Scientific reductionism, the view that higher level functions can be explained by properties at some lower-level or levels, has been an assumption of nervous system analyses since the acceptance of the neuron doctrine in the late 19th century, and became a dominant experimental approach with the development of intracellular recording techniques in the mid-20th century. Subsequent refinements of electrophysiological approaches and the continual development of molecular and genetic techniques have promoted a focus on molecular and cellular mechanisms in experimental analyses and explanations of sensory, motor, and cognitive functions. Reductionist assumptions have also influenced our views of the etiology and treatment of psychopathologies, and have more recently led to claims that we can, or even should, pharmacologically enhance the normal brain. Reductionism remains an area of active debate in the philosophy of science. In neuroscience and psychology, the debate typically focuses on the mind-brain question and the mechanisms of cognition, and how or if they can be explained in neurobiological terms. However, these debates are affected by the complexity of the phenomena being considered and the difficulty of obtaining the necessary neurobiological detail. We can instead ask whether features identified in neurobiological analyses of simpler aspects in simpler nervous systems support current molecular and cellular approaches to explaining systems or behaviors. While my view is that they do not, this does not invite the opposing view prevalent in dichotomous thinking that molecular and cellular detail is irrelevant and we should focus on computations or representations. We instead need to consider how to address the long-standing dilemma of how a nervous system that ostensibly functions through discrete cell to cell communication can generate population effects across multiple spatial and temporal scales to generate behavior. There is extensive debate on reductionism in the philosophy of science , and in Reductionism is not a unitary phenomenon . The bioWhile debate in the philosophy of science has traditionally focused on intertheoretical reduction, constitutive, and explanatory reductionism have come to the fore . These fBroadly speaking, given the controversy over definitions , psychonTo consider constitutive and explanatory reduction in neurobiology, I will start with the basic issue of experimentally identifying component neurons, and then consider how the organization of neurons in neural circuits affects our ability to offer reductive or mechanistic explanations. I will finish by considering claims that our mechanistic knowledge of the nervous system obtained in reductionist analyses is sufficient to be translated into practical uses. These claims extend beyond interventions in traditional areas like neurology and psychopathology to include aspects of normal cognition and behavior, with some claiming that not only can we safely and effectively intervene in the normal brain, but also that we should.The reductionist belief that molecular and cellular properties underlie cognition and behavior has been called the neuron doctrine e.g., . This do1The experimental criteria claimed for a reductive explanation of behavior in neurobiology have been outlined several times , 1x is the only functional connection onto y, and activity of y is evoked/abolished when 1x is activated/inactivated, suggesting that 1x is necessary and sufficient for y. However, degeneracy .Consider the scheme in 1-4x introduce additional issues. In 1x sends parallel feedforward projections to 2–x4x, which sum to activate y. Activating 1x will evoke y, and blocking 1x will block y, suggesting 1x is necessary and sufficient for y, but 1x would not be sufficient if the summed input from 1–x4x was needed to activate y. In a synfire-like chain . However, with feedback excitation from 1y to 1x  increased inhibition from 2x reduces 1y activity; which (2) reduces feedback excitation of 1x; which (3) reduces 2x activation and disinhibits 1y; (4) this increases 1y activity and thus feedback excitation of 1x and 2x activity to reduce 1y activity; and (5): the cycle repeating to cause oscillation. With feedback inhibition from 1y to 1x . This depends on the equilibrium potential for chloride, which in turn depends on the activity of Cl-pumps that determine the intracellular Cl-levels and the membrane potential of the cell, all of which will simultaneously change as the neuron and receptor are activated. Even if a GABAergic neuron was known to hyperpolarize and inhibit a postsynaptic cell this may still not describe its functional effect as inhibition of other inhibitory neurons (disinhibition) will evoke excitation.Relational aspects oppose substantivalist views that see functions represented in components, expressed in references to memory molecules, inhibitory or excitatory neurotransmitters or neurons, and mood or reward neurotransmitters. Neurons and neurotransmitters are not intrinsically inhibitory or excitatory, as can often be claimed e.g., . InhibitRelational aspects are illustrated by homeostatic plasticity where parameter values vary as a function of the variability of other components to maintain an output. In single cells this can reflect variations in different classes of ion channels or synaptic inputs to maintain a certain level of cellular excitability , while i1 v N2…Nn) can realize a single behavior or cognitive process (P1 ↔ N1 v N2…Nn). This could be considered a neurobiological example of multiple realisability, although this is a contentious issue  acting in the environment see . This haIt could be argued that as embodied effects are represented in the molecular transduction of sensory neurons and through various sub-cortical and cortical sensory processing stages, they can be incorporated into the neurobiological explanation. But this would require consideration of heterarchical processing that is continuously altered by internal and external relationships between ongoing functions and behaviors. As sic], viewed as a behaving system, is quite simple. The apparent complexity of his behavior over time is largely a reflection of the complexity of the environment in which he finds himself.” But this can be extended because environments are also continuously modified by ongoing behavior , and the interpretation of system manipulations. Newer techniques like gene knock-outs and optogenetics claim greater precision than traditional approaches , but no matter how surgical a manipulation is, relational effects mean that there will necessarily be changes in the properties of other components . This is of course the aim of a manipulation, to identify a component and its role from how the system changes after its manipulation. In decomposable or nearly-decomposable systems where parts are relatively independent we could relate the resulting system changes to the manipulated component, but relational effects in non-decomposable systems mean that system effect will reflect changes in more than the manipulated component  the properties that we characterize can differ to those in the intact, active system. Claude Bernard wrote, “the phenomena of a living body are in such reciprocal harmony one with another that it seems impossible to separate any part without at once disturbing the whole organism,” quoting Georges Cuvier, “All parts of a living body are interrelated; they can act only in so far as they act all together; trying to separate one from the whole means transferring it to the realm of dead substances; it means entirely changing its essence” see . An examVm) is the difference between the intracellular (Vi) and extracellular potential (Ve), Vm = Vi − Ve. Current flow to or from the local extracellular environment caused by cellular or synaptic activity generates local field potentials that change Ve and thus Vm. These effects are anisotropic, the magnitude and direction of the change in eV reflecting a complex interaction of several variables including the number of active cells, the pattern of their activity, the packing and orientation of neurons and processes, the geometry of the extracellular space, and the properties of the “postephaptic” cell . Ephapses provide a concrete neurobiological example of an emergent effect. While these effects can be modeled, the equations currently rely on assumptions of several unknowns. We know field effects occur, they are measured in EEGs, but are they functionally-relevant signals  this only says how the depression occurred, not why. Knowing why is necessary to determine the optimal intervention; do we act on serotonin levels or address the negative life event? An analogy would be that hemorrhage may cause death through loss of blood volume and blood pressure leading to insufficient oxygen delivery to the brain and heart, but treatment for this would not be continual blood transfusions to maintain blood volume but treating the hemorrhage.Even if neurobiological causation was determined, this still may not necessarily make a neurobiological intervention better than non-biological approaches , smart drugs , or downloading, “straightening out,” and re-uploading information from the brain . These cEducation is a recent focus for translational neuroscience. Neuroeducation claims that neuroscience can inform educational practices. This could reflect multiple approaches , but theBehavioral genetics illustrates a dominant reductive neurobiological focus on cognitive abilities. This is a long-standing and contentious issue that uses heritability estimates derived from family studies of identical and non-identical twins raised together (same or different genetics in the same environment) or after adoption  over education that we know enhances cognition and has benefits beyond job status and salary in improving overall health and quality of life ? ClaiminThis hyperbole is balanced by Reductionist analyses that examine component parts to provide mechanistic schemes have been successful in many areas of science, including neuroscience where over several decades experimental tools have allowed increasingly precise molecular and cellular analyses and manipulations that have given insight into various aspects of nervous system function and dysfunction (e.g., the identification of biomarkers in neurology that have supplemented traditional behavioral descriptions; Knowledge of parts, their organization, and the functions they perform can in principle explain any system, including relational and emergent effects, providing that the necessary parts, interactions, and functions are considered. What constitutes necessary and sufficient detail remains