However, the prognostic significance and function of ECHDC3 in AML remain to be clarified.Enoyl-CoA hydratase domain containing 3 (ECHDC3) increased in CD34+ cells at diagnosis. In the validation set, 351 bone marrow RNA-seq data of non-APL AML were obtained by two independent online datasets (TCGA-LAML and BEAT-AML). LASSO regression model was conducted to a new prediction model of ECHDC3-related genes. In addition, the ECHDC3 signature was further explored by GO, KEGG, GSEA, and immuno-infiltration analysis. By RNA interference, the function of ECHDC3 in mitochondrial DNA (mt-DNA) transcriptome and chemoresistance was further explored, and the GSE52919 database re-verified the ECHDC3 chemoresistance feature.In the training cohort, 24 AML  patients were enrolled in Peking University People’s Hospital and tested for ECHDC3 in enriched CD34high demonstrated inferior overall survival (OS) compared to those with ECHDC3low both in the training  and validation cohorts . In addition, ECHDC3high predicted inferior OS in the subgroup of patients with ELN 2017 intermediated (int) risk  or FLT3+NPM1− adverse (adv) risk . In multivariate analysis, ECHDC3 was an independent risk factor of inferior OS . In the prediction model combining ECHDC3 and nine selected genes  by Lasso regression, patients with high risk showed inferior 5-year OS . Bioinformatic analysis suggested that ECHDC3 alters the bone marrow microenvironment by inducing NK, resting mast cell, and monocyte differentiation. Knocking down ECHDC3 in AML cells by RNAi promoted the death of leukemia cells with cytarabine and doxorubicin.By Kaplan-Meier analysis, patients with ECHDC3These bioinformatic analyses and experimental verification indicated that high ECHDC3 expression might be a poor prognostic biomarker for non-APL AML, which might be a potential target for reverting chemoresistance. Acute myeloid leukemia (AML) is a heterogeneous disease characterized by different molecular subtypes with different prognoses and responses to treatment. Patients up to the age of 60 years reported 5-year survival rates of 30%–35% and <10%–15% for older patients (age 60 years and older) .+ progenitors of chemoresistant AML , age 16–70 years; (ii) no previous antineoplastic therapy; (iii) ECOG performance score ≤ 2, without other major coexisting illnesses such as severe kidney and liver insufficiency.The inclusion criteria are as follows: (i) newly diagnosed BM samples were acquired before the first induction chemotherapy. Cell sorting and mass spectrometry measurements were followed as published study . The dia2.The human AML cell lines K562 were purchased from the American Type Culture Collection (ATCC) agent in China. All the cell lines have passed the STR authentication. Cell lines were cultured in RPMI 1640 medium  containing 10% FBS  and 1× antibiotic–antimycotic . Cells were grown at 37°C in a humidified atmosphere with 5% COhttps://portal.gdc.cancer.gov/) and the Beat AML database (http://www.vizome.org/aml/). We retrieved 154 non-APL AML BM mRNA sequence data (excluded 16 APL and 3 incomplete data) and corresponding clinicopathological characteristics from the TCGA. Additionally, 197 BM mRNA sequence data (excluded 9 APL and 25 incomplete data) were retrieved from the Beat AML database (BEAT-AML). The GSE52919 database (https://www.ncbi.nlm.nih.gov/geo/) was performed to analyze the correlation of ECHDC3 expression with cytarabine (Ara-C) sensitivity in AML samples.The clinical and transcription matrix of AML was downloaded from the TCGA database  were ultimately retained. Then, the hub genes were used to construct a risk signature based on the coefficient for each patient according to the following formula: i) denotes the coefficient, and X(i) is the z-score transformed relative expression level for each DEG. To validate the established model, the prognostic risk score of each patient was calculated using the formula, and, according to the median score, patients were classified into high- and low-risk groups. Then, time-dependent receiver operating characteristic (ROC) curve (tdROC) and Kaplan-Meier survival curve analysis were performed to verify this risk score system (Lasso regression was constructed by the glmnet R package according to the method previously published by our institute . In brieGene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed ECHDC3 were performed using the R package “cluster profile”. GO terms were classified as molecular functions (MFs), biological processes (BP), and cellular components (CCs). Both p- and q-values less than 0.05 were considered to indicate statistically significant differences. Gene set enrichment analysis (GSEA) was performed to find enriched term differences.The gene transcriptome data were used to estimate the content of multiple immune cells infiltrated in the BM microenvironment. CIBERSORT in combination with the LM22 signature matrix was used to estimate the fractions of 22 human immune cell phenotypes between AML samples with high or low ECHDC3 expression. The CIBERSORT analysis was conducted in R software by the CIBERSORT R script (version 1.03). The correlation analysis of the index was completed using the Spearman’s test.ECHDC3 siRNA was designed and synthesized by GenePharma . The plasmid was then transfected into the K562 cell lines. Opti-MEM medium and Lipofectamine L300015 reagent were used for the transfection of siRNA according to the manufacturer’s instructions. After transfection, whole-cell cDNAs were synthesized, and quantitative real-time PCR was performed to verify the ECHDC3 expression level.4 cells/100 μl in a 96-well plate and incubated at 37°C for 48 h. Ten microliters of CCK-8 was added to each well of the 96-well plate, followed by further incubation for 1 h. The absorbance was measured at 450 nm. The cell viability was calculated using GraphPad Prism software.Ara-C (cytarabine) and doxorubicin (DOX) were purchased from Selleck Chemicals . Cell viability was determined by CCK-8 assay . The cells were seeded in a density of 2 × 10Whole-cell cDNAs were synthesized, and quantitative real-time PCR was performed using the FastStart Universal SYBR Green Master mix  with a StepOnePlus real-time PCR system . The threshold cycle (Ct) values of target genes were normalized over the Ct of β-actin in whole-cell lysate. For comparison, the siCT group was set as 1 unless otherwise indicated. Primers used for real-time PCR and quantitative real-time PCR are listed in U-test for continuous variables and the X2 test for categorical data. P < 0.05 marked with *, P < 0.01 marked with **, and P < 0.005 marked with *** are considered significant. The survival functions were