Specifically, we employed a cost-effective and portable smartphone-based platform coupled with customized software that evaluates the response of Ca2+ ions to ratiometric fluorescence probe in bovine serum. The platform consists of a three-dimensional (3D) printed housing and low-cost optical components that excite fluorescent probe and selectively transmit fluorescence emissions to smartphones. The customized software is equipped with a calibration model to quantify the acquired fluorescence images and quantify the concentration of Ca2+ ions. The ratio of the green channel to the red channel bears a highly reproducible relationship with Ca2+ ions concentration from 10 μM to 40 μM in bovine serum. Our detection system has a limit of detection (LOD) of 1.8 μM in bovine serum samples and the recoveries of real samples ranged from 92.8% to 110.1%, with relative standard deviation (RSD) ranging from 1.72% to 4.89%. The low-cost SRFP platform has the potential to enable campesino to rapidly detect Ca2+ ions content in bovine serum on-demand in any environmental setting.Hypocalcemia is a disease that adversely affects the production and reproduction of dairy cows. A portable device for rapid bovine blood calcium sensing has been growing in demand. Herein, we report a smartphone-based ratiometric fluorescence probe (SRFP) platform as a new way to detect and quantify calcium ions (Ca Under the optimal detection conditions, the fluorescence spectra of ratiometric probe solution toward Ca2+ of various concentrations  were determined (as illustrated in 519/I665) was set to be the readout value. The fluorescence intensity ratio I519/I665 was linearly correlated with different Ca2+ concentrations in the range from 10 μM to 40 μM with a decent correlation coefficient of 0.993  with different calcium ion concentrations with time (0~180 min) were studied. As shown in t 250 μM c. Howeve2+ and different kinds of interfering ions  were pretreated according to the recommended procedure. Under the same conditions, the response of Ca2+ and possible interfering analytes to the fluorescence intensity ratio I519/I655 was determined at a concentration of 25 μM and data were normalized in processing. As shown in 519/I655 value changed significantly in the presence of Ca2+, whereas the fluorescence ratio I519/I655 values barely changed in the presence of other interfering ions. In addition, the concentrations of these metal ions were Mg2+ (~0.02 mM), Zn2+ (0.001 mM), Cu2+ (0.001 mM), Fe3+ (0.001 mM), Na+ (3 mM), K+ (0.1 mM), far exceeding their concentrations in bovine serum (after 50-fold dilution) ). As shown in 2+. The linear equation in the range of 6~40 μM is y = 2.358x − 2.273 and R2 = 0.998. According to the methods recommended by the International Union of Pure and Applied Chemistry (IUPAC), 10 blank samples (without Ca2+) were tested in Tris-HCl buffer. The LOD was calculated to be 1.8 μM according to the following formula: 3σ/k . Therefore, we prove here that the portable quantitative detection of Ca2+ can be achieved using just a smartphone.To determine the effective range of calcium concentrations for blood serum detection, the samples of different Cashown in a. We sel2+ to the fluorescence ratiometric probe solution. The fluorescence response value of Ca2+ was about 40 times higher than that of other metal ions.Excellent selectivity to shield from interference are a necessary capacity for a ratiometric fluorescence sensing system. To demonstrate the selectivity of the developed SRFP platform system, a number of potential substances were measured under the same conditions and data were normalized. As shown in 2+ detection in more detail, we performed another perturbation experiment when the presence of other interferences with Ca2+. In the solution in the presence of different interfering ions, Ca2+ of the same concentration was added sequentially. As shown in 2+. At the same time, the other interferences used above did not produce such a significant change in ratiometric fluorescence change in comparison. This phenomenon manifested that the SRFP sensing system has excellent anti-interference for Ca2+, which indicates the wide applications of such system in complex samples. We also compared the SRFP performance with other reported sensors for Ca2+ determination  were added to serum diluted 50-fold with Tris-HCl as test samples. Then, the ratio probe is put into the samples in a 96-well plate. Subsequently, the processed samples are placed in the SRFP platform, and a smartphone is used for fluorescence image capture and concentration analysis. Meanwhile, the fluorescence spectrum is measured and recorded by a fluorescence spectrophotometer, and the results are shown in 2+. We obtained a favorable linear relationship between the ratio of G/R channel (blue line) and the concentrations of Ca2+  ranging from 1.72% to 4.89%. To further verify the accuracy of the method, we also detected the calcium ion concentration in these serum samples by fluorescence spectroscopy. The detection results of the SRFP platform are consistent with the detection results of fluorescence spectroscopy, which proves that the SRFP platform has good accuracy and reliability.Moreover, the data analyzed by the homemade App of the smartphone were compared with the data detected by fluorescence spectra detection. The results of the data show that there was no statistically significant difference in the assay between the method using fluorescence spectra detection and the method