debated (see Aplysia as an exemplar of a psychoneural reduction, saying “we take it to be a sociological fact that Kandel’s theory is widely regarded in the neuroscientific community as the best that neuroscience can now offer in the way of explanation of behavior or the mind in fundamental neuroscientific terms.” They evaluated Kandel’s explanation at some length and concluded that it was not a successful psychoneural reduction because it still relies on psychological concepts. But the claim of a successful neurobiological reduction can be negated on far simpler grounds as it begs the question in only considering the sensory neurons and ignores known and relatively well-characterized changes in motor neurons and interneurons (see Explanations can also selectively use available information. In discussing the neuron doctrine, rons see . A succerons see , but aspHighlighting the limitations and challenges of reductive analyses should not be taken as support for the opposing view that lower-level detail is irrelevant and we should instead focus on higher-level computations and representations . The latDichotomies like that between reductionist and representational approaches have stymied various fields (e.g., sensory vs. centrally driven locomotion, and presynaptic vs. postsynaptic expression of LTP; In addition to being counterproductive, debate, or for Bullock the mutual disparagement, over the relative merits of representational/computational and reductionist approaches seems premature given the lack of necessary detail and clarity of definitions. For example, definitions of representations vary  and numeClaiming that cognitive explanations need to account for state spaces across many spatio-temporal scales e.g.,  repeats Placing representational or computational aspects in neurobiological terms is not impossible: a visual receptive field is a representation of external space that can be reduced, although not yet completely, to the connectivity of retinal neurons; analyses of synaptic information transfer consider representational aspects in neurobiological terms , and gracfLinking lower and higher-level effects nevertheless remains the major open question in neuroscience. Claims to Kuhnian paradigm shifts and scientific revolutions , which aThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hence, maintaining their homeostasis is crucial for plant growth and development. In this study, we showed that MTP10  is an important regulator for maintaining homeostasis of Mg and Ca in Arabidopsis. The mtp10 mutant displayed severe growth retardation in the presence of excess Mg2+, to which the addition of Ca2+ was able to rescue the phenotype of mtp10 mutant. Additionally, the deficiency of Ca2+ in the culture medium accelerated the high-Mg sensitivity of the mtp10 mutant. The yeast complementation assay suggested that AtMTP10 had no Ca2+ transport activity. And the ICP-MS data further confirmed the antagonistic relationship between Ca2+ and Mg2+, with the addition of Ca2+ reducing the excessive accumulation of Mg2+ and high-Mg inhibiting the uptake of Ca2+. We conclude that the Arabidopsis MTP10 is essential for the regulation of Mg and Ca homeostasis.Nutrient antagonism typically refers to the fact that too high a concentration of one nutrient inhibits the absorption of another nutrient. In plants, Ca As a nutrient and signaling molecule, calcium (Ca2+) levels in plant cells are strictly controlled by channels, carriers, and transporters, providing a mechanistic basis for Ca2+ homeostasis and Ca2+ signaling. Both Ca2+ and Mg2+ are absorbed by plants from the soil and then translocated from the roots to the shoots through the xylem.1 Typically, serpentine soils show low Ca2+ but high Mg2+ concentrations, which often cause Mg2+ toxicity, leading to reduced crop yields.2 Ca2+ and Mg2+ are the two most abundant divalent cations, which appear to have antagonistic interactions in the plant cell, and the homeostasis balance between them is crucial for plant growth and development.1 It is widely believed that Ca2+ and Mg2+ can compete for the same enzymes or channels, transporters, etc. Notwithstanding their distinct physiological and biochemical roles, Ca2+ and Mg2+ homeostases in plants appear to be closely linked and may be regulated, at least partially, by a common signaling network. There are several ion channels and transporters that have been identified, and they are localized to the plasma membrane and various intracellular membrane systems to mediate the transport of Ca2+ or Mg2+. For example, the Ca2+/H+ exchange activity of the vacuole in the cax1 mutant is inhibited, thereby causing Ca2+ to be poorly sequestered into the vacuole and leading to elevated Ca2+ concentrations in the cytoplasm. Consequently, the Arabidopsis cax1 mutant is more resistant to high-Mg toxicity compared to the wild-type plants.3 Consistently, it has also been shown that the Mg deficiency phenotypes upon multiple knockouts of Arabidopsis thaliana MRS2 clade B genes, AtMRS2-1 and AtMRS2-5, can be ameliorated by concomitantly reduced Ca2+ supply.4 A Na+-K+ transporter family member OsHKT2;4 in rice is permeable to both Ca2+ and Mg2+ when heterogeneously expressed in both bacterial and oocyte systems. Moreover, the overexpression of OsHKT2;4 in the Arabidopsis mgt6 mutant results in the seedlings being more sensitive to high-Mg stress,6 implying an interaction between Ca2+ and Mg2+. A study on Vicia faba guard cells suggested that Mg2+ can prevent continuous Ca2+-leakage through the channels including CGNCs, thereby assigning Mg2+ an important role in Ca homeostasis and Ca2+-dependent downstream signaling.7 A further example of understanding Ca2+-Mg2+ interactions come from the findings that Ca2+ signaling networks also function in the regulation of Mg homeostasis. In yeast, Mg2+ deprivation elicits rapid Ca2+ uptake and activates Ca2+/calcineurin signaling.8 It has been suggested that high-Mg stress is also capable of activating relevant ion channels, leading to a rapid elevation of Ca2+ concentration in the cytoplasm and stimulating specific Ca2+ oscillation.1 In Arabidopsis, two tonoplast-localized calcineurin B-like (CBL) proteins, CBL2 and CBL3, can perceive the Ca2+ signal and activate a subset of CBL-interacting protein kinases (CIPKs), CIPK3, 9, 23, 26, which in turn phosphorylate the as yet unknown Mg2+ transporters on the tonoplast, ultimately regulating Mg2+ sequestration into the vacuole to reduce the Mg2+ toxicity in the cytoplasm.9Magnesium  belong to the CDF (cation diffusion facilitator) family, and these proteins have been identified as proton/divalent cation transporters, responsible for the efflux of cations from the cytoplasm to the extracellular space or the transport of cations from the cytoplasm to subcellular organelles, consequently making this type of transporters essential to metal tolerance.10 Hitherto, most of the functionally well-defined MTP transporters are capable of transporting divalent metal ions, such as Zn2+, Mn2+, Co2+, and Ni2+.10 Previous studies have shown that MTP10 is capable of transporting Mn2+ in Arabidopsis.11 However, our very recent study showed that plasma membrane-localized MTP10 can transport Mg2+ in the bacterial strain MM281 system, and MTP10 loss-of-function mutant exhibits sensitivity to high-Mg stress.12 In this study, we further provided evidence that MTP10 is an important contributor to the regulation of Ca and Mg homeostasis. The high-Mg-sensitive phenotype of the mtp10 mutant was effectively alleviated when the concentration of Ca2+ was appropriately increased in the medium. Under Ca2+ deficiency conditions, it is instead in a position to exacerbate the high-Mg-sensitive phenotype of the mtp10 mutant. Thus, our study revealed that MTP10 is involved in regulating not only Mg homeostasis but also Ca homeostasis in Arabidopsis.In Arabidopsis thaliana ecotype Col-0 was used in this study. The T-DNA insertion mutant mtp10 (SALK_121470) was obtained from Arabidopsis Biological Resource Center (http://abrc.osu.edu). The Arabidopsis seeds were sterilized using 75% ethanol, and plated on 1/2 strength Murashige and Skoog (MS) medium containing 1% (w/v) sucrose. The pH of the medium was adjusted to 5.7 using KOH and solidified using 1% (w/v) agar. After 2 days of stratification at 4°C, the seedlings were then grown in a growth chamber under 150 µmol/m2/s light intensity with a 16-h light/8-h dark photoperiod at 22°C. For the phenotypic assay, the seedlings were grown in 1/2 strength MS medium for 3 days, and then transferred to a 1/6 MS containing different concentrations of MgCl2 or CaCl2 for a designed time. For the hydroponic culture, seeds of A. thaliana (Col-0 and mtp10) were grown on 1/2 strength MS for 7 days, then transferred to 1/6 strength MS solution and grown for the indicated time period.mtp10 mutant were grown in 1/2 MS agar medium for 7 days and then transferred to 1/6 MS liquid medium for another 7 days. The seedlings were then treated with high-Mg (1/6 MS+10 mM MgCl2) for 10 h. Total RNA was isolated using Trizol reagent (Invitrogen), treated with DNase I (Invitrogen), and used as a template to synthesize first-strand cDNA with M-MLV Reverse Transcriptase (Promega) and an oligo dT primer. The qRT-PCR was performed on a Light Cycler 96 (Roche Diagnostics) with SYBR Green I Master mix (Roche Diagnostics) according to the manufacturer’s instructions. ACTIN2 (At3g18730) was used as an internal standard. The primers are listed in Table S1.Seedlings of wild-type Col-0 and 9 Transformants were then grown in a synthetic dropout medium lacking tryptophan and leucine, and a synthetic dropout medium lacking tryptophan, leucine, histidine, and adenine at different dilutions  cells/ml at 30°C.The non-transmembrane C terminal region sequence of MTP10 was cloned into the vector pGADT7, and the CDS sequence of CIPKs was cloned into the vector pGBKT7, respectively. The fusion constructs were transformed into yeast strain AH109 using the lithium acetate transformation method as previously reported.13 After transfection, N. benthamiana were cultured for 48 h, YFP signals were imaged by the LSM710 META confocal laser scanning microscope (Carl Zeiss). The excitation wavelength for YFP was 514 nm, and the emission wavelength was between 525 and 575 nm.To generate BiFC constructs, the coding sequence of CIPK7 and CIPK26 without the stop codon was in-frame cloned into the pSPYNE(R)173-CAMBIA1300 vector, and the coding sequence of MTP10 was sub-cloned into the pSPYCE(M)-CAMBIA1300 vector.