estimated by the Kaplan-Meier method using the log-rank test with asymmetric 95% confidence intervals (CIs). Variables included in the multivariate analysis (MVA) were selected by the backward elimination process with a criterion of P < 0.10 for retention. Age is linear with estimates of HRs for 10-year difference.R software 4.05 and GraphPad Prism 8.0.2 were mainly used for laboratory data statistical analysis. Comparisons between the two groups were performed using the Mann-Whitney The total study design is illustrated in + cells at diagnosis, patients were divided into “ECHDC3high” and “ECHDC3low” groups, and the baseline characteristics were similar between the two groups  patients were enrolled between September 2019 and September 2020, followed up to July 2022. According to the median protein level of ECHDC3 in enriched CD34o groups Table S2high” and ECHDC3low” groups according to the median expression level of ECHDC3 in BM, and the baseline characteristics were listed in high group than in the ECHDC3low . Patients were divided into “ECHDC3In the validation set, the expression of ECHDC3 was analyzed by age and ELN risk groups. Patients more than 60 showed higher ECHDC3 levels compared to those less or equal to 60  rather than a low percentage of blast cells (p = 0.537) in PB . Adding ECHDC3 expression to age and ELN risk improved the area under the curve (AUC) from 0.657 to 0.671 of ROC  performed 6-fold cross-validation and 9 genes in addition to ECHDC3 were finally found with regression coefficients, namely, RPS6KL1, RELL2, FAM64A, SPATS2L, MEIS3P1, CDCP1, CD276, IL1R2, and OLFML2A , and resting mast cell (p= 0.039) had significant differences in the immune cell fractions between the two groups ECHDC3high and ECHDC3low  were significantly downregulated (p = 0.003).The human leukocyte antigen (HLA) system is an important part of the immune system, and HLA encodes cell surface molecules specialized to present antigenic peptides to the T-cell receptor (TCR) on T cells . Immune In the GSE52919 database, patients with Ara-C resistance showed higher ECHDC3 expression compared with sensitive patients  in the K562 cell lines  as well as to ELN risk stratification (int/adv vs. fav), the question of whether ECHDC3 would be an independent biomarker is raised. Firstly, it is demonstrated that ECHDC3 and age remained the only two independent risk factors for inferior OS in MVA. Secondly, a new prediction model established by integrating ECHDC3 with basic characteristics including age and ELN risk groups further improved the accuracy of survival prediction, which highlighted the possibility of integration of ECHDC3 as an independent risk factor into risk stratification system in the future. Nevertheless, the current study did not follow the latest ELN 2022 criteria; therefore, the meaning of ECHDC3 in AML under ELN 2022 criteria is needed to be further validated by a larger cohort in the future.The present paper presents a set of evidence for proving that ECHDC3 is a poor prognostic biomarker and a therapeutic target of AML through proteomics, genomics, and mitochondrial transcriptomics. Improvement in AML patient stratification is critical in clinical practice. Next-generation sequencing technologies are gradually implemented in the clinical setting to facilitate diagnosis and treatment decisions in AML. Currently, ELN 2017 criteria have been widely applied as the standard risk stratification method to guide treatment in the first complete remission period in AML . In the 9/L at diagnosis), mutated DNMT3A, and genes involved in signaling pathways, which divided int AML into two subgroups that could predict that the high-risk subgroup was associated with inferior OS and relapse-free survival (RFS), whereas allo-HSCT reduced the relapse risk of high-risk patients   concerning both DFS (3-year rates: 32% vs. 22%) and OS (3-year rates: 41% vs. 31%) , it remained controversial whether allo-HSCT should be recommended as a priority in patients with int-risk AML. Several groups addressed this question by adding other gene profiles to the ELN 2017 criteria. Hu et al. established a prognostic nomogram characterized by white blood cell count (≥10×10= 0.010) . Eisfeldroup n = 9 concernConsidering the negative effect of high ECHDC3 expression on the survival of AML patients, it becomes an important question why and how ECHDC3 promotes AML. By bioinformatic analysis and experimental verification, the current study preliminarily supported that ECHDC3 might be associated with chemoresistance in AML treatment. Although ECHDC3 was predicted to be active in the mitochondrion, knocking down ECHDC3 in the current study did not change the mt-DNA transcriptome, which remained to be explored in the future.in vivo (in vitro interleukin 2 (IL-2)–activated NK cells. It has been revealed that the NK function is related to the levels of the fatty acids, and treatment of NK cells with saturated fatty acids or arachidonic acid significantly enhanced killing receptor expressions and decreased inhibitory NK (KIR) receptors, whereas docosahexaenoic and eicosapentaenoic acids increased KIR . Immature myeloid cells are characterized by the ability to suppress immune responses and expand during cancer . As M-MDIn sum, this study firstly described ECHDC3 as a negative AML prognosis predictor, which might refine the risk stratification of AML, especially in int AML. ECHDC3 is also involved in chemoresistance and shaping the immune microenvironment of AML, which could be a potential target for chemoresistance reverting and immune microenvironment remodeling for AML patients in the future.The original contributions presented in the study are included in the article/This study was reviewed and approved by the ethics review board of Peking University People’s Hospital (2022PHB030). The patients provided their written informed consent to participate in this study.YZ, L-JH, and ML participated in the study design. YZ and L-TN participated in experiments. YZ and ML analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by: The national key research and development plan of China (2021YFA1100902), National Natural Science Foundation of China , Beijing Nova Program of Science and Technology , Peking University People’s Hospital Research and Development Funds , Peking University Medicine Fund of Fostering Young Scholars’ Scientific and Technological Innovation and supported by “the Fundamental Research Funds for the Central Universities” (BMU2021PYB005), China Scholarship Council (202106015007).We appreciated Dr. Xingchen Li  providing help in the data analysis.The authors declare 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.


