Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine.

Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.

Dielectric Properties of Materials Used for Microwave-Based NOx Gas Dosimeters.

Nitrogen oxides (NOx), primarily generated from combustion processes, pose significant health and environmental risks. To improve the coordination of measures against excessive NOx emissions, it is necessary to effectively monitor ambient NOx concentrations, which requires the development of precise and cost-efficient detection methods. This study focuses on developing a microwave- or radio frequency (RF)-based gas dosimeter for NOx detection and addresses the optimization of the dosimeter design by examining the dielectric properties of LTCC-based (Low-Temperature Co-fired Ceramics) sensor substrates and barium-based NOx storage materials. The measurements taken utilizing the Microwave Cavity Perturbation (MCP) method revealed that these materials exhibit more pronounced changes in dielectric losses when storing NOx at elevated temperatures. Consequently, operating such a dosimeter at high temperatures (above 300 °C) is recommended to maximize the sensor signal. To evaluate their high-temperature applicability, LTCC substrates were analyzed by measuring their dielectric losses at temperatures up to 600 °C. In terms of NOx storage materials, coating barium on high-surface-area alumina resolved issues related to limited NOx adsorption in pure barium carbonate powders. Additionally, the adsorption of both NO and NO2 was enabled by the application of a platinum catalyst. The change in dielectric losses, which provides the main signal for an RF-based gas dosimeter, only depends on the stored amount of NOx and not on the specific type of nitrogen oxide. Although the change in dielectric losses increases with the temperature, the maximum storage capacity of the material decreases significantly. In addition, at temperatures above 350 °C, NOx is mostly weakly bound, so it will desorb in the absence of NOx. Therefore, in the future development of a reliable RF-based NOx dosimeter, the trade-off between the sensor signal strength and adsorption behavior must be addressed.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

Current updates on arrhythmia within Timothy syndrome: genetics, mechanisms and therapeutics.

Timothy syndrome (TS), characterised by multiple system malfunction especially the prolonged corrected QT interval and synchronised appearance of hand/foot syndactyly, is an extremely rare disease affecting early life with devastating arrhythmia. In this work, firstly, the various mutations in causative gene CACNA1C encoding cardiac L-type voltage-gated calcium channel (LTCC), regard with the genetic pathogeny and nomenclature of TS are reviewed. Secondly, the expression profile and function of CACNA1C gene encoding Cav1.2 proteins, and its gain-of-function mutation in TS leading to multiple organ disease phenotypes especially arrhythmia are discussed. More importantly, we focus on the altered molecular mechanism underlying arrhythmia in TS, and discuss about how LTCC malfunction in TS can cause disorganised calcium handling with excessive intracellular calcium and its triggered dysregulated excitation-transcription coupling. In addition, current therapeutics for TS cardiac phenotypes including LTCC blockers, beta-adrenergic blocking agents, sodium channel blocker, multichannel inhibitors and pacemakers are summarised. Eventually, the research strategy using patient-specific induced pluripotent stem cells is recommended as one of the promising future directions for developing therapeutic approaches. This review updates our understanding on the research progress and future avenues to study the genetics and molecular mechanism underlying the pathogenesis of devastating arrhythmia within TS, and provides novel insights for developing therapeutic measures.

Miniaturized On-Ground 2.4 GHz IoT LTCC Chip Antenna and Its Positioning on a Ground Plane.

This paper presents a very low-profile on-ground chip antenna with a total volume of 0.075λ0× 0.056λ0× 0.019λ0 (at f0 = 2.4 GHz). The proposed design is a corrugated (accordion-like) planar inverted F antenna (PIFA) embedded in low-loss glass ceramic material (DuPont GreenTape 9k7 with ϵr = 7.1 and tanδ = 0.0009) fabricated with LTCC technology. The antenna does not require a clearance area on the ground plane where the antenna is located, and it is proposed for 2.4 GHz IoT applications for extreme size-limited devices. It shows a 25 MHz impedance bandwidth (for S11 < -6 dB), which means a relative bandwidth of 1%). A study in terms of matching and total efficiency is performed for several size ground planes with the antenna installed at different positions. The use of characteristic modes analysis (CMA) and the correlation between modal and total radiated fields is performed to demonstrate the optimum position of the antenna. Results show high-frequency stability and a total efficiency difference of up to 5.3 dB if the antenna is not placed at the optimum position.