Δgdt1, respectively. The wild-type yeast strain BY4741 was set as control. The method of the transformation of the plasmids into the yeast is described in the yeast protocols handbook (Clontech Laboratories). The cells transformed with the indicated constructs were grown on the synthetic medium containing amino acids without uracil (SC-U) and in the presence of 0, 100, 200, or 300 mM Ca2+.For complementation of the yeast mutant, the full-length CDS of MTP10 was cloned into the pYES2 vector. The empty pYES2 vector and the recombinant MTP10-pYES2 plasmid were then introduced to the yeast strain mtp10 mutant and characterized its hypersensitive phenotype under high Mg2+ conditions.12 Based on the antagonistic relationship between Ca2+ and Mg2+, we further conducted a detailed assessment of Ca2+ rescue ability of mtp10 under high Mg2+ conditions. The 1/6 MS medium9 was supplemented with 10 mM MgCl2 and a broad range of Ca2+ concentrations were used for growth assays. In the 1/6 MS medium, it had already contained 0.53 mM Ca2+. On the 1/6 MS medium supplemented with 10 mM MgCl2, the growth of mtp10 mutant was significantly inhibited  family proteins, OsHKT2;4, was characterized as a calcium-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations.5 Further work also revealed that OsHKT2;4 could rescue the growth of MM281 in Mg2+-deficient conditions, suggesting that OsHKT2;4 can transport both Mg2+ and Ca2+.6 Considering that the loss-of-function of MTP10 results in seedlings being sensitive to both high-Mg and high-Ca stresses, we wondered whether MTP10 can also transport Ca2+. We then introduced the MTP10 in the yeast strain ∆gdt1, which had been well used to detect the Ca2+ transport activity of heterogeneously expressed proteins.14 Under 0, 100, and 200 mM Ca2+ conditions, the growth of wild-type BY4741 yeast strain, the yeast mutant ∆gdt1 expressing the pYES2 empty vector, and ∆gdt1 expressing the pYES2-MTP10 showed no difference. Under 300 mM Ca2+ conditions, BY4741 yeast showed resistance to high-Ca stress, suggesting that BY4721 yeast is capable of transporting extra Ca2+ out of the cell, thus avoiding the toxicity of high-Ca. Both the ∆gdt1 expressing the pYES2 and pYES2-MTP10 showed growth retardation under 300 mM Ca2+ condition. The growth of ∆gdt1 strains expressing pYES2 was not significantly distinguished from that of ∆gdt1 strains expressing pYES2-MTP10, indicating that MTP10 had no Ca2+ transport activity .In rice, one of the high-affinity K2+ fluorescent dye that has been used to track extracellular Ca2+ transport and distribution in the leaf.15 We first grew the wild-type Col-0 and mtp10 mutant in the 1/2 MS agar medium for 7 days, then the detached mature leaves were cultured in 1/6 MS liquid medium or 1/6 MS containing 10 mM MgCl2. Meanwhile, the 200 μM OGB-5 N was applied in the medium. The fluorescence signals at the time point 30 min, 1 h, 3 h, and 5 h were monitored, respectively. We found a gradually increased fluorescence signal in the vascular system over time  is a well-established membrane impermeable low-affinity Caver time . Under c0 mutant . Howeverpe Col-0 . Our res2+ being able to rescue the high-Mg sensitive phenotype of mtp10, we measured the major cation contents in wild-type Col-0 and mtp10 mutant under different treatment conditions by using the ICP-MS. The 7-day-old seedlings grown on 1/2 MS medium were transferred to 1/6 MS liquid medium, 1/6 MS supplemented with 6 mM MgCl2, or 1/6 MS supplemented with 6 mM MgCl2 and 3 mM CaCl2 for another 10 days. We separately sampled the roots and shoots of seedlings. Compared with the control group (1/6 MS), we found that both roots and shoots of the wild-type Col-0 and mtp10 mutant seedlings accumulated a large amount of Mg after high-Mg treatment (1/6 MS+10 mM MgCl2), while the shoots accumulated more Mg than that in the roots. When plants were grown in 1/6 MS medium supplemented with 6 mM MgCl2 and 3 mM CaCl2, the seedlings accumulated a relatively lower amount of Mg compared to the treatment with only 10 mM extra MgCl2  inhibited the Ca accumulation in both the roots and shoots of wild-type Col-0 and mtp10 mutant. When plants were grown on 1/6 MS medium supplemented with 6 mM MgCl2 and 3 mM CaCl2, the shoots of wild-type Col-0 and mtp10 mutant accumulated much higher Ca compared to other treatment groups. Moreover, the Ca contents in the root and shoot of the mtp10 mutant were slightly lower than that in the wild-type Col-0 , high-Mg (1/6 MS+ 10 mM MgCl2), or Mg-deficient (1/6 MS-Mg2+) hydroponic solutions. The total RNA of each group of samples was extracted after 10 hours of treatment in the medium, and then the expression of the target genes was detected. CIPK3/9/23/26, CBL2/3, MGT2, and MHX were mentioned previously as transporters associated with Mg2+ transport on vesicles. MGT9 was associated with reproductive development in Arabidopsis, MGT10 was localized in chloroplasts, and MGT6 was associated with Mg2+ uptake in roots under low-Mg environment and resistance to high-Mg stress.9 The expression of these genes was indeed up- or down-regulated in the mtp10 mutant compared to the wild-type Col-0, indicating the disruption of Mg homeostasis in the mtp10 mutant . Under control conditions (1/6 MS), the expression of all our selected genes showed upregulation in the mtp10 mutant, except for the expression of a downregulated MGT9. Among these genes, the CIPK26 and MGT10 exhibited at least a two-fold up-regulated expression in the mtp10 mutant. When seedlings were treated with high-Mg (1/6 MS+ 10 mM MgCl2), all of these genes showed up-regulated expression in the wild -type Col-0 seedlings compared to the control. However, in the mtp10 mutant, the expression patterns of these genes were differentiated. The expression of CIPK3 and CBL2 showed no difference between wild-type Col-0 and mtp10 mutant. The expression of CIPK9, CIPK23, CBL3, and MGT9 showed higher expression in the mtp10 mutant compared to that in the wild-type Col-0 seedlings. While the expression of CIPK26, MGT2, MHX, MGT6, and MGT10 was compromised in the mtp10 mutant . Our qRT-PCR results indicated at the molecular level that Ca2+ signaling is involved in regulating plant responses to high-Mg stress and that MTP10 plays a role in Ca2+ signaling regulated adaptation to high-Mg stress.We also detected the expression of Mg2+ plays irreplaceable roles in increasing cell wall strength, neutralizing the anion potential in the vacuole, and counteracting external stresses. Ca in the form of calcium pectinate forms an intermediate gelatinous layer of the plant cell wall, enabling cells and cells to join together to form tissues and giving the plant organ or individuals a certain mechanical strength.16 Mg2+ is a component of the chlorophyll molecule, among other functions in plants, and therefore essential for photosynthesis. Ca and Mg exhibit similar chemical properties, and both are doubly positively charged in the soil-water phase and at the soil cation exchange sites.17 The importance of Mg and Ca lies not only in the role they play in plant growth but also in their interaction with each other. In Citrus sinensis (L.) Osbeck seedlings, Mg-deficiency induced leaf vein lignification, enlargement, and cracking.18 These findings imply that Ca2+ and Mg2+ may have mutual antagonism in the cell wall, affecting plant growth, development, and coping with environmental stresses. During our study, we found that the mutation of MTP10 resulted in the plants could not efficiently facilitate Mg2+ diffusion from the xylem to shoots.12 The leaf veins in the mtp10 mgt 6 double mutant also showed abnormal morphology, suggesting that MTP10 is involved in vascular development.12 Under high Mg2+ conditions, we found that the Ca2+ accumulation in the vascular region of the leaf of mtp10 mutant was inhibited , phosphorus (P), and potassium (K), but are equally essential for plant growth, although the requirements of those elements are comparatively negligible to that of macronutrients. In plant cells, Canhibited . The ICPnhibited . We specn plants , and thurestored . The absArabidopsis, there are total of 12 MTP genes which had been identified and clustered in the CDF family, and several of which have been found function in metal ions transport and tolerance.19 However, only MTP10 was found to be localized to the plasma membrane of parenchyma cells in vascular bundles and function in unloading of xylem Mg2+ to confer tolerance to high-Mg stress.12 Among these MTP proteins, MTP1 was discovered to be involved in the response to high-Zn stress.19 The MTP11 was found to transport Mn2+ in a proton-antiport manner and thus conferring high-Mn tolerance.20 The tonoplast localized MTP8 was capable of coordinating Mn homeostasis and Fe reallocation.21 Further study suggested that calcium-dependent protein kinases, CPK4/5/6/11, can interact with and function upstream of MTP8, indicating the presence of tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis.22 Many studies have shown that Ca2+ signaling plays important role in plant response to high-Mg stress. By performing the Y2H and BiFC assay, we found that CIPK7 and CIPK26 can interact with MTP10 . In Arabidopsis, its genome contains 34 CPKs, which are essential to growth and development, and function in response diverse to biotic and abiotic stresses.23 It is likely that certain CBL-CIPK complex and CPK kinases interact with MTP10 and act as upstream elements to mediate phosphorylation of MTP10 and act as a calcium signaling network in response to high-Mg stress. Besides MTP10, the CorA-type plasma membrane localized Mg2+ transporter MGT6 was also found to play an essential role in high-Mg tolerance.24 It is also possible that both the CBL-CIPK and CPKs Ca2+ signaling network regulate the activity of MGT6 and thus MTP10 and MGT6 synergistically regulate Mg2+ transport and homeostasis in Arabidopsis.In 2+ and Mg2+, Ca2+ or Mg2+ can also affect the homeostasis of other ions, such K+, and Na+. The mechanisms of interaction between these ions are also intriguing to explore. For example, high-Mg stress can disrupt the K+ homeostasis, and the loss-of-function of two K+ transporters, AKT1 and HAK5, results in the seedlings hypersensitive to high-Mg stress.25 High-Na stress can interfere with K+ metabolism, and the presence of Ca2+ or Mg2+ reduces this interference.26 And if the K+ concentration in the cultured medium gets too high, the Ca2+ and Mg2+ uptake can be inhibited. Therefore, combining these studies and our research, we believe that understanding the relationship between ion interactions and the molecular mechanisms that mediate this relationship is a fundamental effort to achieve efficient nutrient uptake by plants and enhance crop yields.Apart from the interaction between CaClick here for additional data file.