Plutella xylostella) increased more strongly in a linalool-treated group than in an untreated control group. H2O2 and Ca2+, two important early signals that participated in biotic stress, burst after being treated with linalool in Arabidopsis mesophyll cells. Linalool treatment increased H2O2 and intracellular calcium concentrations in mesophyll cells, observed using a confocal microscope with laser scanning, and H2O2 signaling functions upstream of Ca2+ signaling by using inhibitors and mutants. Ca2+ efflux was detected using non-invasive micro-test technology (NMT), and Ca2+ efflux was also inhibited by NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd. To restore intracellular calcium levels, Ca2+-ATPase was activated, and calmodulin 3 (CAM3) participated in Ca2+-ATPase activation. This result is consistent with the interaction between CAM7 and Ca2+-ATPase isoform 8 (ACA8). In addition, a yeast two-hybrid assay, firefly luciferase complementation imaging assay, and an in vitro pulldown assay showed that CAM3 interacts with the N-terminus of ACA8, and qRT-PCR showed that some JA-related genes and defense genes expressions were enhanced when treated with linalool in Arabidopsis leaves. This study reveals that linalool enhances H2O2 and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their resting state. Additionally, JA-related genes and defense genes’ expression may enhance plants’ defense when treated with linalool.Plants produce linalool to respond to biotic stress, but the linalool-induced early signal remains unclear. In wild-type Arabidopsis, plant resistance to diamondback moth ( Ca2+ and H2O2 signals can be induced by exogenous polyamine and jasmonic acid (JA) treatment [2+ and H2O2 participate in many resistance mechanisms in plants.During plant defense, calcium functions as a crucial early signal. When plants perceive external signals of a pathogen attack, such as oral elicitors from insects or bacterial flagella, Caticulum) ,2. Once r system . H2O2, areatment  and are reatment . Thus, C2+ and H2O2 function together in two processes in plants: Ca2+-induced ROS production and ROS-induced Ca2+ release. There are three ways that Ca2+ induces ROS: Ca2+ activates RBOH by binding to the EF region of the RBOH protein in N-terminal; Ca2+-CPKs phosphorylates RBOH; Ca2+ induces the accumulation of phosphatidic acid (PA), which binds to the N-terminus of RBOH and activates RBOH. ROS-induced Ca2+ release is mainly caused by ROS directly activating or inhibiting the activity of calcium channels or pumps regulating the Ca2+ concentration [Cantration . AdditioArabidopsis thaliana) genome, play important roles in plant stress or defense responses [S-nitroso glutathione reductase (GSNOR), an important component of nitric oxide (NO) homeostasis, during the salt-stress response, as revealed by protein–protein interaction assays [2+ signal transduction [2+-CAM3 activates the expression of heat shock protein (HSP) genes in both the absence and presence of heat shock [Calmodulins (CAMs), including the seven CAMs encoded in the Arabidopsis  when injured by weevils  [FaNES1 produce more linalool than wild-type (WT) plants, which makes them smell better but taste worse to insects [Oryza sativa) plants treated with linalool develop significantly shorter Xoo (Xanthomonas oryzae pv. oryzae) lesions. Notably, linalool enhances rice resistance to Xoo without having a direct antibacterial effect on Xoo [When plants are exposed to insect herbivory, they emit volatile organic compounds (VOCs) ,14. The ionidae) . Herbivo insects . Similart on Xoo . The VOCt on Xoo , in mostt on Xoo ,20,21,222O2 and calcium concentrations in leaves. CAM3 then interacts with ACA8 to transfer Ca2+ out of the cytoplasm and reduce cytoplasmic calcium concentrations. Our findings uncover the role of linalool as a signal inducing H2O2 and calcium concentration changes in Arabidopsis leaves.Here, using non-invasive micro-test technology (NMT) and confocal laser scanning microscopy, we demonstrated that linalool treatment enhances intracellular HDetermination of chlorophyll fluorescence is a common method of studying plant stress responses, particularly in photosystem II (PSII). Additionally, the maximal photochemical efficiency, Fv/Fm, shows the degree of damage in the PSII reaction centers . We set P. xylostella in Arabidopsis, we inoculated WT plants with P. xylostella larvae after 24 h of linalool treatment; the control group was treated with ethyl alcohol. After 7 days, we measured the length and weight of P. xylostella larvae. Both growth indicators were significantly lower in linalool-treated plants than in control plants  were lower than WT linalool group after linalool treatment (2O2 levels (about 30–45% above control) in linalool-treated WT cells pretreated with ruthenium red  [tpc1 cells were similar to those of the WT linalool group  ,26 and tol group . These r2+ fluorescence. Ca2+ fluorescence was higher in the WT linalool group versus the other groups from 30 s to 120 s , remained at a similar level at 60 s, and then gradually decreased to the initial level  in calcium concentrations were detected during this period in response to linalool treatment  was significantly reduced following linalool treatment compared to that in WT plants treated with linalool but not pretreated with ruthenium red. The peak of the tpc1 mutant (166 pmol cm−2 s−1) showed the same degree of decline as that of the WT ruthenium red group (−2 s−1) and rbohd (73 pmol cm−2 s−1) mesophyll cells, the peak following linalool treatment was reduced to a level similar to that of the WT ruthenium red and tpc1 groups , the linalool-responsive peak (peak), and the post-linalool response (post) based on the transient processing of Cacm−2 s−1 a. When Wed group b. In bot1 groups c. To fure groups e. The re2+-ATPase might play an important role in this process. We pretreated WT leaves with the Ca2+-ATPase inhibitor Eosin B and examined calcium flux. The peak of calcium flux was lower in the Eosin B-pretreated WT + linalool group (about 109 pmol cm−2 s−1) than in the WT + linalool group (about 281 pmol cm−2 s−1) , the peak of calcium flux was also about 100 pmol cm−2 s−1 in the cam3-1 + linalool group and about 19 pmol cm−2 s−1 in the cam3-2 + linalool group  d, indicaith ACA8 . Becauseol group d. We preN. benthamiana leaf parts injected with CAM3-Cluc/ACA8-Nluc showed stronger fluorescence than the other three (control) leaf parts   of ACA8 as bait and CAM3 as the prey protein, finding that the competent yeast strain AH109 cells with co-transformed plasmid pairs CAM3-AD plus ACA8 (residues 1–180)-BK grew on SD/−Leu/−Trp and SD/−Ade/−His/−Leu/−Trp and grew blue colonies on SD/−Ade/−His/−Leu/−Trp with X-α-gal. This result indicated that CAM3 binds to the N-terminus of ACA8 in the Y2H assay a. CAM3 aA8-Nluc) c. These reatment d. These LOX6, MYC2, JAZ4, and JAZ8, in WT plants following linalool treatment for 5 min, 0.5 h, and 2 h. In addition, we measured the expression of defense genes PDF1.2, THI2.1, VSP1, and VSP2 in WT plants following linalool treatment for 0.5 h, 2 h, and 8 h. Almost all genes are expressed in the WT (PDF1.2 (about 3.2-fold) and THI2.1 (about 22-fold) gene expression was at 8 h (VSP1 (about 4.7-fold) and