The features of their three‐dimensional structures comprise highly oriented [Ga7S12]3− and [K2PbX]3+ frameworks derived from AgGaS2, which display the largest phase‐matching SHG intensities (2.5−2.7 × AgGaS2 @1800 nm) among all SICs. Concurrently, three compounds manifest band gap values of 2.54, 2.49, and 2.41 eV (exceeding the criterion of 2.33 eV), which can avoid two‐photon absorption under the fundamental laser of 1064 nm, along with the relatively low anisotropy of thermal expansion coefficients, leading to improved laser‐induced damage thresholds (LIDTs) values of 2.3, 3.8, and 4.0 times that of AgGaS2. In addition, the density of states and SHG coefficient calculations demonstrate that the Pb2+ cations narrow the band gaps and benefit SHG responses.In contrast to anionic group theory of nonlinear optical (NLO) materials that second‐harmonic generation (SHG) responses mainly originate from anionic groups, structural regulation on the cationic groups of salt‐inclusion chalcogenides (SICs) is performed to make them also contribute to the NLO effects. Herein, the stereochemically active lone–electron‐pair Pb 2PbX][Ga7S12]  are successfully obtained, which display the highest phase‐matching nonlinear optical (NLO) responses in reported SICs and suitable band gaps exceeding 2.33 eV to avoid two‐photon absorption at 1064 nm.By introducing stereochemically active lone–electron‐pair cations in the cationic groups of salt‐inclusion chalcogenides (SICs), three compounds [K However, they suffer from inherent deficiencies, such as the relatively low laser‐induced damage thresholds (LIDTs) of AgGaQ2 or the unexpected multi‐phonon absorption of ZnGeP2, hindering their wide application in high‐power laser field. Therefore, an efficient route to explore IR NLO material with excellent comprehensive performance remains a challenge.Nonlinear optical (NLO) crystals enable the production of coherent lasers owing to their frequency conversion ability, serving as the core component of solid‐state lasers in modern optical science and technology.18 To  Alternately, salt‐inclusion chalcogenides (SICs) [AxXy][MmQn]  have been synthesized and characterized by us and other researchers. Such a compound contains covalent anionic groups that enable polarization ability for remarkable SHG responses as well as strong electropositive alkali  metals and electronegative halogens in cationic groups that are conducive to obtaining wide band gaps for enhancing LIDTs; examples include recently discovered [A3X][Ga3PS8] , [ABa2Cl][Ga4S8] , [ABa3Cl2][Ga5S10] , [K4Cl][CdGa9Q16] , [K3Cl][Mn2Ga6S12], and [NaBa4Cl][Ge3S10]. Furthermore, SICs exhibit structural features that allow the building blocks of the hosts and guests to be flexibly optimized, thereby providing infinite possibilities for NLO material design. As expected, the SIC [Ba4Cl2][HgGa4S10] with enhanced SHG intensities was derived from [Ba4Cl2][ZnGa4S10] by replacing Zn with Hg atoms in its anionic group. By comparison, for tailoring the structural regulation, the incorporation of an A‐free element into the cationic group of the SIC as an NLO material has yet to be reported.In general, the desirable characteristics for prominent IR NLO materials should be technically demanded, including large NLO coefficients, broad band gaps for high laser damage thresholds, broad transmitting spectra, moderate birefringence, and stable physical and chemical properties. Strong NLO coefficients and wide band gaps are two critically important but contradictory properties within an NLO material. From the chemical design point of view, “functional motif theory,” combining the NLO with LIDT functional motifs, has been proven to be an efficient strategy.26 Alt2+, Sn2+, Bi3+, Sb3+, and As3+, as a result of the “additive” effect of these types of polarization, has been intensively investigated in pursuit of high NLO responses. Among these cations, Pb2+ makes considerable contribution to SHG response but adversely affects the band gap. However, crystals with a band gap exceeding 2.33 eV can be efficiently pumped by fundamental 1064 nm laser sources because of the absence of two‐photon absorption and free‐carrier absorption. Therefore, skillfully obtaining a vital band gap wider than 2.33 eV while achieving a giant SHG response in IR NLO material design becomes crucial but challenging. These excellent predictions motivated us to investigate whether we might introduce A‐free active lone‐electron‐pair cations, such as Pb2+, into the cationic group of SICs to generate novel IR NLO materials with the expected strong SHG intensity and band gap. Hence, comprehensive research into the K−Ga−S−PbX2  system, the first lead‐containing SICs, [K2PbX][Ga7S12] , have been successfully designed and synthesized by adding lead element to the cationic groups. Intriguingly, compounds 1−3 have the largest SHG intensities among lead‐containing chalcogenides with band gaps greater than 2.33 eV. This host–guest system not only enhances SHG responses but also exceeds the crucial band gaps for improvement of the LIDT by involving stereochemically active lone–electron‐pair cations in SICs.Over the last decade, introducing stereochemically active lone–electron‐pair cations, such as Pbesponses.40 Amo21−3 were successfully grown in sealed silica tubes by high‐temperature solid‐state method. The powder X‐ray diffraction (XRD) patterns of the compounds are in good agreement with the simulated ones , Ga, and S and displays the molar ratio of each element well similar to chemical formula one  architecture composed of order‐accumulation [Ga7S12]3− host moieties and [K2PbI]3+ guest moieties. Regarding the [Ga7S12]3− polyanionic structure, the Ga atoms are four‐coordinated with the sulfur atoms to create the tetrahedral [GaS4]5− basic units with Ga—S bond lengths ranging from 2.2298(2) Å to 2.3657(1) Å S4, and two Ga(3)S4 are S‐vertex shared to enclose into an [Ga4S10]8− T2 supertetrahedron, and two supertetrahedra further polymerize into an [Ga7S16]11− double supertetrahedra  and I—Pb bond lengths (3.027 Å), which are comparable to those of K2ReI6, K2AgI3, Pb14O8I12, and Pb18O8Cl15I5. Each [IK3Pb] tetrahedral unit gives rise to the [K2PbI]3+ line by sharing K atoms. The [K2PbI]3+ guest moiety then extends in the channels formed by the [Ga7S12]3− network along the b‐axis direction  to compound 3 is depicted in Figure 2S7]8− dimers in AGS as “extension sites” are filled with GaS4 tetrahedra in compound 3. That is, [Ga2S7]8− is substituted by a [Ga7S16]11− unit. At the same time, the [K2PbI]3+ polycation group replaces Ag+ to build novel Pb‐containing SICs. The aforementioned evolution processes can provide additional favorable structural features. Primarily, the substitution of Ag+ with a [K2PbI]3+ unit breaks the all‐covalent structure of AGS and reduces the detrimental effects of photo‐sensitive Ag+ cations on the band gap and LIDT. According to the previously discovered SICs, such structures always exhibit the covalent host moiety and the ionic guest moiety. Pb in derivative compounds 1−3 does not occupy the covalent framework but rather locates the more extensive sites of cationic groups.Moreover, the structural evolution from AGS , [A3Cl][Ga3PS8]  (1.0−1.1 × AGS), [ABa2Cl][Ga4S8]  (0.9−1.0 × AGS), and [K3Cl][Mn2Ga6S12] (0.8 × AGS). In addition to microscopic GaS4 tetrahedrons, Pb2+ in the cationic groups plays vital roles in the SHG intensities of 1−3. The SHG efficiency order is reasonable because they are isostructural, and as the atomic weight of the halogen rises, so does the atomic contribution to the density of states close to the Fermi level, resulting in a larger SHG response. Consequently, the corresponding SHG coefficients deff [deff(sample) = (2I2ω(sample)/2ω2I(AGS))1/2)·deff(AGS)] of 1−3 are calculated to be 14.2, 14.7, and 15.1 pm V−1 with the benchmark AGS (deff = 11.6 pm V−1), respectively.The powder SHG signals of technique48 und1−3 were analyzed using single‐pulse method at 1064 nm, and AGS served as the standard. The LIDT results indicate that polycrystalline samples 1−3 possess comparatively large LIDTs of 2.5, 2.3, and 4.0 times that of AGS, respectively  and are sufficient to prevent the negative effect of two‐photon absorption under the fundamental laser of 1064 nm. In addition, a 2D map of the SHG response and band gap properties of the reported lead‐containing chalcogenides is illustrated  of phases 1−3. As shown in Table δ; defined as δ = max{(αi−αj)/αi}) values of 1, 2, and 3 are 1.65, 2.27, and 1.58, respectively, which are smaller than that of AGS (2.95). Thus, crystals with lower thermal expansion anisotropy values and exceeding vital band gaps are more likely to present higher LIDTs.For the development of high‐power laser systems, high laser‐induced damage is a critical parameter for an NLO crystal. The LIDTs of  1064 nm,50 andd Figure . Compoun1−3. As illustrated in Figure 1−3 are extremely similar: all three are indirect band gap semiconductors with valence band maximum at T point and conduction band minimum at X point. They possess the Perdew–Burke–Ernzerhof (PBE)‐calculated band gaps of 2.12, 2.10, and 2.13 eV for 1−3, respectively, which are acceptable because PBE methods underestimate the band gap. As depicted in Figure 7S12]3− anionic framework but also associated with the [K2PbX]3+ cationic groups because the optical properties are predominantly related to the electronic transitions around the forbidden band edge. This conclusion is quite different from previous findings that the SHG effects were primarily generated by the host moieties and the guest moieties contributed negligibly.The electronic band structures were theoretically calculated to understand further the relationship between the structures and optical properties of 1−3 belong to the point group of class mm2. Moreover, according to Kleinman's symmetry restriction, compounds 1−3 only have three non‐vanishing independent SHG tensors . As the energy‐dependent SHG coefficients of 1−3 are displayed in Figure d113, d223, and d333 are 20.2, −21.3, −7.5 pm V−1 for 1, 21.6, −23.1, −9.3 pm V−1 for 2, and 21.9, −26.2, −13.4 pm V−1 for 3, respectively, under the energy of 0.69 eV (1800 nm). As depicted in Figure deff values of 1−3 are 16.3, 17.7, and 19.7 pm V−1, respectively, at 1800 nm, which are slightly larger than the experimental results of 14.2, 14.7, and 15.1 pm V−1. Through orbital‐ and atom‐resolved SHG coefficient calculations, the atomic NLO efficiency contributions of 1−3 are demonstrated in Figure 1−3 contribute considerably to the largest tensors of d223, and the contributions of the K atom to the SHG effects are negligible. Among them, the SHG contribution of Pb atoms is comparable with that of Ga atoms. The results suggest that the [Ga7S12]3− host moieties dominate the macroscopic SHG efficiencies at 78%, 76%, and 75% for 1, 2, and 3, respectively, whereas the [K2PbX]3+ cationic group contribute more than 20% to the SHG responses due to the introduction of Pb2+ ions. The Pb‐6p state occupies the bottom of the conduction band to narrow the band gaps (still above 2.33 eV) but considerably contributes to SHG responses. These results align with those of the PDOS analysis.The NLO coefficients were calculated on the basis of the first‐principle calculation. The space groups of 32PbX][Ga7S12]  are successfully prepared via high‐temperature solid‐state methods. The 3D frameworks are composed of tetrahedral‐packing [Ga7S12]3− host and [K2PbX]3+ guest moieties derived from AGS. Remarkably, compounds 1−3 exhibit the highest phase‐matching SHG responses (2.5−2.7 × AgGaS2 @1800 nm) in reported SICs and suitable band gaps (2.41−2.54 eV) exceeding 2.33 eV to avoid two‐photon absorption at 1064 nm for high LIDTs (2.3−4.0 × AgGaS2) by introducing stereochemically active lone–electron‐pair Pb2+ in their structures. The theoretical calculations reveal that the origins of SHG effects are associated not only with the host moieties [Ga7S12]3− but also with the guest moieties [K2PbX]3+ (contribution rates > 20%); this finding differs from previous understandings of the guest moieties. Hence, this research provides a new paradigm for designing and developing novel IR NLO materials with pronounced comprehensive properties.In summary, by involving the Pb in polar salt‐inclusion chalcogenides for the first time, [K42PbX][Ga7S12] , the mixtures of K metal , Ga metal , S powder , and PbCl2/PbBr2/PbI2 powder  for 1/2/3, respectively, were weighed and carefully loaded into 11‐mm (inner diameter) quartz tubes. These tubes were flame‐sealed under a vacuum of 10−4 Torr and then placed in the furnace. These vessels were gradually heated at room temperature to 930 °C over 25 h, maintained within 96 h, and cooled to 330 °C at a rate of 3 °C h−1 before they reached ambient temperature naturally. The colors of crystals 1−3 are bright yellow, yellow, and orange, which are mechanically separated from the products that are washed with distilled water and ethanol. All three compounds had unchanged weight and color at ambient temperature for half a year which account for their physical and chemical stability. In addition, a Hitachi S‐3500 SEM spectrometer equipped with an EDS was used to carry out the EDS element analyses of 1−3.All reactants were stored and processed in a glove box filled with high‐purity Argon (99.999%). For synthesis of crystals .The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