Tuberous Sclerosis Complex (TSC) Inactivation Increases Neuronal Network Activity by Enhancing Ca2+ Influx via L-Type Ca2+ Channels.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs, such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurologic impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remains unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2-/-) neurons exhibited elevated neuronal activity with highly synchronized Ca2+ spikes. Notably, TSC2-/- neurons presented enhanced Ca2+ influx via L-type Ca2+ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2-/- neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2-/- neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.SIGNIFICANCE STATEMENT There is a close relationship between elevated mammalian target of rapamycin (mTOR) activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2-/- neurons, we identified elevated Ca2+ influx via L-type Ca2+ channels as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2-/- neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca2+ signaling via L-type Ca2+ channels in human TSC neurons, which could cause epilepsy in TSC.

LTCC Strip Electrode Arrays for Gas Electron Multiplier Detectors.

The Low Temperature Cofired Ceramic (LTCC) technology has proven to be highly suitable for 3D microstructures manufacturing in electronic devices due to its excellent electrical and mechanical properties. In this paper, a novel idea of implementing the LTCC structures into high-energy particle detectors technology is proposed. It can be applied in High Energy Physics (HEP) laboratories, where such sophisticated sensors are constantly exposed to particles of the TeV energy range for many years. The most advanced applications of the concept are based on dedicated gas amplifier systems coupled with readout microstructures. Typically, the readout microstructures are made in the Printed Circuit Boards (PCB) technology and processed in a sophisticated and patent-protected way. This article presents the manufacturing process and parameters of the novel microstructures made in the LTCC technology. The structures were implemented into the high-energy particle detector, and the first results are presented.

Silencing of lipocalin-2 improves cardiomyocyte viability under iron overload conditions via decreasing mitochondrial dysfunction and apoptosis.

This study aimed to investigate the mechanistic roles of LCN-2 and LCN-2 receptors (LCN-2R) as iron transporters in cardiomyocytes under iron overload condition. H9c2 cardiomyocytes were treated with either LCN-2 small interfering RNA (siRNA) or LCN-2R siRNA or L-type or T-type calcium channel (LTCC or TTCC) blockers, or iron chelator deferiprone (DFP). After the treatments, the cells were exposed to Fe3+ or Fe2+ , after that biological parameters were determined. Silencing of lipocalin-2 or its receptor improved cardiomyocyte viability via decreasing iron uptake, mitochondrial fission, mitophagy and cleaved caspase-3 only in the Fe3+ overload condition. In contrast, treatments with LTCC blocker and TTCC blocker showed beneficial effects on those parameters only in conditions of Fe2+ overload. Treatment with DFP has been shown beneficial effects both in Fe2+ and Fe3+ overload condition. All of these findings suggested that LTCC and TTCC play crucial roles in the Fe2+ uptake, whereas LCN-2 and LCN-2R were essential for Fe3+ uptake into the cardiomyocytes under iron overload conditions.

Microfluidic Modules Integrated with Microwave Components-Overview of Applications from the Perspective of Different Manufacturing Technologies.

The constant increase in the number of microfluidic-microwave devices can be explained by various advantages, such as relatively easy integration of various microwave circuits in the device, which contains microfluidic components. To achieve the aforementioned solutions, four trends of manufacturing appear-manufacturing based on epoxy-glass laminates, polymer materials (mostly common in use are polydimethylsiloxane (PDMS) and polymethyl 2-methylpropenoate (PMMA)), glass/silicon substrates, and Low-Temperature Cofired Ceramics (LTCCs). Additionally, the domains of applications the microwave-microfluidic devices can be divided into three main fields-dielectric heating, microwave-based detection in microfluidic devices, and the reactors for microwave-enhanced chemistry. Such an approach allows heating or delivering the microwave power to the liquid in the microchannels, as well as the detection of its dielectric parameters. This article consists of a literature review of exemplary solutions that are based on the above-mentioned technologies with the possibilities, comparison, and exemplary applications based on each aforementioned technology.

An Ultrahigh Sensitive Microwave Microfluidic System for Fast and Continuous Measurements of Liquid Solution Concentrations.

In this paper, we describe a low-cost microwave microfluidic system of ultrahigh sensitivity for detecting small changes in the concentration of polar solutions (liquid dielectrics) in the 2.4 GHz ISM band. Its principle of operation is based on microwave interferometry, which is implemented using planar microstrip lines and integrated microwave components. The key features of this system include small solution intake (<200 µL per measurement), short time of measurement (ca. 20 ms), ultrahigh sensitivity of concentration changes (up to 55 dB/%), and low error of measurement (below 0.1%). The ultrahigh sensitivity was proven experimentally by measurements of the fat content of milk. In addition, it is a user-friendly system due to an effortless and fast calibration procedure. Moreover, it can be made relatively compact (<20 cm2) and features low power consumption (200 mW). Thus, the proposed system is perfect for industrial applications, especially for highly integrated lab-on-chip devices.

