Real-Time Signal Analysis with Wider Dynamic Range and Enhanced Sensitivity in Multiplex Colorimetric Immunoassays Using Encoded Hydrogel Microparticles.

The simultaneous quantification of multiple proteins is crucial for accurate medical diagnostics. A promising technology, the multiplex colorimetric immunoassay using encoded hydrogel microparticles, has garnered attention, due to its simplicity and multiplex capabilities. However, it encounters challenges related to its dynamic range, as it relies solely on the colorimetric signal analysis of encoded hydrogel microparticles at the specific time point (i.e., end-point analysis). This necessitates the precise determination of the optimal time point for the termination of the colorimetric reaction. In this study, we introduce real-time signal analysis to quantify proteins by observing the continuous colorimetric signal change within the encoded hydrogel microparticles. Real-time signal analysis measures the "slope", the rate of the colorimetric signal generation, by focusing on the kinetics of the accumulation of colorimetric products instead of the colorimetric signal that appears at the end point. By developing a deep learning-based automatic analysis program that automatically reads the code of the graphically encoded hydrogel microparticles and obtains the slope by continuously tracking the colorimetric signal, we achieved high accuracy and high throughput analysis. This technology has secured a dynamic range more than twice as wide as that of the conventional end-point signal analysis, simultaneously achieving a sensitivity that is 4-10 times higher. Finally, as a demonstration of application, we performed multiplex colorimetric immunoassays using real-time signal analysis covering a wide concentration range of protein targets associated with pre-eclampsia.

Tripartite Motif-Containing Protein 32 (TRIM32): What Does It Do for Skeletal Muscle?

Tripartite motif-containing protein 32 (TRIM32) is a member of the tripartite motif family and is highly conserved from flies to humans. Via its E3 ubiquitin ligase activity, TRIM32 mediates and regulates many physiological and pathophysiological processes, such as growth, differentiation, muscle regeneration, immunity, and carcinogenesis. TRIM32 plays multifunctional roles in the maintenance of skeletal muscle. Genetic variations in the TRIM32 gene are associated with skeletal muscular dystrophies in humans, including limb-girdle muscular dystrophy type 2H (LGMD2H). LGMD2H-causing genetic variations of TRIM32 occur most frequently in the C-terminal NHL (ncl-1, HT2A, and lin-41) repeats of TRIM32. LGMD2H is characterized by skeletal muscle dystrophy, myopathy, and atrophy. Surprisingly, most patients with LGMD2H show minimal or no dysfunction in other tissues or organs, despite the broad expression of TRIM32 in various tissues. This suggests more prominent roles for TRIM32 in skeletal muscle than in other tissues or organs. This review is focused on understanding the physiological roles of TRIM32 in skeletal muscle, the pathophysiological mechanisms mediated by TRIM32 genetic variants in LGMD2H patients, and the correlations between TRIM32 and Duchenne muscular dystrophy (DMD).

Highly Flexible Deep-Learning-Based Automatic Analysis for Graphically Encoded Hydrogel Microparticles.

Graphically encoded hydrogel microparticle (HMP)-based bioassay is a diagnostic tool characterized by exceptional multiplex detectability and robust sensitivity and specificity. Specifically, deep learning enables highly fast and accurate analyses of HMPs with diverse graphical codes. However, previous related studies have found the use of plain particles as data to be disadvantageous for accurate analyses of HMPs loaded with functional nanomaterials. Furthermore, the manual data annotation method used in existing approaches is highly labor-intensive and time-consuming. In this study, we present an efficient deep-learning-based analysis of encoded HMPs with diverse graphical codes and functional nanomaterials, utilizing the auto-annotation and synthetic data mixing methods for model training. The auto-annotation enhanced the throughput of dataset preparation up to 0.11 s/image. Using synthetic data mixing, a mean average precision of 0.88 was achieved in the analysis of encoded HMPs with magnetic nanoparticles, representing an approximately twofold improvement over the standard method. To evaluate the practical applicability of the proposed automatic analysis strategy, a single-image analysis was performed after the triplex immunoassay for the preeclampsia-related protein biomarkers. Finally, we accomplished a processing throughput of 0.353 s per sample for analyzing the result image.

Tripartite motif-containing protein 32 regulates Ca2+ movement in skeletal muscle.