As we approach the limits of semiconductor technology, the development of new materials and technologies for the new era in electronics is compelling. Among others, perovskite oxide hetero-structures are anticipated to be the best candidates. As in the case of semiconductors, the interface between two given materials can have, and often has, very different properties, compared to the corresponding bulk compounds. Perovskite oxides show spectacular interfacial properties due to the the rearrangement of charges, spins, orbitals and the lattice structure itself, at the interface. Lanthanum aluminate and Strontium titanate hetero-structures (LaAlO Nowadays, almost every modern electronic device, such as computers, music players, mobile phones, as well as opto-electronic devices , is equitructure ; and antnterface .In 2004, A. Ohtomo and H.Y. Hwang at Bell Laboratories discovered that, at the interface between LaAlOsequence . This disequence ,12,13,14uctivity  and ferructivity  to tip-iuctivity . In partuctivity ,19,20.The large variety of properties highlights the key role of the LaAlOcarriers ,22; growcarriers ; and thecarriers  at the iterature ,27,28,29The optical Second Harmonic Generation (SHG) process is essentially the generation of a light wave with the frequency ent wave ,31,32. TG signal . One canThe main objective of this review is an experimental in-depth analysis of the optical non-linearities at the LaAlOWe report here, for the sake of clarity the first part of the SHG theory developed in Ref. , and we Under very general circumstances, the dielectric polarization density, i.e., the dipole moment per unit volume ollowing:P→(t)∼ε0χEquation  that if To be useful, the SHG technique must establish a clear interpretive link between the measurable nterface . It shouThe intensity of the SHG signal is the actually measured quantity in SHG experiments. Depending on the particular experiment, we can change the parameter which drives the changes in the SHG signal, we record SHG Spectroscopy as a function of the incident photon energy z-axis perpendicular to it, in such a way that the incident plane is We now introduce a Cartesian reference system having the interface plane oriented along the p for a direction parallel to the plane of incidence  in E in E7) i in Ref. , togethez direction, and (iii) the effect of the symmetry breaking is not too large, i.e., it can be evaluated within the dipole approximation. These points imply that the parity is broken along z but not along x and y, so that the interfacial electronic states can be either even or odd by inversion of the x,y coordinates. Moreover, we recognize that the full When discussing the resonance structure of p to titanium 3d-The bulk optical gap of STO is known to be associated primarily with a transition from oxygen 2d states. The gap between the HOMO and LUMO states makes SrTiOIn its stoichiometric form, SrTiOThe experimental methods used to prepare the samples and to perform the SHG measurements are different for every work covered in this review. Nonetheless, in this section we will give a very brief overview on the most common and widely used techniques and we would like to remind the interested readers to the cited references for more details. Thin LaAlOn ranging from 0 to 12 mono-layers in the energy range p)→Ti(3d) band-gap transition in STO [p) and Ti(3d) orbitals involved in the local surroundings of the interface. Another important piece of information is obtained by the comparison of different SHG spectra as a function of n, where n is the number of LAO over-layers. The integrated SHG yield is small for n in STO ,39. Howen in STO ,41, this-authors  in the ser works . In orde of LAO) . In the eratures : the degSHG Spectroscopy has been the most important tool in order to address the problem of the microscopic origin of SHG in these materials and its relationship with the physical effect under investigation, i.e., the 2D insulator-to-metal phase transition. SHG indirectly senses the interfacial charge asymmetry. This asymmetry may be induced by a structural reorganization of the interface (in particular by Ti ions displacement) or by the presence of an electric field. In the second case, for instance, when a charged region is created close to the interface, the consequent interfacial electric field polarizes all the orbitals involved in the SHG emission and when the charge injection occurs, the SHG signal is enhanced. Therefore, the fact that the SHG signal jumps at s in STO . Regardln-type doping, TiOp-type conductive interface is predicted based on the polar catastrophe scenario, but the samples are found to be insulating for any LAO coverage and growth condition [The most important result of the SHG spectroscopy is summarized in ondition . SHG Spe In Ref. , a detaip-type doping in LAO/STO hetero-structures, and, in particular, all those based on charge carrier trapping.The almost complete suppression of the 3.8–4.0 eV peak in the a-LAO, panel a) and crystalline-LAO  upon heating (red datapoints) and cooling (blue datapoints). It is also worth noting that during the measurement, the temperature was held at 110 c-LAO structures where conductivity is recovered almost 100% after any number of thermal cycles, in the case of a-LAO the 2D electron gas is lost after the very first cycle, indicating an extrinsic origin of the doping charge, and, in particular, pointing to oxygen vacancies, for the simple reason that heating the sample in air has the same effect as filling the oxygen vacancies at the interface, which, in turn, can be formed by ion bombarding during PLD growth of the samples. The SHG results in c-LAO/STO sample, the behavior of the SHG signal as a function of temperature is qualitatively the same as that observed for the conductivity. The SHG signal is approximately constant during both the heating and cooling phases. After the cooling step, the SHG signal from the c-LAO/STO interface recovers approximately its initial value at room temperature. In the case of a-LAO/STO, a pronounced decrease in the SHG signal is observed, analogous to the conductivity. However, SHG also exhibits some significant features which are not visible otherwise. The decrease in the SHG signal already begins at about 110 a- and c-LAO/STO interfaces, oxygen vacancies in the former case and electron reconstruction driven by interfacial polar discontinuity in the second case. The question then arises naturally, why is a similar SHG signal observed for all the c- and a-LAO/STO hetero-structures?As opposed to the case of different terminations where, according to the polar catastrophe model, the SrO-terminated interface is expected to be conductive and is in fact insulating, there is the case of amorphous-LAO structures in which no polar build-up can be postulated since the polar LAO layer is grown on the STO substrate in an amorphous phase and yet electric conduction at the interface is observed. Additionally, in this case, SHG was found to be capable of helping to address the apparently inconsistent observation . Figure z coordinate. The variation of a-LAO/STO and c-LAO/STO interfaces, with an average of the polarization combinations. All crystalline samples above the conductance threshold have an interfacial a-LAO/STO interface, which could help explain this similarity, is based on the hypothesis that a a-LAO/STO interface. The band diagram of a-LAO/STO would, therefore, be determined by the combination of two basic elements, i.e., the typical “dip” of the bands formed in the vicinity of a c-LAO/STO case, as shown in As already mentioned, SHG indirectly probes the charges and their spatial distribution at the interface. When a space-charge region is created at the interface, this develops an electric field, Equation , where oEquation . By assuAnother interesting case of non-standard LAO/STO hetero-structure is the one with unbalanced chemical composition. In particular, Ref.  shows th In Ref. , SHG hasAnother example of the SHG technique applied to non-standard LAO/STO hetero-structures is provided by interfaces with different in-plane orientations, the LAO/STO (110) interface is an interface formed by the deposition of LAO atomic layers on oriented STO substrates (110). According to the polar catastrophe scenario, the LAO/STO interface (110) is formed by a sequence of  studies  show tha studies  as the i studies  is indeeThe possible presence of local inhomogeneities in the electronic structure of the interface was proposed ,51,52,53magnetic , and chemagnetic  phase semagnetic , the SHG In Ref. , SHG Ima in Ref.  and refet indicates the time-delay between the pump and the probe pulses, the bar indicates the spatial averaging over the z coordinate. The time-dependence is fully included in the second term of the sum, whose components, according to Equation  . Figure terature , it was In this work, we have reviewed more than 10 years of experiments applying SHG to the investigation of oxide hetero-structures with a particular focus on the textbook LAO/STO interface. The results clearly show that SHG is capable of providing complementary information on these material systems compared to the standard tools that are widely used in material science. The advantages of SHG compared to the latter are many-fold: (i) SHG can reach vertical spatial resolution on the subnanometric scale; (ii) it is a contact-free technique; (iii) it can directly sense interfacial polarity; (iv) it can be used in situ and real-time for monitoring the hetero-structure growth without disturbing the particle trajectories; and (v) it can reach temporal resolution on the femtosecond time-scale.Let us summarize here what we believe are the main results in the field. SHG spectroscopy revealed a reorganization of the electronic structure at the interface at On the other hand, it can be difficult to directly link the SHG signal to the microscopic properties of the interface under study, so that often the interpretation of the SHG results remains confined to a qualitative level. Enhancing the diagnostic and predictive capacities of SHG would require a major effort on the microscopic theoretical modeling of SHG, thus allowing it to compete with other powerful tools in material science, as photo-emission spectroscopy or scanning tunnel microscopy. Notwithstanding these limitations, some of the results here summarized have proved that SHG is able to provide valuable information on the physics of these interfaces that cannot be easily obtained with other techniques. The main strength of SHG is its capacity of sensing all the charges injected at the interface, both localized or mobile. In the case of the STO-based interfaces it is clear that SHG is more sensitive to the first kind of charges, thus allowing to reveal a phenomenon that acts as a precursor for the onset of conductivity. This precursory interfacial reconstruction, due to charges that are injected but localized at the interface, cannot be of course highlighted by the electrical measurements routinely used to characterize these interfaces. This is even more true if we consider LAO/STO interfaces with different terminations of the (100) STO substrate. In this case, there are few techniques that may provide a significant comparison between these two interfaces, and SHG is one of the simplest. The results obtained by SHG spectroscopy in this particular case are simply striking. First, they show that SHG is able to provide useful information on the level of a single atomic layer. Second, they prove that, at the SrO terminated LAO/STO interface, charges are not injected, an important piece of information, not possible to obtain with other techniques.Moreover, SHG can directly measure the interfacial polarity and even the interfacial electric field that builds-up on charge injection. In this respect, SHG spectroscopy has unequivocally demonstrated that SrTiOIn addition, SHG spectroscopy as a function of temperature has shown that the polar asymmetry induced by the build-up of the polar electric-field is always accompanied by a structural reorganization of the interface due to the displacement of Ti ions that are very much prone to shift from their equilibrium position because of the paraelectric nature of SrTiOIn conclusion, this review article has provided several evidences to convince the readers that Second Harmonic Generation may be an invaluable experimental tool for investigating buried interfaces (LAO/STO and other oxide hetero-structures) which share common chemi-physical characteristics. In this conclusive section we have summarized the major results that, in our opinion, have been obtained by means of SHG and which make this technique unique in comparison with other standard tools used in this field. Along with the review, we have delivered several examples highlighting the richness of information that SHG can gather, with the aim of providing a good starting point for further developments in this fruitful research field.