Cnaphalocrocis exigua (Butler), which reduces rice output globally, twists and feeds on the young rice plant’s leaves. Protein elicitors are hypothesized to be biological components that promote rice in becoming herbivore resistant. The evolving elicitor protein PeBL2, obtained from Brevibacillus laterosporus A60, was tested for biocontrol against C. exigua. Four distinct PeBL2 doses  were assigned to evaluate the impact of PeBL2 on immature growth, survivability, and lifespan. Adult reproductive efficiency and the interaction between the pest and the disease were assessed against C. exigua. Further, the assessment of active compounds in PeBL2 with multi-acting entomopathogenic effects investigated the direct correlations of PeBL2 with temperature and climatic change in plants of rice (Oryza sativa L.). When compared to controls, PeBL2 treatments reduced the growing population of second- and third-generation C. exigua. Cnaphalocrocis exigua colonized control plants faster than PeBL2-treated O. sativa plants in a host selection test. PeBL2 doses delayed the development of the larval stage of C. exigua. PeBL2-treated seedlings generated less offspring than control seedlings, identical to fecundity. Trichomes and wax formation on PeBL2-treated leaves generated an adverse environment for C. exigua. PeBL2 altered the surface topography of the leaves, preventing colonization and reducing C. exigua reproduction. PeBL2-treated O. sativa seedlings exhibited somewhat increased amounts of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). Systemic defensive processes also included the activation of pathways . Following these results versus C. exigua, the use of PeBL2 in an agroecosystem with integrated pest management and biocontrol appears to be reasonable. These findings shed new light on a cutting-edge biocontrol technique based on B. laterosporus A60.The dangerous insect pest known as rice leaf folder  Pathogens must be capable of recognizing and eliminating host defense mechanisms to succeed. Plants have produced resistance (R) proteins in reaction to pathogens that escape, withstand, or reduce basal defense, resulting in gene-for-gene resistance. Plants include a mix of inducible and constitutive defense systems that aid in disease resistance . Microor2O2 and O2, frequently interact via ion influx. These chemicals act as signaling elicitors [Brassica napus L. (Brassicaceae) reduced immature plant persistence and Plutella xylostella (L.) (Lepidoptera: Plutellidae) population increase [LOX1 (lipoxygenase) and PAL  once aphids feed on them [Elicitors are in charge of boosting the plant’s innate immunity and action strategy in both abiotic and biotic approaches . They helicitors . They arlicitors . To guarlicitors ,16,17,18increase . Plants increase ,21,22. N on them .B. laterosporus can reduce infection in plants. Formerly, a protein elicitor named PeBL2 from the B. laterosporus strain A60 was reported. The 471 bp PeBL2 gene produces a 17.22 kDa protein with 156 amino acids containing an 84-residue signal peptide [B. laterosporus A60 has been identified that could help rice plants develop systemic resistance to C. exigua. Systemic resistances and an early plant protection reaction can both be activated by PeBL2. Regional and systemic defensive responses were seen in the host plants following the administration of recombinant PeBL2 from B. laterosporus A60 [B. laterosporus A60, on the management of C. exigua was investigated in the current research. The findings can aid in understanding the systematic resistance mechanism of B. laterosporus A60. The current investigation examined the function, mechanism, and biological activity of PeBL2 and its effects on C. exigua management. The contents of JA and SA, as well as ET gene expression, were determined following the discovery of trichomes on the leaf surface structure. The purpose of this article is to provide information about PeBL2′s function, method of action, and its impacts on the management of the rice leaf folder (C. exigua).By promoting host immunity,  peptide ,25. Howeorus A60 ,14,26,27E. coli BL21 (DE3) cells (E. coli after successful transformation. The full-length DNA sequence encoding PeBL2 (GenBank accession no. ERM16658.1) was amplified from the B. laterosporus A60 strain. The length of the PeBL2 gene is 471 bp, encoding a protein with 156 amino acids comprising a theoretical molecular weight of 17.22 kDa. It was confirmed that the PeBL2 is a hypothetical protein. The recombinant protein PeBL2 was purified with the column of the His-Trap HP  (The pET28 (a+)-TEV/LIC recombinant vector was transformed into 3) cells A. The exWI, USA) B and wasWI, USA) C.C. exigua was induced by PeBL2 in two separate ways. Predominantly, PeBL2-treated O. sativa seedlings exhibited a substantial C. exigua population loss; Cnaphalocrocis exigua’s daily reproducing ability was lowered when fed PeBL2-treated seedlings; all genera showed slower growth, as indicated by the observations that both the second and third generations showed lower growth rates . With a better surface environment and a more complex wax structure, C. exigua colonization should be more difficult.Compared to control seedlings, PeBL2 substantially impacted the plant growth and surface structure of eedlings . There wC. exigua invasion. Higher levels of JA, SA, and ET were discovered in PeBL2 saplings  and benzothiadiazole (BTH), on the population growth and fitness of different aphid species, an effect confirmed by the present findings [Solanum spp., i.e., seven trichomes with two major defense-related effects, have been tested [Solanum hirsutum, are avoided by C. exigua. Trichomes are also associated with the basic defensive mechanism of the tomato plant, as the surface area covered by the epidermal cell appendages of unicellular or multicellular hairs provide resistance to a variety of pests due to the “pubescence” of a plant. Colorado potato beetle (Leptinotarsa decemlineata) (Coleoptera: Chrysomelidae) settlement with thick trichomes was reduced in soybeans compared with the trichome-removed plants, which attracted more beetles [O. sativa seedlings as well. Compared to the controls, the PeBL2-treated seedlings and leaves had more trichomes. The PeBL2-treated O. sativa seedlings and leaflets were reported to have inhibited the reproduction and settlement of C. exigua with an increased number of trichomes. Another key part of the physical barrier lignin is the cell wall, which underpins plant resistance and is an indicator for the improvement of the cell wall [Arabidopsis and tomatoes, respectively [Brassica napus [O. sativa plants can, therefore, be hypothesized as being related to increased trichome density and the deposition of cuticular wax. In addition, the treatment of the PeBL2 elicitor had adverse effects on C. exigua fecundity. PeBL2-treated O. sativa plants produced significantly fewer C. exigua than the buffered and controlled seedlings. The results were consistent with previous studies showing that exogenous SA and MJ have caused lower mean lifetime fecundity [C. exigua fecundity, with the minimum fecundity at higher temperatures (26 °C) due to a decreased metabolic rate [O. sativa plants, the development time of larvae was extended compared to the control; even at a lower temperature (22 °C), the maximum larval development time was observed, indicating that a one-degree temperature increase affected the life cycle of the insect [O. sativa, in particular its effect on fecundity and larval development time.Elicitors are a novel biological tool for pest management that play a function in the signaling system and crop protection ,28. Seve and RNA . The poshibitors . Resultsp to 40% ,30. Herefindings . The curn tested . First,  beetles . PeBL2 a beetles , and it ell wall . The phyell wall . The useectively . The SA ca napus . Accumulecundity ,35,36. Tlic rate . Similare insect . AdditioM. exigua resistance. After C. exigua infestation in Arabidopsis, the transcript genes LOC_Os12g37350.1, LOC_Os11g39220.1, LOC_Os06g23760.1, LOC_Os08g39850.1, LOC_Os11g15040.4, LOC_Os01g56380.1, LOC_Os03g53200.1, LOC_Os05g41210.1, LOC_Os11g08380.1, LOC_Os03g01130.1, LOC_Os01g10940.1, and LOC_Os03g37710.1 were significantly increased. JA, SA, and ET molecules impart resistance to insect herbivorous diseases and pathogens, which enhances plant defense responses [Oryza sativa grew increasingly resilient to C. exigua after being introduced to PeBL2. Beneficial microorganisms initiated systemic resistance. This reaction is governed by a signaling system that involves the plant hormones SA, JA, and ET, as well as by microorganisms [O. sativa. Secondary metabolite formation can assist plants in resisting infestation by building mechanical obstacles to microbial activity, which drives phenolic metabolism and lignin creation, according to a previous study by Chaerle et al. [B. laterosporus A60 acts in O. sativa to control C. exigua [Additionally, JA, SA, and ET increased marker gene transcriptions, signaling that they play an essential role in rganisms . The rese et al. . Furthere et al. . Systemie et al. . These f. exigua .C. exigua and O. sativa in a regulated growing period before the testing. Cnaphalocrocis exigua were collected from a Triticum aestivum L. field in Guiyang, Guizhou, China, and shifted to O. sativa seedlings. A colony of C. exigua was maintained at ambient temperature for six months before the experiment. Cnaphalocrocis exigua were raised on rice plants at a temperature of 25 ± 2 °C, 80% humidity levels, and a photoperiod of 10 D:14 L. Oryza sativa seeds were rinsed in 75% ethanol for 20–40 s. Before use, the item was water cleansed and submerged for two to three days. Isolate of B. laterosporus strain A60 was maintained on solid media of potato dextrose agar  and LBA agar . The Luria–Bertani (LB) medium was utilized for bacterial B. laterosporus strain A60, which was grown in 200 mL LB medium for 48 h at 30 °C and 180 rpm for protein isolation.This study aimed to raise populations of w/v). The sample was centrifuged at 12,000× g for 20 min at 4 °C and the pellet was collected and dissolved in 100 mL of buffer . It was further dialyzed using the same buffer for 48 h for removing ammonium sulphate and harvesting the precipitate. Insoluble debris was removed by centrifuging and crude protein was finally collected by freeze-drying.Culture broth was centrifuged, and the supernatant was added to ammonium sulfate to 62% relative saturation  (Amersham Biosciences) was used to purify the crude protein. The crude protein was loaded onto a HisTrap SPHF column . Briefly, washing of column was conducted with Buffer A for eluting bound proteins and removing traces of any unbound proteins. An increasing linear gradient NaCl (0–1 M) was used to elute the target proteins. Buffer A was used to monitor the elicitor activity against the dialysis ,25. Furtm/z) was used to acquire data. Samples were digested after lyophilization using 2 μL of a 20% nitrile reconstitution. A 1 μL aliquot of dissolved sample was added directly to the illustration point on the target. After allowing the solvent to dry naturally, 0.5 μL of over-saturated CHCA Matrix Solution  was added to the corresponding target and dried. Nitrogen was blown onto the target sample and then the sample was put into the instrument through the target trough; analysis was completed using a time-of-flight tandem mass spectrometer 5800 MALDI-TOF/TOF, Applied Biosystems SCIEX, Concord, ON, Canada). To test the analysis, the laser source was 355 nm wavelength of Nd: YAG Laser. The acceleration voltage was 2 kV, and positive ion mode and automatic data acquisition were used. The first-order mass spectrometry scan range was 800–4000 Da. A signal-to-noise ratio greater than 50 was selected for the two-stage mass spectrometry of mother ions (MS/MS) analysis. Each illustration click was based on the selection of an 8A mother ion, a two-grade mass spectrometry (MS/MS) cumulative overlay of 2500, time, and a collision energy of 2 kV with CID shut down. Using the NCBI taxonomy database against Brevibacillus laterosporus (40021), a combined (MS + MS/MS) enzyme search was carried out with the following parameters: trypsin-fixed modifications, carbamidomethyl (C); dynamical modifications, oxidation. The mass values were as follows: Monoisotopic Protein Mass, unrestricted peptide; Mass Tolerance, ± 100 ppm fragment; Mass Tolerance, ± 0.4 Da peptide; Charge State, 1+ ax; Missed Cleavages, 1.Using the Triple TOF 5600 TOF MS Analyser , the active segment collected after preparative HPLC was analyzed. MS scanning mode with a scan