Mutations in tripartite motif-containing protein 32 (TRIM32), especially in NHL repeats, have been found in skeletal muscle in patients with type 2H limb-girdle muscular dystrophy (LGMD2H). However, the roles of the NHL repeats of TRIM32 in skeletal muscle functions have not been well addressed. In the present study, to examine the functional role(s) of the TRIM32 NHL repeats in skeletal muscle, TRIM32-binding proteins in skeletal muscle were first searched using a binding assay and MALDI-TOF/TOF. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) was found to be a TRIM32-binding protein. Next, a deletion mutant of TRIM32 missing the NHL repeats (NHL-Del) was expressed in mouse primary skeletal myotubes during myoblast differentiation into myotubes. Ca2+ movement in the myotubes was examined using single-cell Ca2+ imaging. Unlike wild-type (WT) TRIM32, NHL-Del did not enhance the amount of Ca2+ release from the sarcoplasmic reticulum (SR), Ca2+ release for excitation-contraction (EC) coupling, or extracellular Ca2+ entry via store-operated Ca2+ entry (SOCE). In addition, even compared with the vector control, NHL-Del resulted in reduced SOCE due to reduced expression of extracellular Ca2+ entry channels. Transmission electron microscopy (TEM) observation of the myotubes revealed that NHL-Del induced the formation of abnormal vacuoles and tubular structures in the cytosol. Therefore, by binding to SERCA1a via its NHL repeats, TRIM32 may participate in the regulation of Ca2+ movement for skeletal muscle contraction and the formation of cellular vacuoles and tubular structures in skeletal muscle. Functional defects in TRIM32 due to mutations in NHL repeats may be pathogenic toward LGMD2H.

Calsequestrin 1 Is an Active Partner of Stromal Interaction Molecule 2 in Skeletal Muscle.

Calsequestrin 1 (CASQ1) in skeletal muscle buffers and senses Ca2+ in the sarcoplasmic reticulum (SR). CASQ1 also regulates store-operated Ca2+ entry (SOCE) by binding to stromal interaction molecule 1 (STIM1). Abnormal SOCE and/or abnormal expression or mutations in CASQ1, STIM1, or STIM2 are associated with human skeletal, cardiac, or smooth muscle diseases. However, the functional relevance of CASQ1 along with STIM2 has not been studied in any tissue, including skeletal muscle. First, in the present study, it was found by biochemical approaches that CASQ1 is bound to STIM2 via its 92 N-terminal amino acids (C1 region). Next, to examine the functional relevance of the CASQ1-STIM2 interaction in skeletal muscle, the full-length wild-type CASQ1 or the C1 region was expressed in mouse primary skeletal myotubes, and the myotubes were examined using single-myotube Ca2+ imaging experiments and transmission electron microscopy observations. The CASQ1-STIM2 interaction via the C1 region decreased SOCE, increased intracellular Ca2+ release for skeletal muscle contraction, and changed intracellular Ca2+ distributions (high Ca2+ in the SR and low Ca2+ in the cytosol were observed). Furthermore, the C1 region itself (which lacks Ca2+-buffering ability but has STIM2-binding ability) decreased the expression of Ca2+-related proteins (canonical-type transient receptor potential cation channel type 6 and calmodulin 1) and induced mitochondrial shape abnormalities. Therefore, in skeletal muscle, CASQ1 plays active roles in Ca2+ movement and distribution by interacting with STIM2 as well as Ca2+ sensing and buffering.

Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.

Calsequestrin: a well-known but curious protein in skeletal muscle.

Calsequestrin (CASQ) was discovered in rabbit skeletal muscle tissues in 1971 and has been considered simply a passive Ca2+-buffering protein in the sarcoplasmic reticulum (SR) that provides Ca2+ ions for various Ca2+ signals. For the past three decades, physiologists, biochemists, and structural biologists have examined the roles of the skeletal muscle type of CASQ (CASQ1) in skeletal muscle and revealed that CASQ1 has various important functions as (1) a major Ca2+-buffering protein to maintain the SR with a suitable amount of Ca2+ at each moment, (2) a dynamic Ca2+ sensor in the SR that regulates Ca2+ release from the SR to the cytosol, (3) a structural regulator for the proper formation of terminal cisternae, (4) a reverse-directional regulator of extracellular Ca2+ entries, and (5) a cause of human skeletal muscle diseases. This review is focused on understanding these functions of CASQ1 in the physiological or pathophysiological status of skeletal muscle.