Skeletal muscle releases numerous hormones into circulation that interact with other organs, such as the liver, bone or the brain. These hormones, termed myokines, mediate the effects of physical activity in health, aging and disease. Interleukin 6 was the first discovered myokine and is actively investigated due to its participation in inflammation, immunity and metabolism. However, there is little information regarding the mechanisms that induce its release from muscle cells, especially in humans. Our aim was to investigate whether changes in the concentration of calcium ions participate in the stimulated release of interleukin 6 in human muscle cells. Using muscle cultures, we have found that several proteins responsible for the calcium increase during stimulation induce the release of interleukin 6 from the muscle cells. This could help to unveil how interleukin 6 and other myokines are released in pathological conditions such as trauma, infections or cancer.2+ signals. The aim of this study was to characterize the role of the main components of Ca2+ signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca2+ mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca2+ signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca2+ channels blocker La3+ reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type , T-type (NNC55-0396) and Orai1 (Synta66) Ca2+ channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors  and IP3 receptors . Inhibitors of calmodulin  and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca2+ signals, including the mobilization of calcium stores, the activation of store-operated Ca2+ entry, and the subsequent activation of voltage-operated Ca2+ channels and calmodulin/calcineurin pathways.The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca The report of IL-6 release by skeletal muscles during contraction to an extent enough to increase its circulating levels  opened tIL-6 secretion has been shown to be released directly from muscular cells in cell culture experiments but also in in vivo experiments, where IL-6 has been shown to be released from muscle tissue in response to contraction , mechanical stress  and puriConcerning the intracellular pathways involved in IL-6 secretion, reports in human and rodent models have identified the involvement of several transduction pathways, such as Stat3 , JNK/MAP2+ signals in IL-6 secretion. Current evidence indicates that both IP3R [2+ stores are involved. Extracellular Ca2+ ions [2+ signals [2+ influx pathways have not been studied. The participation of Ca2+ independently of signalling mechanisms linked to receptors is also supported by the finding that calmodulin [A few groups have also reported the participation of some elements of the Caoth IP3R  and RyR oth IP3R  Ca2+ stoa2+ ions  and cyto signals ,25 seem lmodulin  in mouselmodulin  and rodelmodulin  can medi2+ signal in the IL-6 release in human skeletal cells in culture.Given that the available information on this aspect is fragmentary and almost inexistent for human muscle, the objective of this study was to fully characterize the role of the main components of the Cahttps://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/, accesed on 31 March 2023), revised in 2013. Human primary cultures were prepared from small fragments of rectus abdomini muscle discarded during laparotomy, collected in a sterile Krebs–Henseleit solution (KH), and cultured on the same day following previously reported methods [The study was approved by the Ethical Committees of the University of Extremadura and Hospital Universitario de Extremadura  (ref IB18025) and followed the rules of the Declaration of Helsinki of 1975 ( methods . The stu2). The medium was replaced every two days, and cells were replated when approaching confluence  and frozen in liquid nitrogen for experiments at passages 3–5.The digestion of the sample was based on previous reports . Briefly2 in a growth medium, and at 80–90% confluence, the medium was changed to differentiation medium and replaced every other day. Under these conditions, the proliferating myoblasts fuse into multinuclear myotubes, the in vitro precedent of adult muscle fibres.For experiments, cells were seeded in 24-well culture plates coated with collagen at 6000–7000 cells/cmThe growth medium composition was DMEM:F12 (Hepes and L-glutamine), 20% fetal bovine serum, insulin (10 µg/mL), bFGF (1 ng/mL), EGF (10 ng/mL), dexamethasone (0.4 µg/mL) and antibiotics . The differentiation medium was similar but fetal bovine serum was replaced by 2% horse serum, and bFGF, FGF and dexamethasone were omitted.For secretion experiments replicating myoblasts were differentiated during 6–8 days. On the day of the experiment, the cells were washed three times with PBS and placed in 0.5 mL serum-free culture medium (DMEM:F12) to collect the IL-6 secretion. The medium contained no further addition, the stimulus, the appropriated inhibitor or a combination of both. In each culture plate, the secretion was collected under basal (unstimulated) and stimulated conditions both in the absence and presence of the inhibitors assayed. Inhibitors were applied for 15 min before the application of the stimulus (20 min in the case of BAPTA-AM). After a subsequent 2 h incubation, the supernatant was collected, centrifuged and frozen for analysis. To normalize the secretion to the cellular population, nuclei were marked with Hoechst 33342, 30 images/well were captured (EVOS-Fl2 automated microscope) and automatically analysed with a homemade water-shedding macro using Fiji-2 .IL-6 was assayed using MabTech IL-6 ELISA kit (ref. 3460-1HP) following the manufacturer’s instructions.2, 1.1 MgCl2, 2.2 NaHCO3, 0.4 NaHPO4 and 5.6 D-glucose, pH 7.3). The coverslip was placed in an experimental chamber mounted on the stage of an inverted microscope  to excite the cells at 340 and 380 nm using a computer-controlled monochromator  at 1 Hz, and the emitted fluorescence images were captured with a CCD camera  and recorded using dedicated software . The ratio of fluorescence at 340 nm to 380 nm (F340/F380) was calculated pixel by pixel as an index of cytosolic Ca2+ concentration. Once the experimental chamber was placed in the microscope stage, the cells were kept under a constant flow of Tyrode solution, and stimulation was achieved by switching to a solution containing the desired concentration of the stimuli (500 µM ATPγS). When used, inhibitors were applied in the same Tyrode solution for 15 min previous to stimulation. When necessary, a low Ca2+ Tyrode was used, substituting 1 mM EGTA for 1.8 CaCl2 (0Ca2+ Tyrode). To study changes in the Ca2+ signals, the peak response was measured as a delta increase in the ratio divided by the previous resting ratio (DF/F0). The integrated or overall response was measured as the area under the curve of the response trace for 180 s after the onset of the Ca2+ transient after subtraction of the previous resting value.Skeletal myotubes were cultured on collagen-coated glass coverslips (0.17 mm thick) following the procedure described above. For experiments, the coverslips were placed in a serum-free culture media containing 4 µM fura 2-AM at room temperature for 45 min, followed by a 20 min period in a physiological Tyrode solution , proteins were resolved using 10% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes for subsequent probing. After blocking residual binding sites , Orai1 was detected through 1 h incubation with anti-Orai1 antibody ) followed by washing  and incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase . For normalization, a primary anti-β-actin antibody was used . Chemiluminescence was assessed with a ChemidDoc Imaging System , and the density of the bands were measured using ImageJ 2.0 software. Data were normalized within each membrane to β-actin.−ΔΔCT method, using the basal, non-stimulated samples as calibrator [After RNA extraction with EZNA total RNA kit II  and DNA transcription , the expression of IL-6 gene and GAPDH as constitutive gene for reference were determined using RT-PCR using Taqman probes with accession numbers Hs00174131_m1 for IL-6 and Hs02758991_g1 for GAPDH . Expression levels were normalized to GAPDH levels following the 2librator .DMEM:F12 was obtained from Corning, and fetal bovine serum, horse serum, antibiotics, TNFα, human EGF and human bFGF was obtained from GIBCO. Nifedipine, methoxyverapamil hydrochloride (D-600), lanthanum chloride, Bapta-AM, caffeine, dantrolene, Stattic, Synta66, EGTA, BSA, cycloheximide and collagenase were obtained from Sigma-Merck; papain was obtained from Worthington Biochemicals. Dexamethasone, 2-APB, 5′-N-Ethylcarboxamidoadenosine (NECA), ATPγS, NNC55-0396, 78c (CD38 inhibitor), xestospongin C, FK506 and ryanodine were purchased from Tocris. Insulin and 8BrcADPr were obtained from Santa Cruz. Fura-2 AM was obtained from Invitrogen. All the lipophilic drugs were dissolved in dimethyl sulfoxide (ethanol in the case of xestospongin C). The final concentration of the solvent was ≤0.1%.5 nuclei. Data are given or represented as average ± standard error of the mean (sem). Comparisons were performed using a Student t-test (paired when appropriated) or ANOVA followed by planned multiple comparisons, as appropriate. When necessary , the data were transformed (log10 or square root), or a non-parametric test was used.IL-6 secretion in response to ATPγS or other stimuli was normalized with respect to unstimulated secretion from the same culture plate, either in the absence or in the presence of an inhibitor, as appropriate. Raw IL-6 secretion is expressed as pg/mL × 102+ signalling in IL-6 release, we focused on the response to ATP because it activates Ca2+ signals in skeletal muscle cultures and has a key role in responses to membrane depolarization and in the differentiation of muscle cells [Human muscle cultures were differentiated for 1 week and released IL-6 into the culture supernatant in response to several stimuli. le cells ,37.2+ signals in many cell types involve an initial and transient increase due to Ca2+ release from internal stores followed by a sustained phase of lower magnitude associated with the entry of extracellular Ca2+. These components can be selectively studied using established manipulations. A classic approach to block calcium entry is the use of media with low Ca2+ concentration  or with submillimolar concentration of La3+, a generic blocker of calcium channels. 2+ signals in response to ATP in control and 0 Ca2+ conditions. Blockade of calcium entry inhibited the sustained phase of the Ca2+ signal but did not modify the initial peak response, reproducing the expected behaviour of calcium-store-based Ca2+ signals in the absence of Ca2+ entry. Therefore, we compared the control of IL-6 response to ATP with the responses both in the absence of extracellular Ca2+ and in the presence of La3+. As shown in p < 0.01), indicating that Ca2+ influx during the stimulation is necessary for IL-6.Intracellular Ca2+ signals can also be blocked or strongly reduced by introducing calcium chelators, such as EGTA or BAPTA, into the cells (loaded as acetoxymethyl esters subsequently cleaved by cellular esterases). This can be seen in the cytosolic Ca2+ signals of p < 0.009), suggesting that secretion requires increases in cytosolic Ca2+. In this series of five experiments using BAPTA-loaded cells, the response to ATP increased IL-6 from 6.47 ± 1.24 (resting) to 11.71 ± 1.67 pg/mL × 105 nuclei, a response statistically smaller  than the matched control response, which was from 8.84 ± 1.28 to 72.04 ± 12.37. The finding that the fast Ca2+ buffer BAPTA is more effective in preventing ATP-induced IL-6 secretion than the blockade of Ca2+ influx strongly suggests that Ca2+ release from intracellular stores plays a relevant role in this process.Ca2+ influx could be due not to the lack of interference with Ca2+ signals but only to the unavailability of Ca2+ ions in the secretory process of IL-6. If this is true, it is likely that the non-stimulated or basal secretion is equally influenced. However, in our experimental conditions, none of the previous procedures inhibited the basal secretion of IL-6. 