range of 250–2000  database, a peptide sequence was obtained. The primers  were designed to amplify the entire coding sequence of elicitor protein-encoding gene of B. laterosporus. The genomic DNA was isolated using the Bacterial DNA Kit D3350-01 (ΩMEGA Biotech). The elicitor-encoding gene was amplified using genomic DNA as a template. The purified fragment was cloned into a pMD18-T simple vector  and then transformed into Trans 1 T1  for sequencing. The gene was constructed into the expression vector, pET-28-a(+), using the BamHI and XhoI restriction sites. The recombinant vector was then transformed into E. coli BL21 (DE3) for protein expression. The recombinant novel protein was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) . The cells were collected by centrifugation and then disrupted by an ultrasonic disruptor to obtain the supernatant. The purification of the recombinant protein consisted of three sequential procedures: (1) affinity chromatography with a HisTrap HP affinity column , (2) ion exchange chromatography with a Resource Q column , and (3) size exclusion chromatography with a Superdex-200 column . By separating on SDS-PAGE and staining the polyacrylamide gel with Coomassie Brilliant Blue, the protein purity and molecular weight were determined. Protein molecular markers  were used to estimate the size of the purified protein.From the results of de novo sequencing and BLAST searches in the NCBI . The pellets were removed, and the supernatant cells were resuspended and sonified by the ultrasonic disruptor. The supernatant was collected and filtered with filter paper (size 0.22 µm) after the solution was centrifuged at 12,000 rpm for 15 min. The Äkta Explorer Protein Purification System , as described by Wang et al., 2015, Jatoi et al., 2019, Javed et al., 2020, and Javed and Qiu, 2020 [PeBL2 was produced with the recombinant vector pET-28-aTEV/LIC in iu, 2020 ,14,24,25C. exigua population that had settled. Oryza sativa plants were soaked in 74.23 μg mL−1 PeBL2 for one day. Four organic seeds were developed (Flora Guard substrate). After seven days, O. sativa seedlings were sprayed with 74.23 μg mL−1 PeBL2 solution and infested with 15 C. exigua female adults 24 h later. Seedlings were treated weekly. Every five days after inoculation, C. exigua were counted. The data fractions were used for data analysis and the conclusion of results. Controls and negative controls were treated with water and 74.23 μg mL−1 of a buffer , respectively. Plants were caged (60 × 60 × 60 cm) in transparent, breathable mesh. Each time, four replicates were used.This experiment was designed to estimate the size of the C. exigua’s intrinsic development. Oryza sativa seedlings were treated the same way (Sec: 2.7). After one day, the seeds were showered with 74.23 μg mL−1 of PeBL2 pure protein solution. A glass tube wrapped with cotton gauze segregated sprouting, and C. exigua mortality on the leaves was confined in a plastic ecological cage  (2.7 × 2.7 × 2.7 cm). The ecological cage’s periphery was sponge-coated to eliminate mechanical damage to the leaf. The C. exigua instars 1st–4th were tested for larval development for 12 h. To avoid overcrowding, newly molted larvae were tallied twice daily to govern the overall aggregate of period and offspring produced. This was repeated on seeds and plants five days afterwards. The experiment employed 30 replicates of each treatment. The intrinsic rate was determined in the following manner:This experiment aimed to examine if nourishing PeBL2-treated or control seedlings increased Md is the number of newborn larvae within a development period of Td, which is the time interval between a C. exigua birth and reproductionC. exigua. On O. sativa plants, PeBL2 was tested against C. exigua at 74.23, 45.53, 22.26, and 11.13 μg mL−1. This was calculated using the Bradford assay [O. sativa plants at the three-leaf stage. Water and buffer were used to treat the controls . Then, 3–6 freshly molted larvae of C. exigua were given to O. sativa plants and desiccated instantaneously. The entire number of descendants formed by all C. exigua instars was used to calculate the overall larval development period, while C. exigua longevity was derived from the number of days they lived. The bioassays were performed in triplicate at three different temperatures .This study examined the larval development time as well as the fecundity of rd assay . Using aC. exigua development and morphology. Oryza sativa seedlings and seeds were treated as intended in This research aimed to determine if PeBL2 influenced The point of the study was to figure out how much SA, JA, and ET were produced in this manner; seeds and seedlings were managed as initially reported. The aerobatic sections of seeds were obtained for SA, JA, and ET ,27,47. TTotal RNA of the treated and control plant leaves after feeding of rice leaf folder was isolated separately using the plant RNA ER301-01 kit , following the manufacturer’s protocol. The concentration of RNA was quantified using a nano-photometer . A One-Step cDNA Removal and cDNA Synthesis AT341-01 kit  was used for cDNA synthesis.18S was considered as endo-reference [LOC_Os12g37350.1, LOC_Os11g39220.1, LOC_Os06g23760.1, LOC_Os08g39850.1, LOC_Os11g15040.4, LOC_Os01g56380.1, LOC_Os03g53200.1, LOC_Os05g41210.1, LOC_Os11g08380.1, LOC_Os03g01130.1, LOC_Os01g10940.1, and LOC_Os03g37710.1 were chosen as key genes [-ΔΔCT methodology was used to determine the relative expression levels of genes [The goal of this experiment was to determine the relative expression levels of genes. To extract RNA, synthesize cDNA, and perform a real-time quantitative polymerase chain reaction (qRT-PCR) , kits from TransGen Biotech  were utilized. qRT-PCR was performed to quantify the expression profile of the key genes associated with JA, SA, and ET pathways. The feeding of rice leaf folder on plants treated with protein elicitors was considered a treatment, and plants treated with buffer were considered controls. For amplification of qRT-PCR, 12 gene primer pairs  were useeference . LOC_Os1ey genes . The keyt-test, whereas data from three or more treatments were compared using the LSD and an ANOVA with Statistix software version 8.1 . Prior to analysis, C. exigua fecundity data were square root transformed. A one-way factorial analysis of variance was performed among treatment factors such as the PeBL2 concentrations and varied temperature regimes to eliminate differences, followed by a 95% probability LSD test. The gene expressions (qRT-PCR) were obtained by the comparative CT (2-ΔΔCT) method; Student’s t-tests (α = 0.05) were used to compare the fold changes in plant samples treated with PeBL2 and buffer.Data from two treatments were compared using an independent Levene’s test and a two-tailed C. exigua resistance in O. sativa, reducing second- and third-generation M. exigua fecundity and its colonization. PeBL2 affected the surface structures of O. sativa leaves. SA, JA, and ET exhibited slight increases in relative expression levels, acting as potential factors engaged in systemic defense responses generated by PeBL2 in O. sativa. Furthermore, PeBL2 exhibited a significant suppression of C. exigua life characteristics in the laboratory; however, more research is required to maximize its effects in the field. Future research should explore whether the wax composition affects C. exigua behavior, how SA and JA work in induced resistance, and whether other plant hormones are involved. We are convinced that PeBL2 could be used as a “vaccine” for O. sativa seeds and seedlings to protect them from C. exigua.PeBL2 enhanced


Ca2+ participates in and plays a crucial role in the energy metabolism, physiology, and pathology of mitochondria. Mitochondria control the uptake and release of Ca2+ through channels/transporters, such as the mitochondrial calcium uniporter (MCU), and influence the concentration of Ca2+ in both mitochondria and cytoplasm, thereby regulating cellular Ca2+ homeostasis. Mitochondrial Ca2+ transport-related processes are involved in important biological processes of tumor cells including proliferation, metabolism, and apoptosis. In particular, MCU and its regulatory proteins represent a new era in the study of MCU-mediated mitochondrial Ca2+ homeostasis in tumors. Through an in-depth analysis of the close correlation between mitochondrial Ca2+ and energy metabolism, autophagy, and apoptosis of tumor cells, we can provide a valuable reference for further understanding of how mitochondrial Ca2+ regulation helps diagnosis and therapy.Mitochondria, as the main site of cellular energy metabolism and the generation of oxygen free radicals, are the key switch for mitochondria-mediated endogenous apoptosis. Ca IP3-depillation . Moreoveeability . Mitochoytoplasm . The uptm signal . The abnm signal .2+ signaling is a key regulator in a series of tumor cell processes, including tumor growth, progression, and metastasis [2+ is a hallmark of many tumors. For instance, Ca2+ is decreased in pancreatic cancer, colon cancer, and prostate cancer, while Ca2+ is increased in breast cancer and hepatocellular carcinoma (HCC) [2+ signaling accelerates lipid accumulation and may promote HCC development [2+ uptake is necessary for the progression of triple-negative breast cancer (TNBC) in vivo and it can also activate the hypoxia-inducible factor-1 alpha (HIF-1α) signal pathway, which contributes to tumor growth and metastasis [2+ signaling is altered in urinary bladder carcinoma cells [2+ entry (SOCE) intracellular Ca2+ oscillation upregulation can activate downstream pathways, stimulate the proliferation and migration of esophageal squamous cell carcinoma (ESCC) cells, enhance their ability to invade other tissues, and promote the formation and growth of ESCC tumors in vitro and in vivo [At present, the incidence and mortality rate of malignant tumors is increasing year by year and it is the primary cause of death from all kinds of diseases. It is estimated that by 2040, 28.4 million new cases of cancer will be diagnosed worldwide, which represents an increase of 47% since 2020 [tastasis . The altma (HCC) . Alteredelopment . Mitochotastasis . In addima cells . Orai1-s in vivo . Meanwhi in vivo .2+ are required for the activity of several mitochondrial enzymes and this, in turn, regulates mitochondria-derived reactive oxygen species (ROS) generation; this is a known driver of pro-tumorigenic redox signaling, resulting in the activation of pathways implicated in cellular proliferation, metabolic alterations, stress adaptations and cell death [2+ homoeostasis is involved in the metabolism, apoptosis, proliferation and other important processes of tumor cells [2+ in the regulation of tumor cell development and its molecular mechanisms, which is conducive to providing a basis for tumor therapy via targeting mitochondrial Ca2+ homoeostasis and regulation.Regulated elevations in Call death ,30,31,32or cells ,34. In t2+, the concentration of Ca2+ in the membrane gap is equivalent to that in the cytoplasm [2+ in cytoplasm is about 100nM. When the cells are excited, the concentration of Ca2+ in the cytoplasm can rise to 1–3 µM. [2+ by mitochondria can be regulated by the one-way transport mechanism or transporter [2+ influx is mainly mediated by the mitochondrial calcium uniporter (MCU), voltage-dependent anion-selective channel (VDAC), and mitochondrial ryanodine receptor transporter. Furthermore, the mitochondrial Ca2+ efflux pathways mainly include leucine zipper/EF hand-containing transmembrane-1 (LETM1), mitochondrial Na+/Ca2+ exchanger (NCLX), and mitochondrial permeability transition pore (MPTP).Due to the outer membrane of mitochondria possessing a high permeability for Caytoplasm . In the  1–3 µM. . In factnsporter . The mit2+ channel ubiquitous in mitochondrial intima [2+ transporter [2+ uptake [2+ uptake triggered by several stimuli in different cell types [2+ uptake through the MCU is driven by an electrochemical gradient. The MCU and related regulating molecules, including the essential MCU regulator (EMRE), mitochondrial calcium uptake (MICU)1, MICU2, MICU3, MCU-dominant negative beta subunit (MCUb), and MCU regulator 1 (MCUR1), form a large complex to manipulate the activities of the MCU [MCU is a Cal intima . It is gnsporter  and sile+ uptake  , MICU1−MICU2 allows Ca2+ to enter the mitochondria through the MCU [2+ flux, decrease mitochondrial oxidative phosphorylation (OXPHOS) and ATP production, and activate AMPK-dependent autophagy [In higher eukaryotes, the EMRE mediates MICU1/MICU2 to regulate Cathe EMRE . The reg the MCU . Down reutophagy . In parautophagy ,51.2+ and the production of ROS, suggesting that the binding of MICU1 to the MCU is necessary for the function of the MCU complex and the entry of Ca2+ into mitochondria is a prerequisite survival factor of cancer cells. MICU1 has been shown to be methylated by protein arginine methyltransferase 1 (PRMT1) in cancer cells, yielding decreased Ca2+ sensitivity and reduced Ca2+ entry. UCP2/3 is fundamental for mitochondrial Ca2+ uptake in cancer cells [2+ sensitivity of MICU1 and re-establishes Ca2+ entry into mitochondria [2+ threshold activated by the MCU. Therefore, MICU2 can inhibit MCU activity at low Ca2+ concentrations [MICU1 is upregulated in ovarian cancer cells and its expression is closely related to the survival of cancer cells and tumor growth . In thiser cells . When itchondria . This mechondria ,56. MICUtrations .2+ [2+ uptake does not depend on the MCUR1, which is only a regulator that sets the Ca2+ threshold of the transition in mitochondrial permeability. Inhibiting the expression of the MCUR1 increases the Ca2+ threshold for inducing MPTP conversion, which can reduce the mitochondrial cell death that is induced by an overload of Ca2+ [Although MICU1, MICU2, and MICU3 belong to the same family, they have different effects on the MCU. MICU2 is the gatekeeper of the MCU, while MICU3 is an MCU activation enhancer. Overexpression of MICU3 causes a 10-fold increase in transient Ca2+ . MCUb di2+ . At pres2+ , is the  of Ca2+ .