Transformation and Evaporation of Surface Adsorbents on a Graphene "Hot Plate".

Dynamic surface modification of suspended graphene at high temperatures was directly observed with in situ scanning transmission electron microscopy (STEM) measurements. The suspended graphene devices were prepared on a SiN membrane substrate with a hole so that STEM observations could be conducted during Joule heating. Current-voltage characteristics of suspended graphene devices inside the STEM chamber were measured while monitoring and controlling the temperature of graphene by estimating the electrical power of the devices. During the in situ STEM observation at high temperatures, residual hydrocarbon adsorbents that had remained on graphene effectively evaporated creating large, atomically clean graphene areas. At other places, dynamic changes in the shape, position, and orientation of adsorbents could be directly observed. The temperature of the suspended graphene sample was estimated to reach up to 2000 K during the experiment, making graphene an efficient high-temperature micrometer-sized electron-transparent hot plate for future experiments in microscopes.

A discrete core-shell-like micro-light-emitting diode array grown on sapphire nano-membranes.

A discrete core-shell-like micro-light-emitting diode (micro-LED) array was grown on a 100 nm-thick sapphire nano-membrane array without harmful plasma etching for chip singulation. Due to proper design for the sapphire nano-membrane array, an array of multi-faceted micro-LEDs with size of 4 µm × 16 µm was grown. Threading dislocation density in the micro-LED formed on sapphire nano-membrane was reduced by 59.6% due to the sapphire nano-membranes, which serve as compliant substrates, compared to GaN formed on a planar substrate. Enhancements in internal quantum efficiency by 44% and 3.3 times higher photoluminescence intensity were also observed from it. Cathodoluminescence emission at 435 nm was measured from c-plane multiple quantum wells (MQWs), whereas negligible emissions were detected from semi-polar sidewall facets. A core-shell-like MQWs were formed on all facets, hopefully lowering concentration of non-radiative surface recombination centers and reducing leakage current paths. This study provides an attractive platform for micro-LEDs by using sapphire nano-membrane.

TRPCs: Influential Mediators in Skeletal Muscle.

Ca2+ itself or Ca2+-dependent signaling pathways play fundamental roles in various cellular processes from cell growth to death. The most representative example can be found in skeletal muscle cells where a well-timed and adequate supply of Ca2+ is required for coordinated Ca2+-dependent skeletal muscle functions, such as the interactions of contractile proteins during contraction. Intracellular Ca2+ movements between the cytosol and sarcoplasmic reticulum (SR) are strictly regulated to maintain the appropriate Ca2+ supply in skeletal muscle cells. Added to intracellular Ca2+ movements, the contribution of extracellular Ca2+ entry to skeletal muscle functions and its significance have been continuously studied since the early 1990s. Here, studies on the roles of channel proteins that mediate extracellular Ca2+ entry into skeletal muscle cells using skeletal myoblasts, myotubes, fibers, tissue, or skeletal muscle-originated cell lines are reviewed with special attention to the proposed functions of transient receptor potential canonical proteins (TRPCs) as store-operated Ca2+ entry (SOCE) channels under normal conditions and the potential abnormal properties of TRPCs in muscle diseases such as Duchenne muscular dystrophy (DMD).

Enhanced optical efficiency and color purity for organic light-emitting diodes by finely optimizing parameters of nanoscale low-refractive index grid.

To extract the confined waveguided light in organic light-emitting diodes (OLEDs), inserting a low refractive index (RI) periodic structure between the anode and organic layer has been widely investigated as a promising technology. However, the periodic-structure-based light extraction applied inside devices has been shown to severely distort spectrum and affect EL characteristics. In this study, a simple light extraction technology using periodic low-RI nanodot array (NDA) as internal light extraction layer has been demonstrated. The NDA was fabricated simply via laser interference lithography (LIL). The structural parameters of periodic pattern, distance, and height were easily controlled by the LIL process. From computational analysis using finite-difference time-domain (FDTD) method, the NDA with 300 nm pitch and 0.3 coverage ratio per unit cell with 60 nm height showed the highest enhancement with spectral-distortion-minimized characteristics. Through both computational and experimental systematic analysis on the structural parameters of low-RI NDA-embedded OLEDs, highly efficient OLEDs have been fabricated. Finally, as representative indicators, hexagonal and rectangular positioned NDA-embedded OLEDs showed highly improved external quantum efficiencies of 2.44 (+29.55%) and 2.77 (+57.38%), respectively. Furthermore, the disadvantage originating from the nanoscale surface roughness on the transparent conductive oxide was minimized.