2+ or La3+ altered the resting secretion . In addition, 2+ signalling in skeletal muscle cells [2+ signals in this model.The effects observed in BAPTA-loaded cells and after the inhibition of Cale cells , but not2+ in terms of IL-6 release, we studied the effect of a moderate level of calcium protonophore ionomycin (0.5 µM), a classic method to increase intracellular Ca2+. 2+ signals are involved in the stimulation of IL-6 secretion and that Ca2+ ions are not just a required or permissive factor for the release of this cytokine.To further test the role of Ca2+ into muscle cells. To assess the participation of voltage-operated Ca2+ channels (VOCC) in IL-6 secretion, we used selective inhibitors for the main types present in these cells: L- and T-type VOCCs (Cav1.x and Cav3.x) [2+ signal elicited by ATP. As can be seen, the inhibition of the L-type channel mainly inhibits the sustained phase of the signal and, to a lesser extent, the initial peak response. This is consistent with the well-known function of L channels, not only as a Ca2+ influx route but as activators of the release from intracellular stores via ryanodine receptor channels.There are multiple channels mediating the entry of Ca Cav3.x) . Figure p < 0.005), similar to the effect of NNC55-0396, indicating that Ca2+ entry through L-and T-type channels contributes to IL-6 release. Note that in the case of the T channel, there was an almost total suppression of the response, indicating that function of this channel is absolutely required for IL-6 secretion.2+ channels, which are opened due to the depletion of internal Ca2+ stores, using the selective inhibitor Synta66 [2+ signal evoked by ATP in matched controls, Synta66-treated cells and shOrai silenced cells. The inhibition of capacitative Ca2+ entry by any of the two treatments clearly impaired the sustained response. The initial peak response was slightly (non-significantly) reduced in Synta66-treated cells but was clearly inhibited in shOrai1 transfected cells (p < 0.005).We also evaluated the participation of Orai1 Ca Synta66  and gene2+ channels and confirms the clear dependence on T channels.In an additional series of experiments shown in B, we det2+ signal, it is likely that ATP-induced IL-6 secretion has a component dependent on Ca2+ release from the intracellular stores. The role of this component has been dissected using inhibitors of ryanodine and IP3 receptors (RyR and IP3R). To inhibit RyR, we used supra-micromolar concentrations of ryanodine and dantrolene, known blockers of the RyR receptor [2+ signal, as expected, for reduced release from intracellular stores. The IL-6 response to ATP was also inhibited by these compounds, as expected if Ca2+ release through RyR was involved in the stimulated secretion. To evaluate if cADPr, the endogenous agonist of the RyR, participates in IL-6 secretion, we treated cultures with an antagonist of this molecule, 8Br-cADPr, and with 78c, an inhibitor of ADP-ribosyl cyclase, the enzyme responsible for its synthesis. Both treatments induced a slight and statistically not significant reduction . Given that calmodulin inhibition impairs the IL-6 response to ATP, we checked whether the inhibition of calcineurin and calmodulin-activated kinase II (CaMKII) also impaired the response. Treatment with the calcineurin inhibitor FK506 resulted in a reduction of ATP-evoked IL-6 release as compared to control cells (p < 0.05). To rule out the possibility that the effect of FK506 was not mediated through calcineurin inhibition but by binding to FKBP proteins, known regulators of RyR, we used rapamycin, an FKBP-binding drug without effect on calcineurin, and found that it has no effect on IL-6 secretion. In the case of CaMKII, the use of a maximal concentration of its specific inhibitor KN93 only induced a slight reduction in the limit of significance compared to untreated cultures or to the inactive analogue of KN93, KN92 (Once established that the secretory response involves Ca93, KN92 C. This i2+ signals evoked by ATP operate at the transcriptional or post-transcriptional level. To test this question, we determined the changes in IL-6 mRNA levels in response to ATP. 2+ chelator BAPTA. In fact, IL-6 transcription was even enhanced in BAPTA-loaded cultures. This result indicates that the role of Ca2+ signals in the IL-6 secretion takes place at the post-transcriptional level. We also tested the effect of the T channel blocker NNC 35-0396 on the IL-6 expression. Similar to BAPTA loading, this treatment was without effect, as expected if T-type calcium channels operate only at the exocytosis process.Previous authors have shown that ATP stimulates IL-6 transcription in rodent muscle . It is p2+ signals are fragmentary, and most of them have been conducted in rodent models. To our knowledge, there are only a few reports in human skeletal muscle, including IL-6 release in response to Ca2+ ionophore [2+ influx via VOCC and Orai1, as well as Ca2+ release from RyR/IP3R-expressing Ca2+ compartments, mediate IL-6 release in response to ATP, a key cellular agonist in the developing skeletal muscle [2+ channels are considered the voltage sensor element in the molecular mechanism coupling excitation of the sarcolemma to contraction [2+ entry [2+ release from internal stores by interacting with RyR receptors of the sarcoplasmic reticulum [Although there is extensive literature on the release of IL-6 and other myokines from skeletal muscle, the scarcity of our knowledge regarding the cellular mechanisms driving this endocrine secretion is surprising, especially considering the pathophysiological role of IL-6. Previous reports on the role of Caonophore , RyR actonophore  and depoonophore . Our worl muscle . The L-ttraction  and to C2+ entry . Cav1.x eticulum . This coeticulum . Moreoveeticulum ,51.2+ release from RyR. To explain the recruitment of RyR in the IL-6 secretion, a likely explanation would be a direct molecular interaction assembling L-channels and RyR during ATP stimulation, given the evidence described above.In keeping with the above precedents, the present data indicate that both L channels and RyR receptors play a role in the stimulated release of IL-6. Although the study was not designed to investigate the regulation of the mechanisms, it is somehow surprising that treatment with antagonists for the cADPr route is without effect on IL-6 secretion because this messenger activates Ca2+ ions, as shown by the effects of low Ca2+ medium, the non-specific blocker La3+, and the inhibitors of VOCC and Orai1 channels. Although it has been claimed that the entry of Ca2+ ions during excitation–contraction coupling in mouse adult muscle is vestigial [2+ entry during activation [2+ signals driven by the entry of extracellular Ca2+ and by the release from RyR and IP3R Ca2+ stores. In muscle cells, ATP mobilizes intracellular calcium stores [2+ channels is firmly established [3R-operated Ca2+ stores, Ca2+ influx via Orai channels might depolarize plasma membranes and trigger VOCC both directly and indirectly via the activation of cationic TRPC channels, which in turn would enhance depolarization [3R blocker 2APB. In addition, it is compatible with the lack of additive effect of inhibitors of IP3R and RyR observed in our experiments (The importance of VOCC channels for IL-6 secretion implies that depolarization is likely also involved. Our results prove that the secretion of IL-6 in skeletal muscle cultures relies on the entry of external Caestigial , there itivation . As a whm stores , and theablished . Though rization ,56. Thiseriments .2+ channels achieved the deepest inhibition while showing no contribution to the whole-cell Ca2+ signal induced by ATP (2+ channels contribute to neurotransmitter release in neurones and chromaffin cells [A remarkable finding is the fact that a specific blocker of the T-type Cad by ATP . This red by ATP . It is kin cells . Interesin cells ,59, and in cells ,61,62.To date, most of the studies on the signal transduction for IL-6 secretion have been performed in rodent models and involve a number of intracellular signalling pathways as mediators for IL-6 release, such as Stat3 , JNK/MAP2+ signals operate at the post-transcriptional level, in contrast to a previous report in rodent muscle [2+ signals or in the positive feedback loop system reported by these authors. They proposed that the Ca2+ signal in response to ATP activates an early IL-6 transcription, which leads to further IL-6 secretion via the autocrine effect of released IL-6. In our experimental system, the initial IL-6 could be rather due to the activation of translation/exocytosis steps. This is in keeping with the strong effect of the specific T-channel inhibitor and the clear inhibitory effect of the translation blocker cycloheximide in IL-6 secretion [Our data indicate that Cat muscle . The ratecretion  (and als2+ signals in IL-6 secretion are fragmentary and, in the case of human cells, almost inexistent. The evidence presented here describes for the first time in a comprehensive way the participation of the main elements of the Ca2+ signalling pathway in the stimulated IL-6 secretion from skeletal muscle cells. In addition to its previously reported participation in contraction, transcription and differentiation, this signal pathway is likely a physiological regulator of IL-6 secretion. Given the obvious importance of this myokine in a broad group of metabolic, immunological and pathophysiological processes, a deeper understanding of this topic deserves further investigation.To our knowledge, previous reports on the participation of Ca2+ signals operating at the post-transcriptional level and involving both Ca2+ entry from the extracellular milieu and release from intracellular stores and the activity of downstream calmodulin/calcineurin pathway. Our data suggest that Ca2+ release from internal stores activates store-operated Orai1 Ca2+ channels, which in turn triggers the opening of voltage-operated Ca2+ channels.The present study shows that in cultured human skeletal muscle, the IL-6 secretion in response to ATP requires Ca2+ signal elements.The conclusions of the study are limited to the stimulation with purinergic agonist ATP. Although physiological muscle contraction is due to depolarization and releases extracellular ATP, it is likely associated with other mechanisms leading to IL-6 release. In addition, the study does not take into account the temporal kinetics of the secretion, which could show a differential contribution of the Ca