+)-dependent Na+−Ca2+ reverse exchange channel and can positively regulate the outflow of Ca2+ in mitochondria [2+ in mitochondria is too high, it will enhance the activity of the NCLX and cause the opening of the MPTP on the inner membrane of mitochondria. Ca2+ is the center that regulates the MPTP. It can directly regulate the MPTP itself and indirectly affect the MPTP by regulating the adenosine diphosphate (ADP)/ATP balance, mitochondrial membrane potential, and ROS/reactive nitrogen level [2+/Ca2+ caused by MPTP opening are reversible [2+ in the mitochondrial matrix is low, LETM1 can transport Ca2+ into the matrix. On the contrary, Ca2+ is transported out of mitochondria. The study also found that silencing LETM1, despite the presence of the MCU, can still inhibit the influx of Ca2+ into mitochondria  Ca2+ exists in a domain formed between the ER and the mitochondria, which is called the mitochondrial-associated membrane (MAM) and controls mitochondrial Ca2+ homeostasis [2+ homeostasis and promote cell survival, migration, invasion, metastasis, autophagy, and inhibit apoptosis [2+ levels in cancer cells [2+) transporter are abnormal. The imbalance of Mg2+ homeostasis may destroy Ca2+ homeostasis [2+ signaling network by changing the expression and function of cation channels, pumps, or transporters [Mitochondrial Caeostasis   In eostasis . (4) Cansporters .2+ participates in almost all physiological activities in cells. Mitochondria were originally considered to be a “Ca2+ pool” with the ability to absorb a large amount of Ca2+, and the uptake of Ca2+ by mitochondria increases significantly when the extramitochondrial Ca2+ is overloaded [2+ can stimulate glycogen decomposition and glucose oxidation, resulting in an increase in ATP supply [2+ concentration is transmitted to mitochondria and Ca2+-activated dehydrogenase is a key rate control enzyme in the tricarboxylic acid cycle (TAC) flux. Ca2+ activation will lead to the increase in pyridine nucleotide reduction and oxidative phosphorylation [2+ uptake can activate matrix enzymes, stimulate ATP production, and regulate energy metabolism by activating pyruvate dehydrogenase, isocitrate dehydrogenase, and ketoglutarate dehydrogenase. This “parallel activation model” provides a mechanism in which Ca2+ stimulates the process of energy consumption caused by physiological activities such as various hormones, muscle contraction, or increased cardiac load [Caerloaded . It has P supply . The incrylation . Mitochoiac load . It alsoMetabolic reprogramming in tumor cells is considered to be a sign of cancer and is involved in tumor growth and development. Compared to normal cells, under the condition of sufficient aerobic supply, tumor cells still obtain energy by aerobic glycolysis and produce a large amount of lactic acid and a small amount of ATP . M2 isof2+ affects the functional changes of mitochondria  and thus, cell energy metabolism, which is closely related to the occurrence and development of tumors  the inositol 1,4,5-trisphosphate receptor (IP3R) mediates Ca2+ to reduce the release of Beclin1 so as to reduce autophagosome production and inhibit autophagy; (2) IP3R mediates Ca2+ activation of calpain, separates autophagy protein 5 from autophagy protein 12, reduces the level of their complex, and inhibits autophagy [2+ released by the ER to the mitochondria enhances the TAC and ATP production and inhibits autophagy [3R mediates Ca2+ into mitochondria, resulting in increased ATP production and the inhibition of AMPK, thereby inhibiting autophagy. Therefore, Ca2+ may have different regulatory effects on autophagy.Intracellular Cautophagy ,91, whic factors . Ca2+ ovse CaMKK . Consisty in HCC . Conversutophagy ; (3) Theutophagy ,97; (4) 2+ exchanges have been associated with autophagy and mitophagy regulation. Therefore, unsurprisingly, some mitochondrial calcium transporters, such as the MCU, have recently been found to be involved in autophagy and mitophagy regulation in tumor cells.As with non-selective autophagy, the role of mitophagy is complex and can depend on tumor type and stage. Since both autophagy and mitophagy are related to mitochondrial function, targeting mitochondrial ion channels may also be an interesting strategy to regulate autophagy or mitophagy in tumors. Ca2+ transporter in the matrix, which is a major mediator of calcium influx into mitochondria. The MAM is an important part of Ca2+ transfer from the ER to the mitochondria to regulate mitochondrial enzymes. Ca2+ flow mainly occurs through IP3R and transient receptor potential cation channel subfamily M member 8 (TRPM8) in the ER membrane [3Rs and the interruption of Ca2+ flow between the ER and the mitochondria break the calcium homeostasis and decrease mitochondrial bioenergetics, which subsequently decreases OXPHOS and activates autophagy [2+ in the MAM in tumor cells, decreasing mitochondrial function and inducing cell death. The MCU has also been found to be altered in tumors from different tissues [2+ and the MCU represent attractive antitumor targets for regulating mitochondrial dysfunction and autophagy/mitophagy in tumors.The MCU is generally considered to be the main Camembrane . Sensitiutophagy ,100. Howutophagy   After cells receive the pro-apoptotic signals, the increase of ROS promotes mitochondrial Ca2+ influx, which may be caused by affecting voltage-dependent Ca2+ channels, non-specific cell membrane Ca2+ permeability changes, and Na+/Ca2+ exchanges [2+ can activate other enzymes to further upregulate the level of oxygen free radicals, so ROS can indirectly produce more oxides and further promote the rise in mitochondrial Ca2+ level [2+ leading to oxidative metabolism impairment and ROS overproduction [2+ and apoptosis of mitochondria [3R production and affect Ca2+ release from the ER into mitochondria [2+ pump and inhibit intracellular or extracellular ER Ca2+ transfer by inhibiting the Ca2+-ATPase pump [2+ can induce MPTP opening. On the other hand, MPTP opening leads to a large increase in ROS [Mitochondria are the central link mediating apoptosis, as well as the main site of ROS generation . With thxchanges ; (2) Inc2+ level . In addioduction . Previouchondria ; (3) ROSchondria ; (4) ROSase pump ; (5) Bote in ROS .2+ signal transduction mechanism, which promotes the occurrence of tumors [2+ signal proteins, including VDAC1, IP3R, and SERCA, which play vital roles in the processes of tumors. VDAC1 plays a significant role in cellular Ca2+ homeostasis and it has also been recognized as a key protein in mitochondria-mediated apoptosis [3R1 in bladder cancer cells prevents mitochondrial Ca2+ overload by decreasing the uptake of ER−mitochondria Ca2+, thereby reducing cisplatin-mediated apoptosis [2+ signal is remodeled in tumorigenesis [Several types of tumor cells have experienced extensive reorganization of their internal Caf tumors . Calciumpoptosis . For exapoptosis . The dowpoptosis . The sigigenesis .3R3 proteins and interfering with the Ca2+ exchange between the ER and mitochondria [3R3, which slows down FBXL-2-mediated IP3R3 proteasome degradation. This represents a new mechanism. The deletion of PTEN enables tumor cells to avoid apoptosis [3R3 impairs the pro-apoptotic mitochondrial Ca2+ transfer. (2) The tumor suppressor protein BRCA1-associated protein 1 (BaP1) is a deubiquitinase that promotes the transfer of ER−mitochondria Ca2+ by stabilizing IP3R3. Under long-term environmental pressure, the function of BaP1 will be seriously disrupted, which is related to the acquired inactivating mutations of the BaP1 gene. The loss of BaP1 will lead to the downregulation of IP3R3, which hinders the effective apoptotic clearance of damaged cells and is conducive to the occurrence of tumors and the survival of malignant cells [2+ signal regulation, such as resistance to apoptosis. Because mitochondrial Ca2+ overload is related to apoptosis and death, modifying ER−mitochondria Ca2+ transfer at the MAM will change the sensitivity of apoptosis, and tumor cells can acquire resistance to cell death accordingly [3R-mediated Ca2+ signaling or increasing the transmembrane distance at the MAM, the efficiency of ER−mitochondria Ca2+ transfer can be reduced, so as to decrease the sensitivity of tumor cells to apoptosis [Recently, it has been found that in several cancer types, the imbalance of two new mechanisms will affect the renewal of the proteasome, so as to regulate the apoptosis sensitivity of tumor cells by affecting IPchondria . (1) Thepoptosis . The downt cells . In addiordingly . For exapoptosis   is an important signal molecule in the pathway of cell survival, apoptosis, and necrosis, which is significantly upregulated in colorectal cancer (CRC) cells. RIPK1 interacts with the MCU to promote CRC cell proliferation by increasing mitochondrial Catabolism . Comparetabolism . Dihydrotabolism . MCU-meding MPTP . The MCUing MPTP . The MCUpoptosis . RY10-4  the MCU . The MCUrognosis . The dater cells .2+ channels play different roles in some cancers due to different regulatory mechanisms. Previous studies have revealed that a highly expressed MCU promotes the metastasis of adrenocortical carcinoma breast cancer cells with poor prognosis. In hepatocellular carcinoma studies, MCU-dependent mitochondrial Ca2+ uptake promotes metastasis of HCC cells [https://tnmplot.com/analysis/; accessed on 31 March 2022) [http://gepia.cancer-pku.cn/index.html; accessed on 31 March 2022) and found that in adrenocortical carcinoma and hepatocellular carcinoma, overall survival is significantly greater in low MCU expression than in high MCU expression   and demopression A,B. On tpression C,D. To s2+ homeostasis through the uptake of Ca2+ into the mitochondria via MCU and the release of Ca2+ from the mitochondria via NCLX, regulating intramitochondrial and intracytoplasmic Ca2+ concentrations. Therefore, the regulation of both is deeply intertwined. Since the reorganization of cytosolic calcium signaling commonly occurs in tumor cells, mitochondrial calcium imbalance causes alterations in cytosolic calcium signaling and thus, affects tumorigenesis and progression [2+-buffering protein parvalbumin significantly inhibits the ROS/Nrf2/Notch pathway, MCUR1-induced epithelial− mesenchymal transition and HCC metastasis [2+ uptake and causes mitochondrial Ca2+ imbalance, which, in turn, promotes CRC cell growth in vitro and in vivo. Ru360 is a highly potent and selective MCU inhibitor that can effectively block MCU-mediated mitochondrial Ca2+ uptake and, ultimately, slow CRC progress. These results may provide a potential pharmacological target for CRC treatment [2+ [2+ uptake.Mitochondria regulate Cagression . Given tgression . Anothertastasis . In a sttastasis . An uprereatment . Saverioment [2+ . Therefo2+ and the MCU play crucial roles in energy metabolism, autophagy/mitophagy, and apoptosis of tumor cells. The discovery of the MCU and its regulatory proteins represents a new era of research on MCU-mediated mitochondrial Ca2+ dyshomeostasis in cancer. Currently, drug candidates targeting the MCU or its regulatory factors are still emerging. Although a flurry of studies has confirmed the correlation between mitochondrial Ca2+ dyshomeostasis and the progression of a variety of tumors, the exact mechanism and targeted therapy remain to be further elucidated. The tumor diagnosis and treatment strategy for mitochondrial Ca2+ homeostasis will bring a new dawn to tumor risk prediction, precancerous lesion screening, clinical targeted therapy, and prognosis assessment.The occurrence and development of tumors is a complex process regulated by multiple signaling networks. In this paper, we summarize, analyze, and discuss that mitochondrial Ca


