Meeting Youth Where They Live: How to Use a QR Code in Safety Planning.

When in extreme distress, it is difficult to remember the coping strategies and resources available to you. The purpose of a safety plan is to make it easier for individuals to make wise choices in moments of crisis. By virtue of this, we should strive to have safety plans as easy and convenient to use as possible. The Stanley-Brown safety plan is a template for this style of intervention and has been adapted and adopted in numerous institutions.1 A pillar of this intervention is ready-at-hand contact information for mental health agencies and crisis resources. Although the classic paper safety plan remains an invaluable tool, integration of digital resources may be necessary to meet our young patients "where they live."2 Generation Z are "digital natives" with great fluency and comfort with smart devices. Expansion of the audio-only National Suicide Prevention Lifeline to the audio, SMS, and Web-based instant messaging capabilities of the 988 Crisis & Suicide Lifeline recognizes this shift.3 At the University of Michigan, we developed a quick response (QR) code to save these resources directly onto youths' smartphones.

Kinetic fingerprinting of metabotropic glutamate receptors.

Dimeric metabotropic glutamate receptors (mGluRs) are abundantly expressed in neurons. In mammals, eight subunit isoforms, mGluR1-8, have been identified, forming the groups I, II, and III. We investigated receptor dimerization and kinetics of these mGluR isoforms in excised membrane patches by FRET and confocal patch-clamp fluorometry. We show that 5 out of 8 homodimeric receptors develop characteristic glutamate-induced on- and off-kinetics, as do 11 out of 28 heterodimers. Glutamate-responsive heterodimers were identified within each group, between groups I and II as well as between groups II and III, but not between groups I and III. The glutamate-responsive heterodimers showed heterogeneous activation and deactivation kinetics. Interestingly, mGluR7, not generating a kinetic response in homodimers, showed fast on-kinetics in mGluR2/7 and mGluR3/7 while off-kinetics retained the speed of mGluR2 or mGluR3 respectively. In conclusion, glutamate-induced conformational changes in heterodimers appear within each group and between groups if one group II subunit is present.

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A'-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A'-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A'-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

Block of Voltage-Gated Sodium Channels as a Potential Novel Anti-cancer Mechanism of TIC10.

Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs. Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells. Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 µM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states. Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Block of Voltage-Gated Sodium Channels by Atomoxetine in a State- and Use-dependent Manner.

Atomoxetine, a neuroactive drug, is approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). It is primarily known as a high affinity blocker of the noradrenaline transporter, whereby its application leads to an increased level of the corresponding neurotransmitter in different brain regions. However, the concentrations used to obtain clinical effects are much higher than those which are required to block the transporter system. Thus, off-target effects are likely to occur. In this way, we previously identified atomoxetine as blocker of NMDA receptors. As many psychotropic drugs give rise to sudden death of cardiac origin, we now tested the hypothesis whether atomoxetine also interacts with voltage-gated sodium channels of heart muscle type in clinically relevant concentrations. Electrophysiological experiments were performed by means of the patch-clamp technique at human heart muscle sodium channels (hNav1.5) heterogeneously expressed in human embryonic kidney cells. Atomoxetine inhibited sodium channels in a state- and use-dependent manner. Atomoxetine had only a weak affinity for the resting state of the hNav1.5 (Kr: ∼ 120 µM). The efficacy of atomoxetine strongly increased with membrane depolarization, indicating that the inactivated state is an important target. A hallmark of this drug was its slow interaction. By use of different experimental settings, we concluded that the interaction occurs with the slow inactivated state as well as by slow kinetics with the fast-inactivated state. Half-maximal effective concentrations (2-3 µM) were well within the concentration range found in plasma of treated patients. Atomoxetine also interacted with the open channel. However, the interaction was not fast enough to accelerate the time constant of fast inactivation. Nevertheless, when using the inactivation-deficient hNav1.5_I408W_L409C_A410W mutant, we found that the persistent late current was blocked half maximal at about 3 µM atomoxetine. The interaction most probably occurred via the local anesthetic binding site. Atomoxetine inhibited sodium channels at a similar concentration as it is used for the treatment of ADHD. Due to its slow interaction and by inhibiting the late current, it potentially exerts antiarrhythmic properties.

Terahertz refractive index-based morphological dilation for breast carcinoma delineation.

This paper reports investigations led on the combination of the refractive index and morphological dilation to enhance performances towards breast tumour margin delineation during conserving surgeries. The refractive index map of invasive ductal and lobular carcinomas were constructed from an inverse electromagnetic problem. Morphological dilation combined with refractive index thresholding was conducted to classify the tissue regions as malignant or benign. A histology routine was conducted to evaluate the performances of various dilation geometries associated with different thresholds. It was found that the combination of a wide structuring element and high refractive index was improving the correctness of tissue classification in comparison to other configurations or without dilation. The method reports a sensitivity of around 80% and a specificity of 82% for the best case. These results indicate that combining the fundamental optical properties of tissues denoted by their refractive index with morphological dilation may open routes to define supporting procedures during breast-conserving surgeries.

Action potentials in Xenopus oocytes triggered by blue light.