In this study, we propose a highly sensitive and selective biosensor platform for Ca2+ detection, which comprises a dual-gate (DG) field-effect transistor (FET) with a high-k engineered gate dielectric, silicon nanowire (SiNW) random network channel, and Ca2+-selective extended gate. The SiNW channel device, which was fabricated via the template transfer method, exhibits superior Ca2+ sensing characteristics compared to conventional film channel devices. An exceptionally high Ca2+ sensitivity of 208.25 mV/dec was achieved through the self-amplification of capacitively coupled DG operation and an enhanced amplification ratio resulting from the high surface-to-volume ratio of the SiNW channel. The SiNW channel device demonstrated stable and reliable sensing characteristics, as evidenced by minimal hysteresis and drift effects, with the hysteresis voltage and drift rate measuring less than 6.53% of the Ca2+ sensitivity. Furthermore, the Ca2+-selective characteristics of the biosensor platform were elucidated through experiments with pH buffer, NaCl, and KCl solutions, wherein the sensitivities of the interfering ions were below 7.82% compared to the Ca2+ sensitivity. The proposed Ca2+-selective biosensor platform exhibits exceptional performance and holds great potential in various biosensing fields.Calcium ions (Ca These ions are responsible for performing various biological functions, such as blood clotting, intercellular adhesion, skeletal integrity maintenance, and cell mobility facilitation. Maintaining appropriate Ca2+ levels within the body is critical for sustaining optimal biological health borate , tetrahydrofuran , sodium chloride , calcium chloride , and potassium chloride . All the materials were used without any further purification.The following materials were used in this study: SiO2, NaCl, and KCl solutions by dissolving CaCl2, NaCl, and KCl, respectively, in DI water. Through sequential dilution, we prepared solutions with concentrations of 100 M, 10−1 M, 10−2 M, 10−3 M, and 10−4 M for each ion.We prepared CaCl2 layer, which was patterned using a lift-off process. Subsequently, PVP nanofibers were deposited as a pattern template via an electrospinning process utilizing a PVP precursor solution under a controlled humidity of 25% and room temperature (25 °C). The PVP precursor solution was prepared by dissolving 200 mg of PVP in 3 mL of ethanol, followed by stirring at 800 RPM for 4 h at room temperature. A SiNW random network channel was obtained using a template transfer method employing electrospun PVP nanofibers. This method offers a straightforward approach for creating a SiNW random network structure through electrospinning and RIE. The formation process of the SiNW channel commenced with the preparation of a silicon-on-insulator (SOI) substrate with defined active regions and n-doped source/drain (S/D) electrodes, as described in 6 plasma ambient. Any residual PVP nanofibers were removed via wet etching using a sulfuric acid–hydrogen peroxide mixture. Finally, the shielding oxide layers were removed using a 30:1 buffered oxide etchant (BOE). After the electrospinning process, conventional thermal annealing (CTA) was performed in a furnace system at 300 °C, which is the melting point of PVP nanofibers. This step was performed to solidify the patterned template and enhance its adhesion to the Si film. The pattern template of the PVP nanofibers was then transferred onto the underlying Si channel layer through RIE in an SF2 p-type (100) SOI substrate with a 100 nm thick top silicon layer and 200 nm thick buried oxide (BOX) layer was prepared. The resistivity and boron doping concentration of the top silicon layer were 1–10 Ω·cm and 1 × 1015 cm−3, respectively. To eliminate surface impurities and contaminants, the substrate was cleaned via a standard Radio Corporation of America (RCA) cleaning process. Active regions with a channel layer width/length of 130/90 μm were formed using photolithography and RIE. A 200 nm thick SiO2 layer was blanket-deposited using RF magnetron sputtering as a dummy oxide for the phosphorus doping process. The source and drain (S/D) areas were patterned using photolithography, followed by the use of 30:1 BOE to etch the dummy oxide on the S/D area. For n+ doping of the S/D regions, a PSG film was spin-coated and thermally diffused using a rapid thermal annealing (RTA) process at 950 °C for 30 s in an O2/N2 ambient. The residual PSG and dummy oxide layers were removed using 30:1 BOE. Next, the SiNW channel formation process was performed, as described in 2 layer and an 80 nm thick Ta2O5 layer were deposited as high-k engineered top-gate oxides using RF magnetron sputtering and a lift-off process. A top-gate electrode of 150 nm thick Al was formed using an electron-beam evaporator and the lift-off process. Simultaneously, a film channel DG FET was fabricated as a reference device without SiNW channel formation. To enhance the overall electrical properties of the fabricated devices, a forming gas annealing (FGA) process was performed at 450 °C for 30 min in a 2% H2/N2 atmosphere in a furnace. A 1 × 1 cm2 was deposited as a sensing membrane. The SnO2 sensing membrane transfers the surface potential of the analyte solution to the transducer unit via the ITO conductive layer. The ITO and SnO2 layers were deposited using an RF magnetron sputtering system. To form a Ca2+-selective membrane, a Ca2+-selective cocktail (100 μL) was drop-casted onto the SnO2 sensing layer and allowed to dry in ambient air at room temperature for 24 h to evaporate the solvent and enhance adhesion. The Ca2+-selective cocktail was prepared by dissolving 1.2 mg of Ca ionophore IV, 0.3 mg of Na-TFPB, 32.5 mg of PVC, and 66 mg of 2-nitrophenyl octyl ether in 660 μL of THF. The mixture was then stirred at 800 RPM for 6 h at room temperature. Finally, a sensing region with a diameter of 0.6 cm was defined by attaching a PDMS reservoir to the center of the Ca2+-selective membrane. The process flow of the Ca2+-selective EG sensing unit is illustrated in The EG sensing unit was fabricated on a glass substrate (1.5 cm × 2.5 cm). A 300 nm thick ITO layer was deposited as the conductive layer, which was electrically connected to the top-gate electrode of the transducer through an electrical cable. Subsequently, a 50 nm thick layer of SnO2, Ta2O5, SnO2, ITO, and drop-casted Ca2+-selective membranes were measured using a Dektak XT Bruker stylus profiler . The electrical characteristics of the devices were measured using an Agilent 4156 B precision semiconductor parameter analyzer . A commercial Ag/AgCl electrode  was used as the reference electrode for pH and Ca2+-selective sensor platforms. To minimize external interference, all the electrical measurements were performed in an electromagnetically shielded dark box. k gate dielectric engineered SiNW and film channel DG FET, respectively. The thickness of the Ca2+-selective membrane was approximately 5.3 μm, as depicted in The thicknesses of Si, Al, SiOk gate dielectric engineered SiNW channel DG FETs. The operation of the fabricated FET transducer units enabled the sensing functions of the entire sensor platform. Therefore, prior to evaluating the sensing performance, the electrical characteristics of the fabricated SiNW and film channel DG FETs were compared. DS-VG) curves for the top-gate operation of the SiNW and film channel DG FETs are presented in DS-VD) curves. Transfer characteristic curves were obtained at a drain voltage (VD) of 1 V. While sweeping either the top- or bottom-gate voltage, the other gate electrode was connected to the ground electrode. To quantitatively compare the measured electrical characteristics of the SiNW channel DG FETs with those of film channel devices, we extracted various electrical parameters, as summarized in TH) and on/off current ratio (ION/OFF) were similar, the SiNW channel DG FETs exhibited better field-effect mobility (μFE) and subthreshold swing (SS) values compared to those of the film channel device. Overall, a comparison of electrical characteristics indicates that the SiNW channel DG FETs possess favorable properties for sensor applications, demonstrating improved field-effect mobility and subthreshold swing values.We fabricated FET-type sensors based on high-k gate dielectric engineered DG FETs. The equivalent electrical circuit of the MOSCAP structure, excluding the parasitic components, is shown in TG) and bottom-gate voltage (VBG) are capacitively coupled based on the capacitances of the top-gate insulator (CTox) and bottom-gate insulator (CBox). Because the depletion capacitance of the Si channel (CSi) is negligible, the relationship between ΔVTG and ΔVBG in the DG mode can be expressed as in Equation (1). Because the top-gate electrode was connected to the EG sensing unit, VTG was equivalent to the surface potential (ψ0) of the analyte solution (VTG = ψ0).The fabricated FET devices comprised two gate electrodes: top-gate and bottom-gate electrodes. In the proposed sensor platform, the top-gate electrode is connected to the EG sensing unit, and the FETs can be operated in either SG or DG modes. Tox/CBox due to capacitance coupling. Notably, a larger amplification factor can be achieved using a larger CTox. We deliberately fabricated DG FETs with different CTox and CBox values by varying the oxide thickness. To achieve a higher CTox/CBox, we adopted a high-k gate dielectric engineered top-gate insulator structure by utilizing stacked oxide layers of SiO2/Ta2O5, which resulted in a higher CTox compared to that of single SiO2 oxide layers with the same thickness. Moreover, when compared to conventional film channel DG FETs, SiNW channel devices exhibit greater CTox owing to the high surface-to-volume ratio of the SiNW channel devices.This relationship indicates that the sensitivity of the proposed sensor platform can be amplified by the amplification factor of Ck gate dielectric engineered SiNW channel DG FETs were investigated in both the SG and DG modes to verify their detection and self-amplification capabilities for the surface potential of the analyte solution. For pH sensing characterization, the SnO2 layer was used as the sensing membrane of the sensor platform without the formation of a Ca2+-selective membrane. REF). The VREF values were obtained at a read current (IR) of 1 nA. The calculated pH sensitivities of the SiNW and film channel devices are presented in The pH sensing characteristics of the high-H) was determined by calculating the difference between the initial and final VREF values. In the SG mode, the resulting VH values for SiNW and film channel devices were 4.85 and 2.22 mV, respectively, while in DG mode, the corresponding values were 12.13 and 12.06 mV, respectively. The drift effects of the SiNW and film channel DG FETs in the SG and DG modes are depicted in D) was measured after the sensing membrane was immersed in a pH 7 buffer solution for 10 h. The fabricated SiNW and film channel devices exhibited RD values of 6.25 and 4.71 mV/h, respectively, in the SG mode. In the DG mode, the corresponding values were 14.37 and 13.38 mV/h, respectively. H, RD, VH-to-pH sensitivity, and RD-to-pH sensitivity. The results indicated that the DG mode operation yielded higher values for both VH and RD, as well as pH sensitivity, compared to the SG mode for both the SiNW and film channel devices. However, when considering the VH-to-pH sensitivity and RD-to-pH sensitivity, the increase in the VH and RD values was notably lower than the increase in pH sensitivity for each device. Furthermore, the SiNW channel device exhibited a more significant reduction in both VH-to-pH sensitivity and RD-to-pH sensitivity in the DG mode compared to the film channel device. These findings suggest that capacitive coupling in the DG mode is an effective approach for amplifying the sensitivity beyond the theoretical limit, resulting in higher sensitivity, stability, and reliability. Consequently, the proposed high-k gate dielectric engineered SiNW channel DG FET demonstrates promising potential as a high-performance sensor platform, offering highly sensitive and stable sensing characteristics.In addition to pH sensitivity, non-ideal effects such as hysteresis and drift can affect the sensing characteristics of FET-type sensor platforms. Direct contact between the EG sensing unit and analyte solution can result in chemical damage to the sensing membrane, leading to a decrease in sensing performance. Hysteresis effects are influenced by the presence of buried OH sites and the transport of defects within the sensing membrane ,32,33. Hk gate dielectric engineered SiNW channel DG FET sensor platform, we applied our device to practical biosensing applications, specifically, the selective detection of Ca2+. To enable the device as a Ca2+-selective sensor, we fabricated a Ca2+-selective EG by forming a Ca2+-selective membrane on a SnO2 layer. The detailed fabrication process of the Ca2+-selective EG sensing unit is described in 2+ concentrations. The corresponding results for the film channel devices are shown in 2 solutions with varying Ca2+ concentrations. As the Ca2+ concentration increased, the transfer characteristic curves shifted in the negative direction. 