black Salt (RBS) and Roussin's Red Ester (RRE) species. We have shown that O2 plays a critical role in controlling the major product of nitrosylation, with RBS-like products favoured under strictly anaerobic conditions and RRE favoured in the presence of trace O2. Moreover, addition of trace O2 to anaerobically nitrosylated samples induces conversion of RBS-like products to RRE. These findings may have implications for mechanisms of iron–sulfur cluster repair following nitrosative stress, suggest a crucial role for trace O2, and provide an important link between nitrosylation chemistry of iron–sulfur proteins and the well-established reactivity of synthetic iron–sulfur clusters

Amphibian Research and Monitoring Initiative (ARMI): A successful start to a national program in the United States

Most research to assess amphibian declines has focused on local-scale projects on one or a few species. The Amphibian Research and Monitoring Initiative (ARMI) is a national program in the United States mandated by congressional directive and implemented by the U.S. Department of the Interior (specifically the U.S. Geological Survey, USGS). Program goals are to monitor changes in populations of amphibians across U.S. Department of the Interior lands and to address research questions related to amphibian declines using a hierarchical framework of base-, mid- and apex-level monitoring sites. ARMI is currently monitoring 83 amphibian species (29% of species in the U.S.) at mid- and apex-level areas. We chart the progress of this 5-year-old program and provide an example of mid-level monitoring from 1 of the 7 ARMI regions

Angiotensin type I receptor blockade in conjunction with enhanced Akt activation restores coronary collateral growth in the metabolic syndrome

We have previously demonstrated that Akt was required for repetitive ischemia (RI)-induced coronary collateral growth (CCG) in healthy rats but was not activated by RI in the metabolic syndrome (JCR:LA-cp rats) where CCG was impaired. Here we hypothesized that failure of angiotensin type I receptor (AT1R) blockers to restore Akt activation is a key determinant of their inability to completely restore CCG in the metabolic syndrome. Therefore, we investigated whether adenovirus-mediated delivery of constitutively active Akt (MyrAkt-Adv) in conjunction with AT1R blockade (candesartan) was able to restore RI-induced CCG in JCR:LA-cp rats. Successful myocardial MyrAkt-Adv delivery was confirmed by a >80% transduction efficiency and an approximately fourfold increase in Akt expression and activation. CCG was assessed by myocardial blood flow measurements in the normal and collateral-dependent zones. MyrAkt-Adv alone significantly increased RI-induced CCG in JCR:LA-cp rats (∼30%), but it completely restored CCG in conjunction with administration of candesartan. In contrast, dominant negative Akt (DN-Akt-Adv) reversed the beneficial effect of candesartan on CCG in JCR:LA-cp rats. We conclude that optimal restoration of coronary collateral growth in JCR:LA-cp rats requires a combination of AT1R blockade with constitutive Akt activation. These findings may carry implications for metabolic syndrome patients in need of coronary revascularization