To assess the visual and refractive outcomes of femtosecond laser-assisted in situ keratomileusis (FS-LASIK) concurrent with accelerated cross-linking (LASIK Xtra) compared with conventional FS-LASIK (convLASIK) for high myopia in Chinese individuals.In this prospective, randomized, fellow-eye comparison study, 25 patients with high myopia were treated randomly with LASIK Xtra in one eye and convLASIK in the other. A 24-month follow-up was conducted, and the main outcome measures included uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction spherical equivalent (MRSE) and corneal tomography.P = .001), but the difference became nonsignificant from 1 week after surgery. The efficacy index was 0.88 ± 0.18 in the LASIK Xtra eyes and 0.99 ± 0.13 in the convLASIK eyes at 24 months (P = .028). Throughout the follow-up period, a slight myopic shift in the MRSE and keratometry values were observed in both groups without significant intergroup differences. The coefficient of determination was 0.9982 in the LASIK Xtra eyes and 0.9987 in the convLASIK eyes. The corneal density was significantly higher, and demarcation lines were visible in the first 6 months in LASIK Xtra eyes, but both signs of cross-linking gradually disappeared during follow-up. No severe complications were detected in either group.The UDVA was 0.09 ± 0.15 logMAR in the LASIK Xtra group, which was significantly worse than that in the convLASIK group 1 day postoperatively (LASIK Xtra showed comparable safety and predictability with convLASIK for high myopia in Chinese, but lower efficacy and no greater stability was observed up to 24-month follow-up. Femtosecond laser-assisted in situ keratomileusis (FS-LASIK) is a common and affordable refractive surgery for a large number of people, especially those with aesthetic or occupational demands. However, the circumferential corneal flap has been proven to weaken the integrity and rigidity of the cornea as much as 14% to 33% , which mMany studies have focused on demonstrating the greater stability achieved with CXL concurrent with LASIK (LASIK Xtra) than with convLASIK, but no consensus has been reached. Some studies have suggested that LASIK Xtra shows reduced myopic drift and better keratometric stability than convLASIK –11, whilThis prospective self-controlled randomized cohort study enrolled 50 eyes of 25 patients with high myopia and/or myopic astigmatism in the Peking University Third Hospital (PUTH) from May 2019 to January 2020. This study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the PUTH.The inclusion criteria were as follows: 1) age > 18 years; 2) preoperative spherical refractive error from -6.00 D to -14.00 D; 3) stable refractive error for at least 1 year; 4) cessation of wearing soft contact lenses for 1 week or rigid contact lenses for 3 weeks; 5) no ocular disease except for ametropia; and 6) no history of ocular surgery or trauma. The exclusion criteria included 1) surgical contraindications such as explicit keratoconus or forme fruste keratoconus; 2) anisometropia of more than 1.00 D; 3) predicted residual stromal bed thickness of less than 280 μm; 4) active ocular inflammation; 5) systemic diseases or medication affecting wound healing; and 6) pregnancy or lactation.All patients signed informed consent forms before the surgery after receiving an explanation of the nature and consequences of the study and underwent convLASIK in one eye (selected randomly) and LASIK Xtra in the fellow eye. The randomization was performed by sealed opaque envelopes.Detailed ocular examinations prior to the surgical procedures included measurements of uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction, cycloplegic refraction and intraocular pressure, slit-lamp examination, dilated funduscope examination, corneal topography , corneal tomography , endothelial cell count  and partial optical coherence interferometry . The topographic parameters from the Topolyzer device were used for iris registration and automatic kappa angle and cyclotorsion compensation during operation, while those from the Sirius device were adopted for follow-up and statistical analysis. The corneal density was measured manually in triplicate by an experienced technician using a Scheimpflug image from the Sirius device, and the average value was expressed as a percentage of the maximum value Fig. . The sam2 for 90 s .All surgeries were performed by two experienced ophthalmologists (YZ and YC) under topical anaesthesia. Flaps were created by a WaveLight FS200 femtosecond laser  with a thickness of 110 μm and diameter of 8.5 to 9.0 mm. The hinge position was superior. Ablation was performed by a WaveLight EX500 excimer laser  with an optical zone of 6.0 to 6.5 mm, and the nomogram supplied by the manufacturer was adopted with a refractive target of plano-correction for all eyes. For the LASIK Xtra group, the bare stromal bed was saturated with 0.22% riboflavin diluted with saline  for 90 s. The flap was carefully protected so as not to contact the Vibex Xtra. The riboflavin was rinsed thoroughly with balanced saline solution afterwards, and the flap was repositioned properly. Irradiation was then accomplished using a KXL system  with a UVA fluence of 30 mW/cmPostoperatively, topical medications were administered as follows in both groups: 0.1% fluorometholone  four times daily and tapering for 1 month, 0.5% levofloxacin  Co Ltd, Jiangsu, China) four times daily for 2 weeks and artificial tears four times daily for 1 month.All patients were asked to test their visual acuity one day and one week after surgery. Further follow-ups were performed at 1, 3, 6, 12 and 24 months. Follow-up examinations included measurements of UDVA and CDVA, manifest refraction, slit-lamp examination, corneal tomography and endothelial cell count (ECC).P value of < 0.05 was considered statistically significant.Visual acuity values measured by a Snellen chart were converted to logarithm of the minimum angle of resolution (logMAR) accordingly. Efficacy and safety indices were calculated as the ratio of postoperative UDVA and CDVA, respectively, over preoperative CDVA. Data were analysed using SPSS . Normality of continuous variables was tested using the Shapiro–Wilk test. Normally distributed data are presented as the mean ± standard deviation, and nonnormally distributed data are presented as the median. Normally distributed variables were compared using the paired t test, while the Wilcoxon signed-rank test was used in the absence of a normal distribution. Categorical variables are presented as frequencies and percentages and were compared using the chi-squared test or Fisher’s exact test. Intragroup difference analysis during the follow-up period was performed using repeated ANOVA or the Friedman test. The Pearson method was used for correlation analysis. A The mean age was 27.7 ± 7.0 range 18 to 42) years, and 22 (88%) were females among the 25 patients. There was no significant difference in baseline characteristics between the two groups  eyes of the LASIK Xtra group at the 1-month follow-up and remained in 12 eyes at 3 months. The haze was barely resolved until 6 months after surgery.P = 0.367 and 0.338, respectively)  eyes of the LASIK Xtra group versus 9 (50%) eyes of the convLASIK group gained 1 Snellen line of CDVA, while 4 (22%) eyes and 1 (6%) eye lost 1 line in the LASIK Xtra and convLASIK groups, respectively . However, the difference became nonsignificant at 7 days postoperatively (P = 0.084) and all subsequent follow-ups between the two groups. At 24 months, the logMAR UDVA was 0.00 ± 0.11 in the LASIK Xtra group and -0.05 ± 0.04 in the convLASIK group (P = 0.119)  was achieved in convLASIK eyes than in LASIK Xtra eyes  , 16 (88.89%), 17 (94.44%) and 17 (94.44%) eyes in the LASIK Xtra group at 1, 3, 6 and 24 months respectively after surgery, while in the convLASIK group, 18 (85.71%), 16 (88.89%), 17 (94.44%) and 18 (100.00%) eyes, respectively, achieved a 20/20 or better UDVA  .The MRSE was measured at each follow-up point, and the results are listed in Table P = 0.034), steep K (P = 0.015) and mean K (P = 0.020) in the LASIK Xtra group and flat K (P = 0.041) in the convLASIK group increased significantly. However, the K values did not vary significantly between the two groups throughout the follow-up period, nor did the changes in K values from 1 month or 3 months to 24 months postoperatively , 16 (88.89%), 16 (88.89%) and 16 (88.89%) eyes, respectively, achieved an MRSE within ± 0.50 D of the attempted MRSE in the LASIK Xtra group, while in convLASIK eyes, 18 (85.71%), 17 (94.44%), 15 (83.33%) and 16 (88.89%) eyes achieved an MRSE within ± 0.50 D, respectively  and from -1.14 ± 0.75 D to -0.32 ± 0.30 D in convLASIK eyes (P < 0.001) (Table P = 0.711)  Fig. .Fig. 6ReBoth groups showed a strong correlation between the attempted and achieved spherical equivalent refraction, as shown in scatter plots Fig. . The coeP = 0.001, 0.001 and 0.023, respectively) .The thinnest corneal thickness was 385 ± 24 μm, 389 ± 23 μm and 389 ± 23 μm at 1, 3 and 6 months, respectively, in the LASIK Xtra group, which was accordingly lower than the 399 ± 25 μm, 406 ± 27 μm and 404 ± 22 μm in the convLASIK group , 3 (P < 0.001), 6 (P = 0.002) and even 24 (P = 0.043) months, but the difference between the two groups gradually narrowed . A similar conclusion was drawn by Chan (P = 0.001) [In regard to efficacy, the LASIK Xtra group may perform worse shortly after surgery. In this study, the LASIK Xtra protocol had inferior refractive outcomes one day after surgery, which did not recover until one week postoperatively. Tomita  and coll= 0.001) , and in = 0.001) , 14, 16.= 0.001) , 14–16. = 0.001) , we obseIn general, LASIK with and without CXL both achieved good predictability with a strong correlation between the attempted and achieved MRSE. Low J.R . reporteP = 0.09), but which did not differ significantly from LASIK Xtra (P = 0.86). Kanellopoulos made similar conclusions of the nonsignificant difference concerning myopic regression between the two procedures through both 12-month (P = 0.063) [P = 0.065) [P = 0.032 [Stability is the core focus in the evaluation of LASIK Xtra compared to convLASIK; however, previous studies failed to report evidence sufficient enough to support LASIK Xtra. In the present study, the stability concerning both MRSE and K values in the LASIK Xtra group showed no advantage over that in the convLASIK group throughout the 24-month follow-up. In researches by Tomita  and Tan = 0.063)  and 24-m = 0.032 , respectThe innovation of the flap creation technique using a femtosecond laser rather than a microkeratome has improved the predictability of flap thickness and reduced the risk of severe flap complications , 26. In The limitations of our study included the relatively high rate of loss to follow-up due to the implementation of COVID-19 pandemic prevention and control guidelines; also, there is a lack of consecutive biomechanical examinations and accurate densitometry provided by Oculus Pentacam, especially at the early postoperative stage, when they are more likely to differ. Moreover, the 24-month follow-up is greater than the average level of previous studies, but a longer follow-up is still needed, considering that post-LASIK ectasia may occur from 1 week to several years postoperatively , 30.In conclusion, we conducted a randomized fellow-eye controlled study in a higher myopic population with a longer follow-up period than previous studies. The present study confirmed LASIK Xtra to be as safe as convLASIK, and favourable predictability was also achieved. However, no superior stability was observed in LASIK Xtra eyes over convLASIK eyes, which was the initial motivation for combining CXL with the standard LASIK procedure. In addition, simultaneous CXL may weaken the advantages of LASIK, such as rapid visual rehabilitation, and increase relift difficulty for enhancement. We therefore tend to regard CXL as a treatment for post-LASIK ectasia rather than a prophylaxis unless more compelling evidence with long-term follow-up can be published.