2+ sensitivities of the SiNW and film channel devices, respectively. The VREF values were obtained at an IR of 1 nA. In the SG mode, the Ca2+ sensitivities of the SiNW and film channel devices were 37.44 and 34.45 mV/dec, respectively. In the DG mode, the corresponding Ca2+ sensitivities were 208.25 and 139.41 mV/dec, respectively. While both devices exhibited similar Ca2+ sensitivities in the SG mode, the Ca2+ sensitivities of the SiNW and film channel devices were amplified by factors of 5.51 and 4.04, respectively, in the DG mode. This result is consistent with the findings observed in the pH sensing operations described in 2+ sensor based on the high-k gate dielectric engineered SiNW channel DG FET demonstrated highly sensitive characteristics, suggesting its potential for various biosensing applications.After successfully demonstrating the high-performance sensing capabilities of the fabricated high-2+ sensor, we conducted hysteresis and drift effect measurements during the Ca2+ sensing operations. 2 solutions in the SG and DG modes, respectively. We measured the hysteresis effects of the Ca2+ sensing operations for 45 min, changing the Ca2+ concentration every 5 min according to the following CaCl2 concentration loop: 10−4 − 10−3 − 10−2 − 10−1 − 100 − 10−1 − 10−2 − 10−3 − 10−4 M. VREF values were extracted from the transfer characteristic curves measured every 1 min. In the SG mode, the VH values of SiNW and film channel devices were 3.65 and 3.06 mV, respectively. In the DG mode, the corresponding values were 13.60 and 12.76 mV, respectively. 2+ operation. The RD values of the SiNW and film channel devices were monitored for 10 h, while the Ca2+-selective membrane of EG was immersed in a 10−4 M CaCl2 solution. In the SG mode, the RD values of SiNW and film channel devices were 7.59 and 6.69 mV/h, respectively. In the DG mode, the corresponding values were 13.22 and 13.38 mV/h, respectively. 2+-sensing characteristics of the high-k gate dielectric engineered DG FETs. Although the VH and RD values increased in the DG mode compared to the SG mode, the increase in these non-ideal effects was much smaller than the increase in the Ca2+ sensitivity of both devices. Moreover, in the DG mode, the SiNW channel device significantly reduced the VH and RD to Ca2+ sensitivity from 9.74% to 6.65% and 20.27% to 6.34%, respectively. The observed enhancement in stability is consistent with the pH sensing results and is attributed to the high amplification capability of the SiNW channel device. Thus, we verified the stable Ca2+ sensing characteristics of the fabricated high-k gate dielectric engineered SiNW channel DG FETs.To verify the stability and reliability of the fabricated Ca2+-selective sensing characteristics, we measured the pH, Na+, and K+ sensitivities of Ca2+-selective EG using pH buffer, NaCl, and KCl solutions, respectively. 2+-selective sensing characteristics of the SiNW and film channel devices, respectively, in the SG mode. Among the interfering ions, including H+, Na2+, and K+, the highest interfering-ion sensitivities were only 7.95% and 7.72% for the Ca2+ sensitivities of the SiNW and film channel devices, respectively. 2+-selective sensing characteristics of the SiNW and film channel devices, respectively, in the DG mode. The maximum interfering-ion sensitivities measured in the DG mode were 7.82% and 12.62% for the Ca2+ sensitivities of the SiNW channel and film channel devices, respectively. The interfering-ion sensitivities are assumed to be amplified along with the Ca2+ sensitivities, proportional to the amplification factor, as the interfering-ion sensitivity arises from the transfer of the ion’s surface potential (ψ0) to the sensing membrane [2+ sensitivity, this corresponds to a negligible value that does not significantly hinder the selective Ca2+ sensing operation. Therefore, these findings suggest that the fabricated Ca2+-selective sensor, constructed with high-k gate dielectric engineered SiNW channel DG FETs, exhibits extensive versatility as a high-performance biosensor platform, owing to its ultrasensitive and highly selective sensing characteristics for the selective detection of Ca2+. +, K+, and Ca2+ sensing characteristics of the high-k gate dielectric engineered DG FETs.To establish Camembrane ,39,40,41k gate dielectric engineered SiNW random network channel DG FETs for the selective detection of Ca2+. The proposed sensor platform combines the advantages of high-k gate dielectric engineered SiNW channel DG FETs as transducer units and separate EG as a sensing unit. The template transfer method using PVP nanofibers enabled the fabrication of SiNW channels. Due to the high surface-to-volume ratio of the SiNW channel structure, top-gate oxide capacitance of the SiNW channel device could be larger, thereby enhancing the self-amplification capability of capacitively coupled DG FETs. In addition to the SiNW channel device, a conventional film channel device was fabricated to validate the improved characteristics of the sensor platform. The electrical characteristics and pH-sensing capabilities of the sensor platform were thoroughly evaluated to lay the foundation for Ca2+ detection. The integration of a Ca2+-selective membrane to the fabricated sensor platform resulted in remarkable Ca2+ sensitivity, with the SiNW channel device achieving a sensitivity of 208.25 mV/dec, surpassing that of the film channel device by 149%. The assessment of non-ideal effects, such as hysteresis and drift, demonstrated that the fabricated SiNW channel device effectively mitigated these effects, with the VH and RD values remaining below 6.53% despite the enhanced Ca2+ sensitivity. To further assess its selective sensing capabilities, the sensitivity of the platform to interfering ions, including H+, K+, and Na+, was evaluated using a pH buffer, KCl, and NaCl solutions. Although the interfering-ion sensitivities were also amplified in proportion to the Ca2+ sensitivity, the SiNW channel device exhibited a sensitivity of less than 7.82% of the amplified Ca2+ sensitivity. These results confirm the successful application of the proposed sensor platform as a high-performance biosensor. Therefore, the proposed high-performance biosensor platform based on high-k gate dielectric engineered SiNW random network channel DG FETs demonstrated highly sensitive and selective characteristics with reliable sensing operation. These exhibit promising potential for broad applications in various biosensing fields, highlighting applicability and versatile capabilities in biomedical diagnostics, environmental monitoring, and food safety analysis.In this study, we present a high-performance biosensor platform based on high-


Here, we examined the role of NCX1 in the development of multiple myeloma (MM), with a special focus on the underlying effects involved osteoclast differentiation. Firstly, we detected NCX1 protein highly expressed in BM tissues of MM patients, and its expression was positively correlated with serum calcium and the percentage of BM CD138+ cells. In vitro, NCX1 suppression with the inhibitor KB-R7943 reduced cell viability of MM cells and caused apoptosis. Extracellular high Ca2+ environment increased the level of intracellular Ca2+ in MM cells through gating the calcium influx, with subsequently promoting the expression of NCX1 and osteoclastogenesis-related genes (receptor activator of nuclear factor-κB (RANKL), nuclear factor of activated T cell cytoplasmic 1 (NFATc1), and proto-oncogene Fos (c-Fos). This phenomenon could be reversed by KB-R7943 or calcium chelation. Furthermore, NCX1 overexpression in MM cells accelerated osteoclastogenesis, while NCX1 knockdown or suppression resulted in the opposite effect. Mechanistically, we further investigated the related mechanisms of NCX1 regulating osteoclast differentiation using RNA sequencing, western blotting and Enzyme linked immunosorbent assay, and found that NCX1 modulated osteoclast differentiation in MM though JNK/c-Fos/NFATc1 signaling pathway. In conclusion, the Ca2+/NCX1-mediated signaling participates in the osteoclasts-myeloma cell interactions, which represents a promising target for future therapeutic intervention in MBD.Although several types of calcium channels abnormalities have been shown to promote myeloma bone disease (MBD), the relationship between NaThe online version contains supplementary material available at 10.1007/s10238-022-00905-1. Briefly, cells were cultured with RPMI-1640 for 48 h and washed 3 times in Ca2+-free D-Hanks Balanced Salt Solution (Ca2+-free HBSS). Then, the cells were incubated for 40 min at 37 °C in the dark with 3 μmol/l Fluo-4 AM. They were then washed 3 times in Ca2+-free HBSS to remove extracellular Fluo-4 AM. Replace the solution with Ca2+-containing HBSS 1 min before the test. The fluorescence intensity of [Ca2+]i was detected by flow cytometry, excited with 488 mm, and the fluorescence signal was collected by the FLI-H fluorescence channel. 1 × 104 cells were collected, and the [Ca2+]i concentration was expressed as the mean fluorescence.The o could increase [Ca2+]i levels after stimulation of 2, 4, 6, and 8 mM CaCl2, respectively. Then, we investigated the expression of NCX1 protein in response to increased [Ca2+]o concentration. The results showed that [Ca2+]o significantly increased NCX1 expression  related to osteoclastogenesis.Due to the close relationship between hypercalcemia and OC hyperactivity , we alsoto RANKL . Moreoveway Fig. c–e. Alto2+]i level in MM cells induced by CaCl2 when compared with the CaCl2 group .Next, RPMI8226 and KMS11 cells were exposed to BAPTA-AM  to test whether the decrease in [Ca2+]o . Based on these findings, NCX1 appears to regulate the expression of osteoclastogenesis-related genes by disturbing calcium homeostasis.Furthermore, western blotting showed that MM cells cultured with KB-R7943 for 48 h could reverse the increase in RANKL, NFATc1 and c-Fos protein expression induced by high [Ca2+]o, we next investigated the effect of NCX1 on osteoclastogenesis in MM. Since murine macrophage cell line RAW264.7 has been widely used in osteoclast differentiation research o. To verify the impacts of NCX1 on RANKL secretion and NFATc1 and c-Fos activity, we performed western blots to determine whether NCX1 expression level affects RANKL expression, NFATc1 nuclear translocation, and c-Fos nuclear translocation. Compared to the corresponding control cells, NCX1-knockdown in MM cells strongly decreased the expression of RANKL, and blocked nuclear translocation of NFATc1 and c-Fos Below is the link to the electronic supplementary material.