VSP2 (about 5-fold) gene expression was at 0.5 h . Therefore, we measured H2O2 levels in ruthenium-red-pretreated wild-type and tpc1 leaves following linalool treatment. The H2O2 burst was not inhibited in these cells, indicating that the linalool-induced H2O2 burst occurs before the increase of Ca2+ levels in Arabidopsis leaf mesophyll cells. (E)-2-hexenal can also increase the Ca2+ concentration in leaves of Arabidopsis thaliana. When leaves were pretreated with ruthenium red, calcium ions could not be induced by (E)-2-hexenal, suggesting that intracellular calcium store is involved in the increase in the (E)-2-hexenal-induced intracellular Ca2+ concentration [When Arabidopsis recognizes linalool, Hnt rbohd . Similarll cells . Increasntration .2+ and H2O2 function together in two processes in plants: Ca2+-induced ROS production and ROS-induced Ca2+ release [2+ and H2O2 signaling function in ROS-induced Ca2+ release. The recognition of different stimuli occurs via different processes during early signaling, making the sequence of early signaling events a special ’language’ in plant resistance.Ca release . Here, w2+ binding to the TPC1 cytosolic EF-hand domain triggers conformational changes to activate TPC1 [2+ treatment increase vacuolar Ca2+ release, while vacuolar Ca2+ efflux was strongly suppressed by H2O2 in an earlier study [2O2 burst is upstream of the enhancement of calcium concentration in Arabidopsis mesophyll cells. Therefore, there may be other ways to slightly increase intracellular calcium concentration in the cytoplasm before linalool-induced calcium is released from the intracellular calcium store, thereby activating TPC1 and releasing large amounts of calcium from the intracellular calcium store into the cytoplasm. At the same time, a more detailed study of how TPC1 is activated with linalool treatment should be undertaken in a separate investigation.TPC1 activation is regulated by many factors. Caate TPC1 . Additioer study . In our 2+ plays an important role in many signaling transduction processes, but high concentrations of calcium in the cytoplasm are toxic to cells. After calcium has functioned as a second messenger, it is transported into calcium stores. The P-type ATPase, Ca2+-ATPase, located in the plasma membrane and the intracellular calcium store membrane, participates in the process of calcium efflux. Studies have shown that only II B Ca2+-ATPase can bind to CAMs. When the N-terminal of Ca2+-ATPase binds to Ca2+, CAM also binds to the N-terminal CAM binding site [2+ flux, we showed that linalool-treated mesophyll cells exhibit strong Ca2+ efflux. Therefore, we investigated whether the Ca2+-ATPase inhibitor Eosin B would inhibit this Ca2+ efflux. Our findings indicate that Ca2+-ATPase participates in linalool-induced Ca2+ efflux in mesophyll cells and that Ca2+ efflux is inhibited in cam3 plants (CAM3 and CAM7 share high sequence similarity (up to 98.9%), so we suggested that CAM3 may also interact with ACA8. Y2H, LCI, and in vitro pulldown assays indicated that CAM3 binds to the N-terminus of ACA8 and functions in the Ca2+ efflux process , tpc1 SALK_125658 (AT4G03560), cam3-1 SALK_149754C (AT3G56800), and cam3-2 SALK_042391C (AT3G56800) mutants were used in this study. The seeds were vernalized at 4 °C for 2 days in the dark. After vernalization, the seeds were surface-sterilized for 4 min in 75% ethyl alcohol, washed four times in sterile water, sown in the autoclaved soil mixture, and placed in an incubator . Plants were grown in soil at 21–23 °C and 70% relative humidity with a light intensity of 80–110 μmol m−2 s−1 under long-day (16 h light/8 h dark) conditions. The plants used for insect inoculation were grown for 4 weeks, and the plants used in the other experiments were grown for 2 weeks before treatment.Wild-type (Col-0) Arabidopsis, Plutella xylostella (diamondback moth) eggs were hatched in an incubator at 25 ± 1 °C with a relative humidity of 50 ± 10%. After 1–2 days, larvae that were identical in length and weight were chosen for inoculation onto Arabidopsis plants; each plant was inoculated with three larvae. WT control and WT linalool-treated plants were prepared for analysis. Seven days later, the insects were removed, and their lengths and weights were measured. Each group contained 25–40 insects.WT control and WT linalool-treated plants were used to measure leaf chlorophyll fluorescence. After 0.5 h dark adaption, the maximum photochemical efficiency Fv/Fm was of PSII, obtained with a modulated fluorometer IMAGING PAM . Every group contained 6–8 Arabidopsis plants.Arabidopsis plants were treated with linalool  via fumigation in glass bell jars  in insect growth test. Cotton balls 1 cm in diameter were soaked in linalool dissolved in ethyl alcohol  and hung in the bell jars; cotton balls soaked in the same volume of ethyl alcohol were used for the control. After adding the cotton balls, the bell jars were immediately sealed with Vaseline petroleum jelly.Arabidopsis plants treated with ethyl alcohol and linalool  were used to measure fresh weight. Additionally, the samples were then dried in an oven at 80 °C for 24 h to constant weight and weighed as dry weight. Every group contained 6–8 Arabidopsis plants.2+ flux in mesophyll cells was measured using non-invasive micro-test technology (NMT) . Before testing, a small cut was made in the leaves of 2-week-old Arabidopsis plants. The leaves were soaked in test solution  for approximately 30 min. Silanized glass micropipettes (2–4 μm aperture) were filled with electrolyte solution (100 mmol L−1 CaCl2) and front-filled with a selective liquid ion exchange (LIX) cocktail to approximately 10 μm. The glass micropipettes were connected to the NMT system with a silver chloride wire, and electrodes with Nernstian slopes of 28 ± 5 mV log−1 were used.Ca2+ concentration in the mesophyll cells was measured near the cells and 30 μm away from the cells at 0.2 Hz. Each leaf was tested for 2 min before linalool treatment . After linalool was quickly added to the test solution to a final concentration of 100 μM, data were collected for approximately 2.5 min as the linalool response peak (peak) group. Data were then collected from the post-linalool response (post) group to indicate the end of the reaction. The final flux values are reported as the mean of eight individual plants per treatment. The Ca2+ flux was calculated as:J is the flux of Ca2+ (pmol cm−2 s−1), D is the diffusion coefficient (0.79 × 10−5 cm2 s−1), ΔC is the difference between the concentrations near and far from the cells, and ΔX is 30 μm. Each group contained 6–8 replicates.The Ca2+ flux experiment). The leaves were then soaked in Fluo3-AM solution to label Ca2+ ions for 1 h in the dark in an incubator. The leaves were washed and placed in the fresh test solution. Ca2+ fluorescence was detected under a confocal laser scanning microscope . The excitation wavelength was 488 nm, and the emission wavelength was 530 nm. Before linalool  addition (0 min), the basal level of Ca2+ fluorescence in mesophyll cells was measured. Following the addition of linalool, fluorescence was measured every 30 sec for 150 s. Each group contained 20–40 cells.Before treatment with Fluo3-AM (10 μM), Arabidopsis leaves were soaked in a test