A muscular hypotonia-associated STIM1 mutant at R429 induces abnormalities in intracellular Ca2+ movement and extracellular Ca2+ entry in skeletal muscle.

Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.

Probing Distinctive Electron Conduction in Multilayer Rhenium Disulfide.

Charge carrier transport in multilayer van der Waals (vdW) materials, which comprise multiple conducting layers, is well described using Thomas-Fermi charge screening (λTF ) and interlayer resistance (Rint ). When both effects occur in carrier transport, a channel centroid migrates along the c-axis according to a vertical electrostatic force, causing redistribution of the conduction centroid in a multilayer system, unlike a conventional bulk material. Thus far, numerous unique properties of vdW materials are discovered, but direct evidence for distinctive charge transport behavior in 2D layered materials is not demonstrated. Herein, the distinctive electron conduction features are reported in a multilayer rhenium disulfide (ReS2 ), which provides decoupled vdW interaction between adjacent layers and much high interlayer resistivity in comparison with other transition-metal dichalcogenides materials. The existence of two plateaus in its transconductance curve clearly reveals the relocation of conduction paths with respect to the top and bottom surfaces, which is rationalized by a theoretical resistor network model by accounting of λTF and Rint coupling. The effective tunneling distance probed via low-frequency noise spectroscopy further supports the shift of electron conduction channel along the thickness of ReS2 .

Soft-type trap-induced degradation of MoS2 field effect transistors.

The practical applicability of electronic devices is largely determined by the reliability of field effect transistors (FETs), necessitating constant searches for new and better-performing semiconductors. We investigated the stress-induced degradation of MoS2 multilayer FETs, revealing a steady decrease of drain current by 56% from the initial value after 30 min. The drain current recovers to the initial state when the transistor is completely turned off, indicating the roles of soft-traps in the apparent degradation. The noise current power spectrum follows the model of carrier number fluctuation-correlated mobility fluctuation (CNF-CMF) regardless of stress time. However, the reduction of the drain current was well fitted to the increase of the trap density based on the CNF-CMF model, attributing the presence of the soft-type traps of dielectric oxides to the degradation of the MoS2 FETs.

Disturbing Effects of Chronic Low-dose 4-Nonylphenol exposing on Gonadal Weight and Reproductive Outcome over One-generation.

4-Nonylphenol (NP) is a surfactant that is a well-known and widespread estrogenic endocrine disrupting chemical (EDC). Although it has been known that the affinity of NP to ERs is low, it has been suggested that low-dose NP has toxicity. In the present study, the endocrine disrupting effects on reproduction, and the weight of gonads, epididymis, and uterus were evaluated with the chronic lower-dose NP exposing. This study was designed by following the OECD test guideline 443 and subjected to a complete necropsy. In male, NP had an effect on the weight of the testis and epididymis in both F0 and F1. In females, NP decreased the weight of ovary and uterus in F0 but not in pre-pubertal F1 pubs. Fertility of male and female in F0 or F1 was no related with NP administration. The number of caudal-epididymal sperm by body weight (BW) was not different between groups in both F0 and F1. Besides, the difference of the sperm number between generations was not detected. The number of ovulated oocytes was similar between groups in F0, but significantly decreased in NP 50 group of F1. The litter size and sex ratios of offspring in F1 and F2 were not different. The accumulated mating rate and gestation period were not affected by the NP administration. Those results shows that chronic lower-dose NP administration has an effect of endocrine disruptor on the weight of gonads and epididymis of F0 and F1 but not in reproduction. Based on the results, it is suggested that chronic lower-dose NP exposing causes endocrine disruption in the weight of gonad and epididymis but not in the reproductive ability of next generations.

Synergistically Enhanced Electrochemical Performance of Hierarchical MoS2/TiNb2O7 Hetero-nanostructures as Anode Materials for Li-Ion Batteries.