This article describes a new method to evaluate the actomyosin activity in living cardiomyocytes during beating based on nonlinear scattering microscopy, which will be useful for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin–actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a  The S1 region converts chemical energy from ATP hydrolysis into mechanical energy by changing its structure corresponding to the nucleotide state, called “lever arm swinging.” The LMM is responsible for rod filamentation, whereas the S2 region acts as a “spring” between the converter and the rod (right). Several groups have reported their success in discriminating SHG anisotropy between rigor and relaxed states in rabbit psoas fibers .Here, we prepared spontaneously beating cardiomyocytes differentiated from human iPSCs (hiPSCs), 253G1 strain. The sarcomeres in chemically permeabilized cardiomyocytes were arranged along the employed line-and-space pattern and selectively visualized via SHG . The staconstant . The parge state , was estγ in a field of view (FOV) of 128 × 512 pixels (25 × 100 μm) clearly decreased from γrigor = 0.73 ± 0.02 (mean ± SD) in the rigor state to γrelax = 0.43 ± 0.03 in the relaxed state (red and green). These values were consistent with the values reported in previous studies, where γrigor = 0.68 ± 0.01 and γrelax = 0.46 ± 0.03 for rabbit psoas fibers  (blue). Although AMPPNP was thought to induce myosin dissociation from actin, sarcomere length (SL) was not increased by AMPPNP treatment but was increased by ATP (P = 1.3 × 10−37 in t test)  , blue. A t test) .γ-value. The total SHG intensity in the FOV decreased after the decrease in γ after treatment with ATP, but not with AMPPNP (red), the intensity was negatively correlated with the γ-value within the rigor or AMPPNP data (green and blue), indicating the presence of a factor contributing to intensity but not related to the γ-value. Thus, SHG anisotropy measurement, apart from intensity measurement, could be meaningful for probing actomyosin activity.According to h AMPPNP . Although AMPPNP , red, thγ synchronized with sarcomere contraction was successfully measured during spontaneous beating in living cardiomyocytes derived from hiPSCs (γ at the resting state γrest and the contracting state γcont could be estimated by fitting the histogram of γ with a double Gaussian distribution (γrest = 0.43 obtained corresponded to the γrelax (0.43) obtained in permeabilized cardiomyocytes, whereas the γcont = 0.50 was smaller than γrigor (0.69). This tendency was the same as that previously reported for isometric force generation in frog muscle fibers  × 0.25 + 0.43 = 0.495, because the population of myosin contributing to force generation during contraction is only between 20–30% , indicating that the relationship between SL and γ was nonlinear during the transition process. Average sarcomere lengths in the contracting and resting states, denoted as SLcont and SLrest, respectively, were measured using the average SHG intensity profiles during the contracting and resting states with reference to the γ-value (SLrest and SLcont were determined to be 1.94 ± 0.10 μm and 1.77 ± 0.10 μm (N = 25 FOVs), respectively, which were consistent with commonly known values measured in myocardial cells . This method lost the length information and the synchronization timing between local sarcomere contraction and the γ-value because the motion of sarcomeres in a FOV was affected by those outside the FOV. Nevertheless, the contraction duration and relaxation delay during sarcomere motion can be obtained without care of defocusing because of muscle contraction.Plots of the average  γ-value  and S3: al cells . The corng state . Insteadupper). BS is known to retain the myosin ATPase cycle in the ADP.Pi state by inhibiting the release of phosphate  (red and magenta). By inactivating the BS effect via blue laser irradiation . Because not all sarcomeres showed recovery of pulsation, no significant difference was observed in the histogram of γave before and after the irradiation, but a small decrease was confirmed in the mean value of γave, 0.47 ± 0.11 (magenta and blue). Treating cardiomyocytes with 10 μM omecamtiv mecarbil (OM), a cardiac-specific myosin activator and a potential pharmaceutical cure for heart dysfunction  upon treatment with 10 μM OM (green). The increase in γave after OM treatment was significantly larger than that by BS, both before and after inactivation . In summary, the dynamic measurement of SHG polarization anisotropy provided quantitative parameters to assess actomyosin crossbridge formation in living cardiomyocytes.By treating cardiomyocytes with 10 μM blebbistatin (BS), a widely used inhibitor of actomyosin ATP hydrolysis , pulsatihosphate . The aveadiation , cardiomfunction , remarkafunction . Unlike function . In view10 μM OM , green. MYBPC3 gene encoding cardiac myosin-binding protein C (cMyBPC) are among the most common gene mutations found in hypertrophic cardiomyopathy (MYBPC3 mutation (Mybpc3t/+). For comparison, hiPSC lines with a homozygous mutation (Mybpc3t/t) as a positive control or without mutations (Mybpc3+/+) as a negative control were also established with a genome editing technique. We then measured the time course of SHG polarization emitted from sarcomeres in cardiomyocytes derived from these cell lines  and mean γcont (0.57 ± 0.06) of Mybpc3t/t were significantly higher than those of Mybpc3t/+  (red and blue). Mybpc3+/+ exhibited a lower γcont  than Mybpc3t/+, whereas there was no significant difference in γrest between the two cells  (green). The difference between γcont and γrest, denoted as dγ = γcont − γrest was thought to reflect the population of myosin actively interacting out of the inhibitory control. We found no significant difference between Mybpc3t/+ and Mybpc3t/t in dγ (red and blue). Meanwhile, significant differences were observed between Mybpc3+/+ and the other cell lines (P = 3.9 × 10−4 in t test) (green). Between Mybpc3t/t and Mybpc3+/+, there were significant differences in all γcont, γrest, and dγ . Thus, the differences in γ measurement represent the effect of heterozygous and homozygous MyBPC3 deficiency or the repair of actomyosin activity.Spontaneous pulsations were observed in cardiomyocytes differentiated from all established hiPSCs . We obta t test) , green.  t test) , green. MyBPC3 deficiency  (lower). The mean SLrest and SLcont were, respectively, 1.96 ± 0.11 μm and 1.79 ± 0.11 μm for Mybpc3t/+, decreased after homozygous mutation , and did not change after rescuing the phenotype (SLrest = 1.91 ± 0.12 μm and SLcont = 1.75 ± 0.12 μm) (upper). Analysis of variance (ANOVA) for the three groups exhibited no statistically significant difference in SL measurements (P = 0.03 for SLrest and 0.08 for SLcont). Although there might be a trend for homozygous mutations in shortening the SL, we were not able to show it statistically in this analysis.Although we expected that the contraction yield of sarcomeres would increase based on a previous experimental report that cellular shortening increased with ficiency , obviousbpc3+/+) , lower. 0.12 μm) , upper. γ–SL correlation (top and bottom). Because a heterozygous MyBPC3-deficiency increases the probability of myosin–actin interaction during contraction . Relaxation of the γ-value was not affected by MyBPC3 deficiency regardless of its homozygosity or heterozygosity (left), indicating that cMyBPC is not directly responsible for the kinetics of the association–dissociation cycle of actomyosin corresponding to ATP hydrolysis. Meanwhile, the average trace of sarcomere movement exhibited a delay of relaxation in Mybpc3t/+ and Mybpc3t/t in contrast to Mybpc3+/+ cells (right), which was consistent with previous results obtained in whole-cell shortening , some ESCs partially survive and maintain their trilineage differentiation potential in humans and mice . In micerelation . HiPSCs and Oct4 . These s2, and they became almost undetectable after UV irradiation at 62.25 mJ/cm2 (bottom). The γave calculated, including both pulsating and non-pulsating sarcomeres, were 0.46 ± 0.05, 0.57 ± 0.11, 0.75 ± 0.10, and 0.57 ± 0.09 upon UV irradiation at 0.00, 31.25, 62.50, and 125.00 mJ/cm2, respectively  and γave changed depending on the irradiation power (2 increased both the slope and the intercept of the liner γave–SLave correlation (second left); the slope and the intercept were further increased at 62.5 mJ/cm2 irradiation (second right); at 125 mJ/cm2 irradiation, the slope returned to that for no irradiation, whereas maintaining the increase in intercept (right). SHG intensity decreased upon UV irradiation  -sn-glycerol) in the inner mitochondrial membrane , which exhibits Barth syndrome-related phenotypes, such as having a significant reduction in cardiolipin, mitochondrial abnormalities, and the degradation of muscle activity  . TAZ defmembrane . In thisactivity . A thirdactivity . DrosophDrosophila prepupae without uncovering the puparium, regardless of the presence or absence of TAZ , except during the times when sarcomeres move out of the focal plane during observation . Occasionally, clear state transitions from resting to contracting or from contracting to resting could be pursued (lower). The SL and γ-values within a single FOV were negatively correlated in both control and TAZ−/− flies .SHG microscopy visualized the sarcomeres of the body wall muscles in e of TAZ . Unlike  pursued , lower. /− flies , as in t/− flies . Plottinrrelated . Meanwhirelation , magentaSL (∼7 μm) for the control prepupae . Then, we extracted the traces that included obvious contracting–resting transitions and estimated the γrest, γcont, and the difference between them, dγ. Although no statistically significant difference was observed, the mean γrest for TAZ−/− flies was higher than that of the control, whereas the mean γcont remained unchanged (left and center). The mean dγ was significantly lower in TAZ−/− group than in the control group . Thus, the present method was applicable for investigating muscle activity in a Drosophila disease model.The value of prepupae  was consprepupae  and was prepupae , which dcy model . Meanwhieen them , but fouues of γ . A slighDrosophila disease model. Disease-derived and genome-edited cardiomyocytes provided information about the parameter γ obtained from SHG anisotropy measurements, reflecting myosin force generation. The sarcomere length SL was simultaneously measurable, and the correlation