solution (the same solution used for the Ca2+ fluorescence = (X − a)/a × 100%, where X is the fluorescence of linalool-treated samples, and a is fluorescence at 0 s. The value for each group was calculated separately.The increase in Ca2DCF-DA was used to measure changes in H2O2 levels in Arabidopsis mesophyll cells. Following incubation in the test solution, the leaves were incubated in 50 μM H2DCF-DA for 15 min. Confocal laser scanning microscopy  was used to detect H2O2 fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 510–530 nm. H2O2 fluorescence measurements were obtained at 0 min  and every 3 min for 15 min. The increase in H2O2 fluorescence was calculated as Ca2+, and linalool’s final concentration was 100 μM. Each group contained 20–40 cells.HThe sequences encoding CAM3 were cloned into pGADT7, and the sequences encoding amino acid residues 1–180 of ACA8 (ACA8 (residues 1–180)) were cloned into pGBKT7, respectively. The plasmid pairs CAM3-AD plus ACA8 (residues 1–180)-BK were co-transformed into competent yeast strain AH109 cells. The cells were grown on SD/−Leu/−Trp medium. After 4 days, growing colonies were transferred to SD/−Ade/−His/−Leu/−Trp for further verification. Growing colonies on SD/−Ade/−His/−Leu/−Trp were transferred to SD/−Ade/−His/−Leu/−Trp with X-α-gal. Cell growth on SD/−Leu/−Trp and SD/−Ade/−His/−Leu/−Trp and blue colonies on SD/−Ade/−His/−Leu/−Trp with X-α-gal indicate protein–protein interactions between the two proteins.CAM3 was cloned into the Cluc plasmid, and ACA8 was cloned into the Nluc plasmid, respectively. Additionally, the plasmids were transformed into Agrobacterium strain GV3101, respectively. Fluorescence from luciferase in Nicotiana tabacum leaves infected with pairs of Agrobacterium was imaged with a molecular imaging system . Each leaf was divided into four quadrants before injection with the following combinations: Cluc/Nluc, CAM3-Cluc/Nluc, Cluc/ACA8-Nluc, and CAM3-Cluc/ACA8-Nluc, respectively.E. coli Rosetta (DE3) cells for protein expression. Protein-GST was used as the bait protein. Glutathione beads containing 50 µg ACA8-GST or GST were incubated with 50 µg CAM3-HIS in pulldown buffer  at 4 °C for 2 h, respectively. The protein beads complexes were washed with pulldown buffer, then centrifuged 500× g for 5 min, the supernatant was removed, and the whole process was repeated 4 times; at last, the SDS sample buffer was boiled for 5–10 min. The bindings of CAM3-HIS with ACA8-GST were detected by immunoblot analysis using anti-GST and anti-HIS antibodies.The genes encoding ACA8 (residues 1–180) were cloned into pGEX-4T-1, and the genes encoding CAM3 were cloned into pET28A. ACA8-GST and CAM3-HIS were transformed into LOX6 (forward primer sequence (5′–3′): GCTTGAAATCAATGCTCGTGCG; reverse primer sequence (5′–3′): GTCAATCACAAGCCTCACGCCAC), MYC2 (forward primer sequence (5′–3′): GATGAGGAGGTGACGGATACGGAA; reverse primer sequence (5′–3′): CGCTTTACCAGCTAATCCCGCA), JAZ4 (forward primer sequence (5′–3′): TCTCCGATAACTGTGAAGGA; reverse primer sequence (5′–3′): CTTGCTGACTGGAACCTG), JAZ8 (forward primer sequence (5′–3′): AATTGTGACTTGGAACTTCG; reverse primer sequence (5′–3′): TTGCCTGAAGATGGGTAAC); PDF1.2 (forward primer sequence (5′–3′): CCATCATCACCCTTATCTTCGC; reverse primer sequence (5′–3′): TGTCCCACTTGGCTTCTCG), THI2.1 (forward primer sequence (5′–3′): GTTCGTATACGTGAAGGGAGTA; reverse primer sequence (5′–3′): CACACACTACATATTATCGACT), VSP1 (forward primer sequence (5′–3′): CGTATCCGTCACCTCCTCAT; reverse primer sequence (5′–3′): CGCCAAAGGACTTGCCCTAA), VSP2 (forward primer sequence (5′–3′): GGCCTTGCATCTTTACCAAAAC; reverse primer sequence (5′–3′): GTAGTAGAGTGGATTTGGGAGC). ACTIN2 (forward primer sequence (5′–3′): AGTGGTCGTACAACCGGTATTGT; reverse primer sequence (5′–3′): GATGGCATGAGGAAGAGAGAAAC) and EF1α (forward primer sequence (5′–3′): TCCAGCTAAGGGTGCC; reverse primer sequence (5′–3′): GGTGGGTACTCGGAGA) were used as references [−ΔΔCt method was used to calculate relative gene expression levels [For quantitative RT-PCR, total RNA was extracted from the samples using an RNA extraction kit, and cDNA was generated using a reverse transcriptase kit (Takara). The expression levels of several important genes in the JA biosynthesis pathway were detected, including the following: ferences ,36,37,38n levels .t-test and Dunnett’s C (variance not neat) at the level of p < 0.05 was significant. Error bars denote ± standard error of mean (SEM).Student’s The GenBank numbers and Genome Initiative numbers of all genes used in this article are as follows: RBOHD , TPC1 , CAM3 , ACA8 , LOX6 , MYC2 , JAZ4 , JAZ8 , PDF1.2 , THI2.1 , VSP1 , VSP2 , ACTIN2 , EF1α .2O2 burst mainly because of NADPH oxidase RBOHD, then calcium ions were released into the cytoplasm through intracellular calcium stores, and the excess calcium ions in the cytoplasm were pumped out through the interaction between CAM3 and ACA8. In addition, linalool may enhance insect resistance by activating the JA pathway in Arabidopsis plants, which requires further study.In this study, we revealed the early signaling process induced by linalool. H


Therefore, plant cells have developed complex mechanisms of Ca2+ communication across the membrane, receiving the message from their surroundings and transducing the information into cells and organelles. A wide range of biotic and abiotic stresses cause the increase in [Ca2+]cyt as a result of the Ca2+ influx permitted by membrane-localized Ca2+ permeable cation channels such as CYCLIC NUCLEOTIDE-GATE CHANNELs (CNGCs), and voltage-dependent HYPERPOLARIZATION-ACTIVATED CALCIUM2+ PERMEABLE CHANNELs (HACCs), as well as GLUTAMATE RECEPTOR-LIKE RECEPTORs (GLRs) and TWO-PORE CHANNELs (TPCs). Recently, resistosomes formed by some NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT RECEPTORs (NLRs) are also proposed as a new type of Ca2+ permeable cation channels. On the contrary, some Ca2+ transporting membrane proteins, mainly Ca2+-ATPase and Ca2+/H+ exchangers, are involved in Ca2+ efflux for removal of the excessive [Ca2+]cyt in order to maintain the Ca2+ homeostasis in cells. The Ca2+ efflux mechanisms mediate the wide ranges of cellular activities responding to external and internal stimuli. In this review, we will summarize and discuss the recent discoveries of various membrane proteins involved in Ca2+ influx and efflux which play an essential role in fine-tuning the processing of information for plant responses to abiotic and biotic stresses.Calcium (Ca Exposure to various sources of abiotic stresses, including heat, metals, salt, wounding, cold, and hypoxia, cause the increase in cytosolic Ca2+ ([Ca2+]cyt). Activation of PATTERN RECOGNITION RECEPTORs (PRRs) by extracellular patterns and NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT (NLR) receptors by cytosolic pathogenic effectors induces PATTERN-TRIGGERED IMMUNITY (PTI) and EFFECTOR-TRIGGERED IMMUNITY (ETI), respectively. Either layer of the immune system also evokes cytosolic Ca2+ signals as a conserved overlapping cell signaling event. The magnitude and pattern of the increases of [Ca2+]cyt vary upon the different stresses .Plants often survive in constantly changing environments that are stressful for their normal growth. To recognize and cope with these stress