As potential high-performance anodes for Li-ion batteries (LIBs), hierarchical heteronanostructures consisting of TiNb2O7 nanofibers and ultrathin MoS2 nanosheets (TNO@MS HRs) were synthesized by simple electrospinning/hydrothermal processes. With their growth mechanism revealed, the TNO@MS HRs exhibited an entangled structure both for their ionic and electronic conducting pathways, which enabled the synergetic combination of one- and two-dimensional structures to be realized. In the potential range of 0.001-3 V vs Li/Li+, the TNO@MS HR-based LIBs exhibited high capacities of 872 and 740 mAh g-1 after 42 and 200 cycles at a current density of 1 A g-1, respectively, and excellent rate performance of 611 mAh g-1 at 4 A g-1. We believe that the fabrication route of TNO@MS HRs will find visibility for the use of anode electrodes for high capacity LIBs at low cost.

Schottky nanocontact of one-dimensional semiconductor nanostructures probed by using conductive atomic force microscopy.

To develop the advanced electronic devices, the surface/interface of each component must be carefully considered. Here, we investigate the electrical properties of metal-semiconductor nanoscale junction using conductive atomic force microscopy (C-AFM). Single-crystalline CdS, CdSe, and ZnO one-dimensional nanostructures are synthesized via chemical vapor transport, and individual nanobelts (or nanowires) are used to fabricate nanojunction electrodes. The current-voltage (I -V) curves are obtained by placing a C-AFM metal (PtIr) tip as a movable contact on the nanobelt (or nanowire), and often exhibit a resistive switching behavior that is rationalized by the Schottky (high resistance state) and ohmic (low resistance state) contacts between the metal and semiconductor. We obtain the Schottky barrier height and the ideality factor through fitting analysis of the I-V curves. The present nanojunction devices exhibit a lower Schottky barrier height and a higher ideality factor than those of the bulk materials, which is consistent with the findings of previous works on nanostructures. It is shown that C-AFM is a powerful tool for characterization of the Schottky contact of conducting channels between semiconductor nanostructures and metal electrodes.

Surface Modulation of Graphene Field Effect Transistors on Periodic Trench Structure.

In this work, graphene field effect transistors (FETs) were fabricated on a trench structure made by carbonized poly(methylmethacrylate) to modify the graphene surface. The trench-structured devices showed different characteristics depending on the channel orientation and the pitch size of the trenches as well as channel area in the FETs. Periodic corrugations and barriers of suspended graphene on the trench structure were measured by atomic force microscopy and electrostatic force microscopy. Regular barriers of 160 mV were observed for the trench structure with graphene. To confirm the transfer mechanism in the FETs depending on the channel orientation, the ratio of experimental mobility (3.6-3.74) was extracted from the current-voltage characteristics using equivalent circuit simulation. It is shown that the number of barriers increases as the pitch size decreases because the number of corrugations increases from different trench pitches. The noise for the 140 nm pitch trench is 1 order of magnitude higher than that for the 200 nm pitch trench.

Exciton Recombination, Energy-, and Charge Transfer in Single- and Multilayer Quantum-Dot Films on Silver Plasmonic Resonators.

We examine exciton recombination, energy-, and charge transfer in multilayer CdS/ZnS quantum dots (QDs) on silver plasmonic resonators using photoluminescence (PL) and excitation spectroscopy along with kinetic modeling and simulations. The exciton dynamics including all the processes are strongly affected by the separation distance between QDs and silver resonators, excitation wavelength, and QD film thickness. For a direct contact or very small distance, interfacial charge transfer and tunneling dominate over intrinsic radiative recombination and exciton energy transfer to surface plasmons (SPs), resulting in PL suppression. With increasing distance, however, tunneling diminishes dramatically, while long-range exciton-SP coupling takes place much faster (>6.5 ns) than intrinsic recombination (~200 ns) causing considerable PL enhancement. The exciton-SP coupling strength shows a strong dependence on excitation wavelengths, suggesting the state-specific dynamics of excitons and the down-conversion of surface plasmons involved. The overlayers as well as the bottom monolayer of QD multilayers exhibit significant PL enhancement mainly through long-range exciton-SP coupling. The overall emission behaviors from single- and multilayer QD films on silver resonators are described quantitatively by a photophysical kinetic model and simulations. The present experimental and simulation results provide important and useful design rules for QD-based light harvesting applications using the exciton-surface plasmon coupling.