Here, however, we show that these functions are clearly segregated by molecular dynamics.In olfactory transduction, Ca 2+ concentration increases through the opening of CNG channels, a phenomenon that underlies two major functions, namely, signal boosting and olfactory adaptation. Signal boosting is achieved by an additional opening of the Ca2+-activated Cl− channel whereas adaptation is regulated by Ca2+ feedback to the CNG channel. Thus, the influx of Ca2+ and the resultant increase in cytoplasmic Ca2+ levels play seemingly opposing effects: increasing the current while reducing the current through adaptation. The two functions could be interpreted as compensating for each other. However, in real cells, both functions should be segregated. Ca2+ dynamics in olfactory cilia need to be directly measured, but technical difficulties accompanying the thin structure of olfactory cilia have prevented systematic analyses. In this study, using a combination of electrophysiology, local photolysis of caged cAMP, and Ca2+ imaging, we found that free Ca2+ in the local ciliary cytoplasm decreased along with a reduction in the current containing Ca2+-activated Cl− components returning to the basal level, whereas Ca2+-dependent adaptation persisted for a longer period. The activity of Cl− channels is highly likely to be regulated by the free Ca2+ that is present only immediately after the influx through the CNG channel, and an exclusive interaction between Ca2+ and Ca2+-binding proteins that mediate the adaptation may modulate the adaptation lifetime.Olfactory signal transduction is conducted through a cAMP-mediated second messenger cascade. The cytoplasmic Ca Olfactory cilia exhibit a fine cylindrical structure with a 100–200-nm diameter and measuring several tens of micrometers in length . Within ncreases  through ncreases  and olfancreases . Signal  channel , whereas channel . Thus, tl origin . Therefo2+-activated Cl− channels is reduced while Ca2+-dependent adaptation is maintained  recording pipette. By simultaneously monitoring membrane currents, we could set the stimulus strength within the dynamic range of the signal transduction machinery and directly compare the profiles of the currents with the dynamics of the [Ca2+]i changes. [Ca2+]i locally (<1 μm) increased upon the opening of the CNG cation channels, which was mediated by laser photolysis of cytoplasmic caged cAMP. We also observed that the Ca2+ signal returned to the basal level after the termination of local UV stimuli with a time course similar to that for the cAMP-induced current that contained a Cl− component. By contrast, Ca2+-dependent adaptation persisted within the same restricted area for a longer period. It is generally known that the WC recording configuration needs to use exogenous Ca2+ buffers to maintain a low cytoplasmic Ca2+ level. However, extrinsic EGTA may cause side effects that are distinct from the endogenous Ca2+ buffers, especially when the focus is on the kinetics of Ca2+. In this study, we also succeeded in recording cell responses without adding exogenous Ca2+ buffers, presumably before the intrinsic Ca2+ buffers were washed out, and we observed essentially the same results. It was indicated in the results that the seemingly two opposing functions of Ca2+ are clearly segregated by molecular dynamics, even in submicron spaces in native olfactory cilia. The activity of Cl− channels is highly likely regulated by free Ca2+ and is only available immediately after the influx through the CNG channel. In addition, an exclusive interaction between Ca2+ and Ca2+-binding proteins that mediate the adaptation may modulate the adaptation lifetime.Specifically, the reason why the activity of Cained see  is puzzlThe experiments were conducted under the Osaka University Regulations on Animal Experiments established by the Animal Experiment Committee at Osaka University in Japan. Approval number: FBS-19-002. Date of approval: May 20, 2019.Cynops pyrrhogaster) because of considerable ORC sizes. The animals were chilled on ice and double pithed. After decapitation, the olfactory epithelia were removed and incubated at 37°C for 5 min in 1% collagenase media containing (in mM) 110 NaCl, 3.7 KCl, 10 HEPES, 15 glucose, 1 pyruvate, and 0.001% phenol red, with pH adjusted to 7.4 using NaOH. Then, the ORCs were mechanically isolated by trituration using a 5-ml measuring pipette. The cells were adhered onto the surface of concanavalin A-coated glass coverslips and placed at the bottom of Petri dishes. The cells were maintained at 4°C before use in normal Ringer’s solution containing (in mM) 110 NaCl, 3.7 KCl, 3 CaCl2, 1 MgCl2, 10 HEPES, 15 glucose, 1 pyruvate, and 0.001% phenol red, with pH adjusted to 7.4 using NaOH.The method was essentially the same as in previous studies , 2018. Bh = −54 mV) as previously described (+ solution containing (in mM) 119 CsCl, 1 CaCl2, 5 EGTA, 10 HEPES, and 0.001% phenol red (pH 7.4 adjusted using CsOH), as well as 1 mM caged cAMP  and 50 µM Fluo-4 . Current signals were I-V converted using a 200B amplifier (Molecular Devices LLC), and data were sampled using pCLAMP ver.10 (Molecular Devices LLC) at 10 kHz, after being low-pass filtered at 2 kHz. For curve drawings of the membrane current, some data were low-pass FFT-filtered at 0.02 kHz. Care was taken to avoid saturation of response, particularly when evaluating adaptation. All experiments were performed at room temperature (23–25°C).Ciliary membrane currents were recorded from single ORCs with a WC recording configuration  under thescribed ; 2019. TFor the stocks, caged cAMP was dissolved in DMSO  and stor2+ imaging  area was raster-scanned for Ca imaging . For all2+ images simultaneously with UV stimulation. However, one limitation of this technique was that the movement of the beam was constant. The efficacy of the laser beam was determined by the time integral of the applied laser for both photolysis and imaging. In the present experimental system, the intensity required for photolysis was generally higher than that required for imaging. Therefore, a fast scan for imaging could not be achieved using this experimental mode. As an alternate method, we used bleach mode . A beam splitter (HFT 488) and a long-pass filter (LP 505) were selected. Fluo-4 was initially dissolved in dimethyl sulfoxide and stored at −20°C in complete darkness. The stock was diluted with a Cs+-containing pipette solution together with caged cAMP before each experiment. The final concentration of Fluo-4 in the recording pipette was 50 µM. The addition of Fluo-4 did not cause remarkable changes in the membrane current response induced by the cytoplasmic photolysis of caged cAMP, either in time course or degree of adaptation and recovery from adaptation, which is influenced by cytoplasmic Ca2+ dynamics. This observation was made based on data comparisons from this study with those from 2+ dynamics are thought to be nearly identical to standard conditions, which have been used for studying ORCs. We did not lower the concentration of Fluo-4 because some cilia were not remarkably stained, even with 50 µM (see Results). Moreover, the concentrations of caged cAMP and Fluo-4 in the cilia may not have been similar to those in the pipette because the recording pipette was placed on the terminal swelling (olfactory knob) or dendrite. It was possible that the concentration in the cilia was lower than the concentration in the pipette.An LSM system equipped with an α Plan Fluar  100×/1.45 numerical aperture (oil immersion) objective lens and an argon laser beam  was used for the visualization of i sensitivities in both systems, (2) differences in K1/2 values, and (3) differences in the time course and spatial distribution. More detailed analyses are needed to understand these possibilities.In the data in 2+ buffer were used ; if this number is extruded in 10 s, then the average current will be 7.6 × 10−6 pA (7.6 aA)/μm cilium. If the length of the cilium is 10 μm and the total number of cilia is 10, then the current amplitude in an ORC becomes 0.76 fA, which cannot be detected using conventional electrophysiology.Therefore, one may speculate that charge movement can be detected using an electrophysiological technique. Charge movements accompanying CaThus, olfactory signal transduction and its modulations are efficiently performed with a very small number of molecules. An extremely high surface-to-volume ratio unique to nanoscale tubular structures such as cilia is highly likely to be at least partially responsible for such specific purposes . Methodo