conditions caused by various biotic and abiotic factors, plants have evolved sophisticated mechanisms to use intracellular signaling molecules as second messengers in alerting cells . The calstresses , and the2+ channels and pumps. Recent advances in Ca2+ signal analysis with high-throughput genetics screens has facilitated the validation of the numbers of Ca2+ channels and pumps which are essential contributors to the Ca2+ signature. However, the specific functions of each member of the Ca2+ channels and pumps in stress signaling are only beginning to emerge. In addition, as more research progresses on Ca2+ signaling upon exposure to diverse stresses, additional unanswered questions continued to be revealed. In this review, we discuss the recent studies of Ca2+ channels and pumps which have focused on elucidating their functional roles in plant stresses.The advance of genomics and molecular biology has accelerated and enabled genome-wide studies, which have resulted in the identification of various CaCYCLIC NUCLEOTIDE-GATED CHANNELs (CNGCs), first discovered in barley  has 20 CNGCs (Oryza sativa (rice) has 16  and hinge region with a CALMODULIN-BINDING DOMAIN (CaMBD) at its C-terminus. Most CNGCs have been demonstrated to localize to the plasma membrane  has 16 . CNGCs a) has 16 . The chaIN CNBD; . The CNBmembrane . Howevermembrane . It is pCALMODULIN-BINDING DOMAIN is located in a site overlapping with the C-terminal α-helix of the CNBD, which allows CALMODULIN (CaM) to compete with cyclic nucleotide monophosphate (cGMP/cAMP) as a ligand in allosteric gating of channel conductance (CaM-BINDING SITE (CaMBS) than initially reported (via “ISOLEUCINE-GLUTAMINE” (IQ) domains adjacent to but not overlapping an α-helix in the CNBD , displayed impaired cell death upon exposure to avirulent bacteria, indicating that AtCNGC2 and AtCNGC4-mediated Ca2+ signaling is critical for plant disease resistance (PATHOGEN RESISTANCE (PR) genes (2+ into cells and the dnd1 mutant (atcngc2) without functional AtCNGC2 lacks this cell membrane Ca2+ current and does not display cell death (Nicotiana benthamiana support the hypothesis that AtCNGC2 and AtCNGC4 are likely part of the same heterotetrameric channel complex (2+ accumulation in response to H2O2 were reduced in atcngc2 and atcngc4 mutants (PATHOGEN-ASSOCIATED MOLECULAR PATTERN (PAMP)-induced REACTIVE OXYGEN SPECIES (ROS) production was impaired in atcngc2 mutant , was identified as the fusion of two tandemly repeated AtCNGC11 and AtCNGC12 genes  genes . It was ll death . Bimolec complex . Ca2+ ac mutants  and PATH2 mutant , suggest12 genes , 2006. Toscngc9 , displayed a lesion mimic phenotype and impaired resistance to Magnaporthe oryzae  and Pseudomonas fuscovaginae  and atcngc4 (dnd2) also show temperature-sensitive cell death phenotypes  containing a COILED-COIL (CC) domain or a RESISTANCE TO POWDERY MILDEW 8 (RPW8)-like CC domain function as Ca2+ channels to induce cell death and defense responses (Z-ACTIVATED RESISTANCE 1 (ZAR1) represents a CNL, which is one of the best structurally characterized sensor NLR recognizing pathogen effectors  domain, TNLs, have been confirmed to possess oligomerization-dependent NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD) HYDROLASE (NADase) activity within their TIR domain (RECOGNITION OF PERONOSPORA PARASITICA 1 (RPP1), creates the active site for catalysis after RPP1 recognizes and directly binds Hyaloperonospora arabidopsidis effector, A. thalianaRECOGNIZED 1  TNL, RECOGNITION OF XANTHOMONASOUTER PROTEIN Q 1 , after it interacts with Xanthomonas effector protein, XopQ 1 , and N REQUIREMENT GENE 1.1 (NRG1.1) subfamilies, belong to the RNL family  are known to mediate the majority of excitatory neurotransmission in the central nervous system (GLUTAMATE (Glu)-gated cation channels that are selective for Na+, K+, and Ca2+ ions. Plant GLUTAMATE RECEPTOR-LIKE RECEPTORs (GLRs) have a high similarity to their animal counterparts in respect to their channel structure, ligand binding domain, and amino acid sequence  GLR in Arabidopsis triggered greater Ca2+ influx after glutamate treatment and conferred enhanced resistance to a necrotic fungal pathogen, Botrytis cinerea (FLAGELLIN 22 (flg22), ELONGATION FACTOR TU 18 (elf18), and fungal chitin, involved apoplastic Ca2+ influx via iGluR-like channels, suggesting that GLRs are related to the induction of defense response after PAMP recognition , and its interaction with 14–3-3 proteins participates in the regulation of cytosolic Ca2+ concentration under salt stress  and their mutants became more sensitive to cold the stress  functions as a nonselective cation channel co-regulated by voltage and Ca2+, and generates a slow vacuolar current  activation for additional production of apoplastic ROS , localized at vacuolar membrane, functions as a Caelopment , 2012. Aelopment . In Arabmulation . Howevernd flg22 . Therefond flg22 . AtTPC1 ncreases . In the  and ROS , AtTPC1 stic ROS . A feed- to cell , suggestDEPOLARIZATION-ACTIVATED Ca2+CHANNELs (DACCs) have been found in various plant species including Arabidopsis, tobacco, and Zea mays (maize), although DACCs in plants have not been associated with any gene yet  were first found in Solanum lycopersicum (tomato) responding to fungal infection  mediated Ca2+ influx in root epidermal cells, which is activated by extracellular •OH and H2O2  and [Ca2+]cyt  ion channels that serve to sense and respond to changes in membrane tension have been known to directly couple mechanical stimuli to ion flux (2+ channels (SENSITIVE CHANNEL OF SMALL CONDUCTANCE (MSCS)-LIKE CHANNELs (MSLs). It has been predicted that there are 10 and five MSL genes in Arabidopsis and rice, respectively (AtMSL10 triggered cell death and wound-induced hyperaccumulation of JA (AtMSL10 exhibited reduced susceptibility to infection by P. syringae pv. tomato DC3000 and showed an accelerated induction of Arabidopsis PATHOGENESIS-RELATED PROTEIN 1 (AtPR1) expression compared to wild-type plants, indicating that mechanical signals are important.Plant cells sense mechanical stimuli in nature, such as touch, gravity, and the stretching of membranes. ion flux . There achannels . One of ectively . Arabidoectively . A gain-on of JA . Recentlon of JA . Finallyon of JA . Overexp2+ channel is a MID1-COMPLEMENTING ACTIVITY (MCA), exhibiting 10% identity to yeast MATING-INDUCED DEATH 1 (Mid1). AtMCA1 and AtMCA2 have been identified in Arabidopsis via functional complementation of the yeast mutant mid1  were first identified as an osmosensors in Arabidopsis  genomic database revealed a total of 11 OsOSCAs genes  and low capacity Ca2+ transporter  or P2b-type AUTOINHIBITED CA2+-ATPASEs (ACAs) which have 10 transmembrane domains , which are known as transporters of Ca2+, Mn2+, and Zn2+ -induced acquired resistance  ACAs, MsRCI2s resulted in increased tolerance to alkaline and salt stress (SOYBEAN CA2+-ATPASE (SCA1) was rapidly induced by NaCl or the fungal elicitor treatment, but not by KCl or mannitol treatment. The regulation of SCA1 activity was shown to be Ca2+-dependent and CaM-binding-dependent  ACA, PCA1, is localized within small vacuoles, and was shown to be up-regulated by drought, salt, and abscisic acid. A knockout of PCA1 resulted in alteration