between γ and SL represented the phenotype of actomyosin dysfunction in genetically diseased cardiomyocytes. Furthermore, the present method was applicable for detecting acquired muscle failure because of radiation exposure and functional decline in Drosophila intravital imaging.In this study, we demonstrated the use of SHG anisotropy to investigate myocardial dysfunction in cardiomyopathy by constructing an effective assay system based on our highly sensitive SHG microscope with a fast polarization-controllable device , and theγ-values to well-aligned skeletal muscles. To remedy this, we employed a line-and-space pattern substrate, which linearly align the myofiber orientation in cardiomyocytes . Controlling the beating cycle needs the installation of an electrical stimulation system, buffer exchange system, and a temperature control system into a tiny space between two objectives with short working distance.Two practical problems needed to be solved in measuring SHG anisotropy during myocardial beating. The first problem was the random orientation of myofibrils in cardiomyocytes, which limited the measurement of myocytes , allowin and γrigor (0.69) obtained in permeabilized cardiomyocytes (γrest (0.43) obtained in a living cell corresponded to the γrelax in permeabilized cells, and the γcont (0.50) obtained was also a reasonable value, considering the active percentage (20–30%) of myosin population binding to actin during contraction (γrest = 0.30 and γcont = 0.64)  . This di = 0.64) . In coll voltage , which iies used , and alshortened  and thathortened . It is mγ, which has been controversial, is discussed here following the present results. SHG from muscles is dominated by SHG from myosin rather than actin. Plotnikov et al. argued that SHG from myosin is derived from the LMM and not from the S1 or S2 regions, according to their finding of small differences in SHG polarization in scallop muscles between upon AMPPNP treatment and that in the rigor state. They also did not observe any difference in SHG polarization from C. elegans muscles against the scallop muscle, whose ratio of paramyosin (headless myosin) to myosin is 15-folds higher in C. elegans muscles . It is unlikely that the stable helical structure of LMM undergoes such a large change. This means that the primary cause of the changes in SHG polarization dependence is simply the angular change in the dipole orientation. Conclusively, it is reasonable to assume that S1 and S2 are the primary sources of change in SHG anisotropy.The attribution of  muscles . Meanwhi muscles . In thisγ and SL , which agrees with the previous result wherein OM increased Ca2+ sensitivity in cardiomyocytes, causing more myosin to interact with actin , another myosin inhibitor that suppresses force generation without interfering with myosin binding to actin, caused a decrease in γ . It can be interpreted that the γ-value in Mybpc3t/t was positively offset from that in Mybpc3t/+ because of the constant residual myosin interacting with actin. Rescue experiments caused a decrease in the slope of the γ-SL correlation without increasing the γrest (green). There are two possible causes for the increase in γrest according to the interpretation of the present result shown in MyBPC3 deficiency that the heterozygous mutation inhibited the super relaxation of myosin in relaxed state  at 37°C and 5% CO2. The medium was exchanged every day, and passaging was performed twice per week.The 253G1 human iPSC line was purchased from the Riken Cell Bank (HPS0002) and was adapted to feeder-free conditions as described previously . The Mybreported . The cel4 cells/well, and then acclimated to the mTeSR1 culture medium  until differentiation began. Differentiation was induced 4 d after EB formation using the PSC Cardiomyocyte Differentiation Kit , following the differentiation time course described in the manufacturer’s instructions. 2 d after the induction, EBs were transferred from the 96 well plate to a gelatin/iMatrix/fibronectin triple-coated dish  and allowed to differentiate up to 14 d after the induction.For cardiomyocyte differentiation, hiPSCs were harvested with TrypLE select , transferred to ultra-low attachment U-bottom 96 well plate  at a density of 1 × 102/min. Under these conditions, ∼92%, 10.5%, 4.5%, and 0.5% cells survived, respectively (n = 2). These cells were cultured for more than two weeks after UV irradiation and used for subsequent experiments. We confirmed that more than 90% cells remained to express both pluripotency markers Sox2 and Oct4 after 10 passages with immunostaining analysis  (HiPSCs on culture dishes were irradiated with 254 nm ultraviolet light (UV) for 0, 2.5, 5.0 or 10 min. The UV dose was measured with an S425C thermal power sensor equipped on a PM100D monitor (Thorlabs Japan) and was calculated to be 12.5 mJ/cmctively) .Immunostaining was performed 14 days after the induction of differentiation. Cultured cells were washed twice with PBS(−), dissociated by trypsinization , and replated on iMatrix511-coated cover glass  in a culture medium consisting of DMEM  with 10% FBS (DMEM/10% FBS). 4 d after the plating, cells were fixed with 4% PFA for 15 min at RT. After washing, the cells were permeabilized with 0.3% Triton X-100 in PBS(−) and then incubated with anti-α-actinin mouse monoclonal antibody  in CAS-Block reagent  at 4°C overnight. After washing, immunoreactive cells were determined using the appropriate fluorescently labeled secondary antibodies, Alexa Fluor 488 conjugated goat anti-mouse IgG . The cells were washed, stained with DAPI , and mounted in Vectashield mounting medium .A strong dependence of myofibril orientation in cardiomyocytes on cell shape has been previously reported . Cell sh2) in PBS(−) at 4°C overnight, and then washed three times with PBS(−). For Mybpc3t/+, Mybpc3t/t, and Mybpc3+/+-derived cardiomyocytes, the patterned glass substrates were coated with 0.1% gelatin instead of iMatrix 511-silk in PBS(−) at 37°C for more than 30 min, and the excess gelatin solution was aspirated before seeding. On 14 d after the differentiation induction, the cardiomyocytes differentiated from hiPSCs were dissociated with 0.25% trypsin/EDTA for 10 min and resuspended in DMEM/10% FBS. Large cell aggregates were removed using a cell strainer . The remaining cells were seeded on an iMatrix-coated or gelatin-coated line-and-space patterned glass substrate at a density of 4 × 104 cells/cm2. The culture medium was exchanged every other day until 18 d after the induction, when the SHG analysis was performed.For seeding of 253G1-derived cardiomyocytes, the line-and-space patterned glass substrates were placed in six well cell culture plate  with the cell adherent side up, coated with iMatrix 511-silk  for 4 min on ice, then washed twice with PBS(−). The permeabilized cells were then treated on ice with the reported solutions to induce rigor and relaxation (5 mM ATP) states  states . The AMP(S)-(−)-Blebbistatin (BS) was purchased from Toronto Research Chemicals Inc. , dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, and stored at −80°C. Omecamtiv mecarbil (OM) was purchased from Toronto Research Chemicals Inc. , dissolved in DMSO to make a 10 mM stock solution, and stored at −80°C. Cardiomyocytes differentiated from 253G1 hiPSCs through EB formation were plated on a patterned substrate coated with iMatrix, as mentioned above, and were incubated at 37°C in the DMEM/10% FBS with 10 μM of BS or 10 μM of OM with 0.1% DMSO for 60 min. As the control sample against drug-treated cells, cardiomyocytes differentiated from 253G1 hiPSCs were incubated at 37°C with 0.1% DMSO for 60 min. All biological assays were repeated at least thrice.TAZ−/− strain was established following the previous report  chromosome tagged with GFP. The pupae were raised at 25°C until the third instar larval stage. GFP-negative larvae were counter-selected as homozygous flies for TAZ. SHG images were acquired during the prepupal stage at a controlled room temperature of 23°C. A prepupa was placed on a glass slip such that its dorsal surface was in contact with the glass surface. The sample was then immobilized from the ventral surface using transparent adhesive tape , passed through a tube lens and a band-pass filter. Optical parts composing the microscope, including silver-coated mirrors, dielectric-multilayered dichroic mirrors, oxide-coated half-mirrors, anti-refection-coated lens, and so on, cause polarization distortion on the sample plane. We conducted the calibration of incident polarization dependence on detected intensity with an estimation of the phase retardation parameters without a sample  were repeatedly acquired with a pixel dwell time of 1 ms and no interval time between images. In the dynamic measurement of UV-treated cardiomyocytes, the rectangular region was set to 0.195 × 4.88 μm2 (2 × 50 pixels). The total acquisition time per 500 frames was 40 s. For the dynamic measurement in a Drosophila prepupa, the rectangular region was set to 25 × 20 μm2 with the spatial resolution of 1 μm/pixel and the temporal resolution of 1 ms. The polarization sweeping condition was the same as that used for cardiomyocytes. A total of 120 polarization-resolved image sets were acquired within 60 s.A set of polarization-resolved SHG images was acquired by synchronous control of the SL measurement was not possible because of the deterioration of the SHG image during contraction. To estimate sarcomere motion in such cases, we used the correlation between images because SHG images in the resting state are similar, whereas those during contraction differ from resting SHG images. The correlation was calculated by first calculating and averaging Pearson’s correlation between the given time t and the others. Then, resting time was identified using Otsu’s binarization method and Pearson’s correlation was calculated and averaged for images at a given time t and resting time images to give a correlation value.In some experiments, Research plan related to the hiPSCs used in this study was approved by the Ethics Committee of RIKEN Center for Biosystems Dynamics Research . The use of patient-derived samples and genomic analysis were approved by the Ethics Committee of Osaka University Hospital, and written informed consent was obtained from all patients. This study conforms to the ethical guidelines for medical and health research involving human participants in Japan and all principles outlined by the Declaration of Helsinki.