LTCC-Integrated Dielectric Resonant Antenna Array for 5G Applications.

A millimeter-wave dielectric resonator antenna array with an integrated feeding network and a novel alignment superstrate in Low Temperature Cofired Ceramics (LTCC) technology is presented. The antenna array consists of 16 cylindrical DR antenna (CDRA) elements operating at 28 GHz for mm-Wave 5G applications. The array is fed by an inverted microstrip corporate feeding network designed and built in the same LTCC stack as the resonators. A grooved and grounded superstrate is introduced to facilitate the alignment of the individual array elements while enhancing the overall performance of the antenna array. The performance of the proposed stack is evaluated numerically and compared with measured data. Measured results show an impedance bandwidth of 9.81% at 28.72 GHz with a maximum realized gain of 15.68 dBi and an efficiency of 88%, and are in excellent agreement with simulations.

New aspects in cardiac L-type Ca2+ channel regulation.

Cardiac excitation-contraction coupling is initiated with the influx of Ca2+ ions across the plasma membrane through voltage-gated L-type calcium channels. This process is tightly regulated by modulation of the channel open probability and channel localization. Protein kinase A (PKA) is found in close association with the channel and is one of the main regulators of its function. Whether this kinase is modulating the channel open probability by phosphorylation of key residues or via alternative mechanisms is unclear. This review summarizes recent findings regarding the PKA-mediated channel modulation and will highlight recently discovered regulatory mechanisms that are independent of PKA activity and involve protein-protein interactions and channel localization.

Fluorescence Sensing Platforms for Epinephrine Detection Based on Low Temperature Cofired Ceramics.

A novel fluorescence-sensing pathway for epinephrine (EP) detection was investigated. The ceramic-based miniature biosensor was developed through the immobilization of an enzyme (laccase, tyrosinase) on a polymer-poly-(2,6-di([2,2'-bithiophen]-5-yl)-4-(5-hexylthiophen-2-yl)pyridine), based on low temperature cofired ceramics technology (LTCC). The detection procedure was based on the oxidation of the substrate, i.e., in the presence of the enzyme. An alternative enzyme-free system utilized the formation of a colorful complex between Fe2+ ions and epinephrine molecules. With the optimized conditions, the analytical performance illustrated high sensitivity and selectivity in a broad linear range with a detection limit of 0.14-2.10 nM. Moreover, the strategy was successfully used for an EP injection test with labeled pharmacological samples.

Biomaterial Embedding Process for Ceramic-Polymer Microfluidic Sensors.

One of the major issues in microfluidic biosensors is biolayer deposition. Typical manufacturing processes, such as firing of ceramics and anodic bonding of silicon and glass, involve exposure to high temperatures, which any biomaterial is very vulnerable to. Therefore, current methods are based on deposition from liquid, for example, chemical bath deposition (CBD) and electrodeposition (ED). However, such approaches are not suitable for many biomaterials. This problem was partially resolved by introduction of ceramic-polymer bonding using plasma treatment. This method introduces an approximately 15-min-long window for biomodification between plasma activation and sealing the system with a polymer cap. Unfortunately, some biochemical processes are rather slow, and this time is not sufficient for the proper attachment of a biomaterial to the surface. Therefore, a novel method, based on plasma activation after biomodification, is introduced. Crucially, the discharge occurs selectively; otherwise, it would etch the biomaterial. Difficulties in manufacturing ceramic biosensors could be overcome by selective surface modification using plasma treatment and bonding to polymer. The area of plasma modification was investigated through contact-angle measurements and Fourier-transform infrared (FTIR) analyses. A sample structure was manufactured in order to prove the concept. The results show that the method is viable.

LTCC Flow Sensor with RFID Interface.

The idea of battery-less flow sensors and their implementation in wireless measurement systems is presented in this research article. The authors take advantage of their latest achievements in the Low Temperature Co-fired Ceramic (LTCC) technology, RadioFrequency Identification (RFID) technique, and increasing availability of low power electronics in order to get rid of the need to use electrochemical cells in a power supply unit of the elaborated device. To reach this assumption, special care has to be put on the energy balance in such an autonomous sensor node. First of all, the new concept of an electromagnetic LTCC turbine transducer with a signal conditioner which only draws a current of around 15 µA, is proposed for measuring a flow rate of fluids. Next, the autonomy of the device is showed; measured data are gathered in a microcontroller memory and sent to a control unit via an RFID interface which enables both information exchange and power transfer. The energy harvested from the electromagnetic field is used to conduct a data transmission, but also its excess can be accumulated, so the proposed sensor operates as a semi-passive transponder. The total autonomy of the device is achieved by implementing a second harvester that continually gathers energy from the environmental electromagnetic field of common active radio systems (e.g., Global System for Mobile Communications (GSM), wireless network Wi-Fi).