of the responses to [Ca2+]cyt, which in turn altered the expression of stress-induced genes and interfered with the tolerance response to abiotic stress  in Arabidopsis roots , especially CA2+/H+ EXCHANGERs (CAXs), largely contribute to Ca2+ homeostasis in plant cells and they are activated by the transport of counter cations. Three subfamilies of CaCAs, NA+/CA2+ EXCHANGERs (NCXs), CATION/Ca2+ EXCHANGERs (CCXs) and CAX, were confirmed to exist in land plants  protein was confirmed to have Ca2+ binding activity and its expression was upregulated by salt stress. Furthermore, the mutant plant of AtNCL exhibited reduced sensitivity to salt stress  and high capacity Ca2+ transport. CAXs restore [Ca2+]cyt levels via removal of [Ca2+]cyt  GmCAX1, rice OsCAX4, Malus x domestica (apple) MdCAXs and Gossypium hirsutum (cotton) GhCAX1 were transcriptionally regulated by abiotic stress and are known to function in process of cold acclimation with key roles related to salt, drought and freezing tolerance  grant funded by the Korea government (MSIT) (NRF-2020R1A2C1007778 to C-JP) and a RIKEN CSRS Innovative Plant Biotechnology Collaboration Project (RIKEN CSRS to RS).The authors declare 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.


It is my privilege to have a mentor cum friend like Prof. Randall Cohrs, who played a significant role in mentoring me and moulding me into the scientist and mentor that I am today. I first met Randy at the 9th International Symposium on Neurovirology held in 2009 in Miami, FL, USA. Cohrs’s then mentor and dear friend Dr. Donald H Gilden introduced me to him. Randy being a kind and generous human, we became friends in no time. Although by training and practice, he was a Herpes virologist and I was a Corona virologist, we often found ourselves discussing science over coffee. Our friendship made me realize that friendship knows no boundaries of age, ethnicity, language, and the branch of science you chose to pursue.I had the chance to meet Randy again in 2010 when I was invited to deliver a grand round lecture at the Department of Neurology, University of Colorado by Dr. Gilden. Randy was a very compassionate human being; and this meeting further strengthened our friendship.PLoS ONE [Clinical Immunology [Randy was a mentor to me; he taught me an essential skill for a scientist in this era: networking and conducting meetings. In 2013, Randy encouraged me to apply for the Indo-US science and technology forum (IUSSTF) American Society for Microbiology (ASM) professorship award. We wrote the grant together, which was funded, and Randy served as my mentor for the program. This was our first venture into collaboration together and a very successful one, highlighted on the International Society of Neurovirology (ISNV) and Indo-US website . This coBox 1I had the privilege to meet Prof. Randall J Cohrs when I was a Ph.D. student, during the Indo-US symposium on Viral Infections of the Nervous System, 2014 at Gurgaon, India. I was working in the laboratory of Prof. Jayasri Das Sarma and was in my final year of Ph.D. As he was eager to interact with the young minds, an exclusive interaction session was arranged for us where we got the opportunity to have a direct meeting with him on a round table. Prof. Cohrs interacted with all thestudents and research scholars with curiosity and patience. He illuminated us with his profound knowledge of viruses and mentored us to a career in science. When I look back, I want to thank him for his thoughtful gesture, which also reflects a part of his persona. All throughout my PhD career his scientific publications on viruses acted as a reference and guiding path to my work. He had also provided his scientific insights into my PhD dissertation. I am proud to share a publication with him as a co-author [In 2014, one of my BS-MS students, Mr. Jibin Sadasivan , receiveBox 2I met Randy in 2014 when I was doing a shortinternship in his lab at the University of Colorado. Moving to a new country for the first time wasn’t easy, but Randy made sure that I had a smooth transition and that I did not feel left alone during my time there. He droveme to the lab every day and took me on short trips to Denver and Boulder on weekends. He made sure that I got souvenirs and pictures from the places we visited together. After my internship, we stayed in touch, and he continued to mentor me as I moved to grad school. Randy was a passionate mentor, a fantastic friend, and an inspiration to many of us, personally and professionally. I will be forever honoured and grateful to have had the opportunity to know himand work with him.Prof. Gilden urged me to host the Indo-US bilateral symposium in India; this would facilitate cultural exchange in addition to science communication. With his support, we hosted a symposium in 2014 in New Delhi with Dr. Pankaj Seth (Indian Host), myself (Indian Co-Host) and Randy (US-Host), and Dr. Lynn Pullium (US Co-Host). The meeting received a great response. Dr. Randall Cohrs, Dr. Ravi Mahalingam, and others presented their work. Dr. Donald Gilden delivered a plenary lecture on temporal arteritis, which the audience received with overwhelming enthusiasm. My students got a chance to interact with Randy here for the first time, and they were overwhelmed with Randy’s easy-going nature and the vital perspective that he gave them on their research projects. After this meeting, our group of scientists took a golden triangle tour to the cities of Agra, Jaipur, and Delhi to experience the culture of these Indian cities .Prof. Donald Gilden was also looking forward to the next Indo US bilateral symposium I hosted in 2016, but he could not attend the meeting due to his sad demise. Dr. Cohr continued to support my endeavours to conduct meetings. He was the Co-PI for the Bioanalytical tools and techniques workshop (BAW) 2018, held around the Indian festival of Colours, Holi . This feRandy was certainly a great leader. His leadership quality is evident from how he established and successfully conducted meetings for the Colorado Alpha Herpesvirus Latency Society (CALS) and led the European Society for Translational Medicine as the Vice-president and Rocky Mountain Virology Association (RMVA) as president.In 2020, amidst the pandemic, we hosted the Indo US bilateral symposium in virtual mode on COVID biology, which hosted many experts from the field and led to interesting discussions on the outbreak and evolution of coronaviruses. He taught me that to be a good scientist, it is crucial to be a good mentor. The last meeting that I hosted with his guidance was the National Association of Biology Teachers (NABT) meeting in 2021, titled “Tips for Teaching Science in a Pandemic: Ways to Lessen the Stress on both Students and Educators”. This series was presented under the banner of NABT and supported by the Indian Institute of Science Education and Research (IISER Kolkata), the Indian Institute of Science , the University of Colorado-School of Medicine and Northwestern Connecticut Community College, USA. Randy was very enthusiastic about having insights regarding the rich Indian tradition of learning and education that prevails since ancient times, The Gurukul System. The Gurukul System is a residential schooling system whose