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary ß1 subunit of voltage-gated Na+ channels. The resulting fusion construct, ß1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and ß1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing ß1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

Pilot study of freshly excised breast tissue response in the 300-600 GHz range.

The failure to accurately define tumor margins during breast conserving surgery (BCS) results in a 20% re-excision rate. The present paper reports the investigation to evaluate the potential of terahertz imaging for breast tissue recognition within the under-explored 300 - 600 GHz range. Such a frequency window matches new BiCMOS technology capabilities and thus opens up the opportunity for near-field terahertz imaging using these devices. To assess the efficacy of this frequency band, data from 16 freshly excised breast tissue samples were collected and analyzed directly after excision. Complex refractive indices have been extracted over the as-mentioned frequency band, and amplitude frequency images show some contrast between tissue types. Principal component analysis (PCA) has also been applied to the data in an attempt to automate tissue classification. Our observations suggest that the dielectric response could potentially provide contrast for breast tissue recognition within the 300 - 600 GHz range. These results open the way for silicon-based terahertz subwavelength near field imager design, efficient up to 600 GHz to address ex vivo life-science applications.

Corrigendum: Quantifying the cooperative subunit action in a multimeric membrane receptor.

This corrects the article DOI: 10.1038/srep20974.

Corrigendum: Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

This corrects the article DOI: 10.1038/srep29378.

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Neurometabolic Disorders: Potentially Treatable Abnormalities in Patients With Treatment-Refractory Depression and Suicidal Behavior.

OBJECTIVE: Treatment-refractory depression is a devastating condition with significant morbidity, mortality, and societal cost. At least 15% of cases of major depressive disorder remain refractory to treatment. The authors previously identified a young adult with treatment-refractory depression and multiple suicide attempts with an associated severe deficiency of CSF tetrahydrobiopterin, a critical cofactor for monoamine neurotransmitter synthesis. Treatment with sapropterin, a tetrahydrobiopterin analogue, led to dramatic and long-lasting remission of depression. This sentinel case led the authors to hypothesize that the incidence of metabolic abnormalities contributing to treatment-refractory depression is underrecognized. METHOD: The authors conducted a case-control, targeted, metabolomic evaluation of 33 adolescent and young adult patients with well-characterized histories of treatment-refractory depression (at least three maximum-dose, adequate-duration medication treatments), and 16 healthy comparison subjects. Plasma, urine, and CSF metabolic profiling were performed by coupled gas chromatography/mass spectrometry and high-performance liquid chromatography electrospray ionization tandem mass spectrometry. RESULTS: CSF metabolite abnormalities were identified in 21 of the 33 participants with treatment-refractory depression. Cerebral folate deficiency (N=12) was most common, with normal serum folate levels and low CSF 5-methyltetrahydrofolate (5-MTHF) levels. All patients with cerebral folate deficiency, including one with low CSF levels of 5-MTHF and tetrahydrobiopterin intermediates, showed improvement in depression symptom inventories after treatment with folinic acid; the patient with low tetrahydrobiopterin also received sapropterin. None of the healthy comparison subjects had a metabolite abnormality. CONCLUSIONS: Examination of metabolic disorders in treatment-refractory depression identified an unexpectedly large proportion of patients with potentially treatable abnormalities. The etiology of these abnormalities remains to be determined.

Influence of the coronary calcium score on the ability to rule out coronary artery stenoses by coronary CT angiography in patients with suspected coronary artery disease.

BACKGROUND: Recent guidelines for the workup of patients with chest pain and suspected coronary artery disease include coronary computed tomography angiography (CTA). However, its diagnostic value may be limited in patients with severe coronary calcification. OBJECTIVE: We investigated the relationship between the extent of coronary calcium and the ability of coronary CTA to rule out significant stenoses in a series of consecutive patients with suspected coronary artery disease. METHODS: 2614 consecutive patients with suspected coronary artery disease in whom coronary calcium scoring and coronary CTA had been performed by Dual Source CT were analyzed. The ability of coronary CTA to rule out coronary artery stenoses (fully evaluable coronary arteries and absence of any luminal stenosis >75%) was analyzed relative to the coronary calcium score. RESULTS: The median coronary calcium score was 12, with calcium present in 60.5% of all patients. Coronary CTA ruled out stenoses in 82% of patients, while in 18% of patients at least one stenosis was found or could not be excluded. The threshold above which coronary CTA permitted to rule out stenoses in less than 50% of patients was an "Agatston Score" of 287. This threshold was significantly lower for male patients (213 vs. 330), for patients with a heart rate >65 beats/min (157 vs. 317) and for patients with a body mass index ≥25 kg/m(2) (208 vs. 392). The evaluability of coronary arteries decreased with increasing amounts of calcium and differed significantly between heart rates ≤65 beats/min and >65 beats/min (p < 0.0001). CONCLUSION: In the largest patient series evaluated so far, we identified an "Agatston Score" of 287 to represent a threshold above which coronary CTA permits to rule out coronary artery stenoses in less than 50% of cases.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

Quantifying the cooperative subunit action in a multimeric membrane receptor.

In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.