Calmodulin Mutations Associated with Heart Arrhythmia: A Status Report.

Calmodulin (CaM) is a ubiquitous intracellular Ca2+ sensing protein that modifies gating of numerous ion channels. CaM has an extraordinarily high level of evolutionary conservation, which led to the fundamental assumption that mutation would be lethal. However, in 2012, complete exome sequencing of infants suffering from recurrent cardiac arrest revealed de novo mutations in the three human CALM genes. The correlation between mutations and pathophysiology suggests defects in CaM-dependent ion channel functions. Here, we review the current state of the field for all reported CaM mutations associated with cardiac arrhythmias, including knowledge of their biochemical and structural characteristics, and progress towards understanding how these mutations affect cardiac ion channel function.

3-Aryl-4-nitrobenzothiochromans S,S-dioxide: From Calcium-Channel Modulators Properties to Multidrug-Resistance Reverting Activity.

Our research groups have been involved for many years in studies aimed at identifying new active organic compounds endowed with pharmacological properties. In this work, we focused our attention on the evaluation of cardiovascular and molecular drug resistance (MDR) reverting activities of some nitrosubstituted sulphur-containing heterocycles. Firstly, we have examined the effects of 4-nitro-3-(4-methylphenyl)-3,6-dihydro-2H-thiopyran S,S-dioxide 5, and have observed no activity. Then we have extended our investigation to the 3-aryl-4-nitrobenzothiochromans S,S-dioxide 6 and 7, and have observed an interesting biological profile. Cardiovascular activities were assessed for all compounds using ex vivo studies, while the MDR reverting effect was evaluated only for selected compounds using tumor cell lines. All compounds were shown to affect cardiovascular parameters. Compound 7i exerted the most effect on negative inotropic activity, while 6d and 6f could be interesting molecules for the development of more active ABCB1 inhibitors. Both 6 and 7 represent structures of large possible biological interest, providing a scaffold for the identification of new ABCB1 inhibitors.

Ring Electrode Geometry for Microfluidic Electrochemistry.

We developed a ring electrode sensor for down-stream electrochemical sensing of microfluidic ELISA assay. The sensor is designed to easily integrate into the flow environment. Noble metal inks on Low Temperature Co-fired Ceramic provide straight forward fabrication at the mesoscale yielding robust sensors with very low sensing volumes. Two different sensor geometries were modeled. The best design was fabricated and tested in both static and flowing solutions. The sensor exhibits both macroelectrode and microelectrode behavior depending on fluid flow rate. Sensors of this type may be ideal for applications where electrochemical detection is desired in a flowing solution since the ring electrode has low propensity for bubble trapping.

Monolithic Microwave-Microfluidic Sensors Made with Low Temperature Co-Fired Ceramic (LTCC) Technology.

This paper compares two types of microfluidic sensors that are designed for operation in ISM (Industrial, Scientific, Medical) bands at microwave frequencies of 2.45 GHz and 5.8 GHz. In the case of the first sensor, the principle of operation is based on the resonance phenomenon in a microwave circuit filled with a test sample. The second sensor is based on the interferometric principle and makes use of the superposition of two coherent microwave signals, where only one goes through a test sample. Both sensors are monolithic structures fabricated using low temperature co-fired ceramics (LTCCs). The LTCC-based microwave-microfluidic sensor properties are examined and compared by measuring their responses for various concentrations of two types of test fluids: one is a mixture of water/ethanol, and the other is dopamine dissolved in a buffer solution. The experiments show a linear response for the LTCC-based microwave-microfluidic sensors as a function of the concentration of the components in both test fluids.

An LC Wireless Microfluidic Sensor Based on Low Temperature Co-Fired Ceramic (LTCC) Technology.

This work reports a novel wireless microfluidic biosensor based on low temperature co-fired ceramic (LTCC) technology. The wireless biosensor consists of a planar spiral inductor and parallel plate capacitor (LC) resonant antenna, which integrates with microchannel bends in the LTCC substrate. The wireless response of the biosensor was associated to the changes of its resonant frequency due to the alteration in the permittivity of the liquid flow in the microchannel. The wireless sensing performance to different organic liquids with permittivity from 3 to 78.5 was presented. The measured results are in good agreement with the theoretical calculation. The wireless detection for the concentration of glucose in water solution was investigated, and an excellent linear response and repeatability were obtained. This kind of LC wireless microfluidic sensor is very promising in establishing wireless lab-on-a-chip for biomedical and chemical applications.