origin dates back to around 5000 BC in the Indian subcontinent. It was more prevalent during the Vedic age where students were taught various subjects and about how to live a cultured and disciplined life. Gurukul was actually the home of the teacher or Acharya and was the centre of learning where pupils resided until their education was completed. At the Gurukul, all were considered equal, and the guru (teacher) as well as shishya (student) resided in the same house, following a system of stages of life discussed in Hindu texts of the ancient and medieval eras. These four stages are: Brahmacharya (student), Gṛhastha (householder), Vanaprastha , and Sannyasa (renunciate). The Asrama system is one important facet of the Dharma concept in Hinduism. Randy was overwhelmed with the practicality of this four stages of life.Azadirachta indica A. Juss (Neem) bark extract in restricting β-Coronaviral infection and replication. Randy played an active role in this project, and his experience in virology helped us throughout the project [In 2020, IUSSTF forum commenced a call-for-proposal for a collaborative effort involving scientists from India and US to explore the therapeutic targets to reduce SARS-CoV-2 infectivity. With the Principal Investigator from the US, Dr. Maria Nagel, I formed a group of scientists including Randy, who worked together to explore the efficacy of  project   bark extract and its Nimbin isomers restrict β-Coronaviral infection and replication”, and drafting our manuscript together with other collaborators from India, USA, and also from Sweden. However, we suddenly lost such an eminent scientist and a humble soul one year before our paper was published in Virology, Elsevier, 2022. Dr. Cohrs has made one of the most tangible contributions to my research life. We miss you, Sir.During my Ph.D. days, in February 2018, whileworking under Prof. Jayasri Das Sarma at IISER Kolkata, I had the privilege of interacting with Prof. Randall J. Cohrs in an International INDO-US Biological and Analytical Workshop, Gangtok, India. Before that, I had heard a lot about his work, personality, knowledge, and sense of humour from my mentor Prof. Das Sarma. Undoubtedly, he had a greatlove not only for Science but also for different cultures and traditions. He used to resemble our Bengali polymath and Nobel Laureate Rabindranath Tagore. He was always enthusiastic about meeting young research minds and take partin cultural programs like dance or music. He also encouraged me to participate in cultural programs like NABT and other national and international platforms. I was fortunate enough to work in an IUSSTF-funded virtual networking grant on COVID-19 since the pandemic struck the world.Since April 2020, we have been working hard on our project “Dr. Abhishek Bose, Post-Doctoral Fellow, Department of Genetic Medicine at Weill Cornell Medicine, New York, USA.During the PhD period, I was fortunate enough to get several chances to meet Dr. Randall Cohrs and it was my absolute privilege and honour to know him from a close distance. We met in the Indo -US science and technology forum (IUSSTF) conference and the BAW-bioanalytical methods workshop organized by Prof. Das Sarma in 2018. In the meeting, I not only got a chance to discuss Science but also interacted with Prof. Cohrs like a friend. Not only was he a renowned virologist and neurologist, but also one who would make us feel at home with his warm smile and jovial nature. He took active part in the cultural program organized as a part of the conference and also met and dressed like the local people of Sikkim. Later, when I joined a post-doctoral position in IUSSTF networking project on COVID-19 with Prof. Das Sarma, we enjoyed Prof. Cohrs’s presence as a collaborator of the team, where he provided thoughtful scientific insights to steer the project at its inception. Also, he made an appearance in our spiritual forum “Kathanubhuti”, where he listened to the glories of Indian cultural heritage and slokes from Srimad Bhagavat Gita with deep interest and shared his spiritual knowledge and love for the Hindu religion. His inclusive and kind nature will keep him alive bright as sunshine, among all of us, who remain inspired by him. We miss you Prof. Cohrs (fondly known as Randy), as a teacher, friend and philosopher, as someone I will always look up to. My prayers for you and for your loving family.


The relevant approximate spontaneous magnetization relations for the simple-cubic and body-centered-cubic Ising lattices have recently been obtained analytically by a novel approach that conflates the Callen–Suzuki identity with a heuristic odd-spin correlation magnetization relation. By exploiting this approach, we study an approximate analytic spontaneous magnetization expression for the face-centered-cubic Ising lattice. We report that the results of the analytic relation obtained in this work are nearly consistent with those derived from the Monte Carlo simulation. J is the coupling strength, Concerning the phase transition theory , the Isisymmetry . The Hamy, Ising . The fory, Ising . Though y, Ising , they ney, Ising ,14,15,16The three-dimensional (3D) Ising model, although subjected to a number of notable attempted solutions ,21,22,23Considering the spontaneous magnetization of the 3D Ising model, a most recent approach in the context of effective field theory  given byIt has been pointed out ,27,101 tT and q denotes the number of nearest neighbors, which is equal to 12 for the FCC lattice. The Let us begin by writing down the Callen–Suzuki identity asSubstituting Equation  into Equdistance . It willσ=12A(K)σFor the proof, the derivation, and further discussions for the Callen–Suzuki identity and its odd-spin correlation expansion for the six neighboring spins , similar to what we detailed in this paper, see Refs. ,65,88,90k on the subscript is an odd number that refers to the order of the odd-correlations, r labels the groups, We now proceed by introducing the heuristic Kaya relation as given in Refs. ,85,86 infinition ,85,86. Aσj1σj2…σjNow, let us cancel Equation , then, iAs these Within this approximation, we reduce the number of unknown parameters to only one, which is Once the approximation procedure given in Equations  and 7b)7b) is apTo determine the Equation  with the in Ref. , which i in Ref. ,47, neveEquation  vanishesEquation  and thenThe analytic relation given in Equation  has been2630(22)  for the To obtain the spontaneous magnetization of the FCC Ising lattice by a different and reliable method, we have also performed an MC simulation by employing the Wolff  cluster K point, we ran the simulation on a relatively larger N is the number of spins on the lattice, In this process, at each In this work, we have studied spontaneous magnetization for the FCC Ising lattice and have obtained an expression as given in Equation . To show in Ref. . TherefoEquation  in the fK is approached to the As Once Considering the fact that the spontaneous magnetization expression of the FCC lattice Ising model is a long-standing open problem, we would like to emphasize that the relation we have obtained herein is quite relevant and remarkable regardless of whether it requires a better approximation at the stage of its derivation.