proposed in this work. Recovery experiments were performed to test all bovine serum sample varieties by adding five levels of Ca2+ in bovine serum with high accuracy and cost-effective. It is obvious that a better linear relationship between concentration and intensity is obtained on our SRFP platform. Moreover, the method allows better quantitative analysis compared to fluorescence spectroscopy. The results demonstrate that the smartphone-based platform can detect different concentrations of Ca2+ concentrations detection in bovine serum by employing a smartphone-based ratiometric fluorescent probe to achieve early warning and initial diagnosis of hypocalcemia. Under the optimal conditions, the anti-interference and selectivity performance of the smartphone-based ratiometric fluorescent sensor for Ca2+ detection is excellent in presenting other interfering ions. Utilizing the developed smartphone-based platform, a clear fluorescent color change can be observed when the Ca2+ concentration level is abnormal. Quantitative detection of Ca2+ was achieved with a minimum detectable concentration of 1.8 μM in bovine serum samples. The test results of real samples demonstrate the feasibility and sensitivity of the smartphone-based ratiometric fluorescence sensing platform for Ca2+ concentration detection. The designed ratiometric fluorescent probe-based sensing platform exhibit good sensitivity and practicality, proves great potential in the early screening detection of hypocalcemia, and further accelerate the development of the real-time/on-site detection equipment research field. Moreover, due to the specific luminescence properties of the analyte, this setup can be extended to different fluorescence detection applications, such as water quality testing, and food processing.In conclusion, we propose a novel sensing platform for Ca


Beta human papillomavirus (beta HPV) infections are common in adults. Certain types of beta HPVs are associated with nonmelanoma skin cancer (NMSC) in immunocompromised individuals. However, whether beta HPV infections promote NMSC in the immunocompetent population is unclear. They have been hypothesized to increase genomic instability stemming from ultraviolet light exposure by disrupting DNA damage responses. Implicit in this hypothesis is that the virus encodes one or more proteins that impair DNA repair signaling. Fluorescence-based reporters, next-generation sequencing, and animal models have been used to test this primarily in cells expressing beta HPV E6/E7. Of the two, beta HPV E6 appears to have the greatest ability to increase UV mutagenesis, by attenuating two major double-strand break (DSB) repair pathways, homologous recombination, and non-homologous end-joining. Here, we review this dysregulation of DSB repair and emerging approaches that can be used to further these efforts. The human papillomavirus (HPV) family is big, with over 450 HPV types already identified ,2. This Unlike tumorigenic “high risk” alpha genus HPVs, beta genus HPV (beta HPV) infections occur in cutaneous epithelia ,12. In pBeta HPV infections are also common in the general population ,21,22,23Here, we focus our discussion on the current understanding of how beta HPV hinders the repair of double-strand breaks in DNA (DSBs). The concentration on DSBs is based on the consensus that they are the most mutagenic type of DNA lesion. Further, failure to repair DSBs increases genomic instability and would be consistent with the hypothesized manner in which beta HPV infections promote NMSC formation. We also provide a survey of novel approaches that could be used to expand this knowledge base. Because beta HPV infections occur in the skin, ultraviolet radiation or UV is the most biologically relevant source of DNA lesions. The cellular response to UV-induced DNA damage is well characterized . The mosHigh-risk alpha HPV encodes two primary oncogenes (E6 and E7) that facilitate extensive manipulation of the host cell environment. Studies of beta HPV biology often focus on the homologs of oncogenes . This woThe E6 and/or E7 from these beta HPVs also show transforming properties in vivo following UV radiation ,51,52,53The interference with UV damage repair by HPV5 and HPV8 E6 causes a more frequent collapse of replication forks into mutagenic DSBs. Erroneous DSB repair can cause the loss of large regions of DNA, chromosome translocation/rearrangements, and aneuploidy ,68. Two Homologous recombination is an error-free DNA-repair pathway. It occurs in S/G2 phase so that a sister chromatid can be used as a homologous template. HR is dependent on DNA resection. ATM kinase and its targets are responsible for DSB sensing, cell cycle arrest, and DNA resection ,71,72,73A GFP-based HR reporter demonstrated that HPV5, 8, and 38 E6 decrease HR efficiency ,85. The NHEJ is responsible for DSB repair throughout interphase and G1 when homologous templates are not available. NHEJ initiates with localization of 53BP1 to the DSB. This helps prevent HR factors from promoting DNA resection ,88,89. NHPV5 and 8 E6 delay 53BP1 repair complex resolution . FurtherIt should be noted that neither HPV5 E6 nor HPV8 E6 completely abrogates DSB repair. Rather, these viral proteins delay DSB repair. This suggests that DSB repair is still occurring and motivated ongoing efforts to identify the pathway(s) by which it was happening. To address this knowledge gap, a recent effort tracked the persistent HR and NHEJ foci found in earlier work ,99. ThisAt least some beta HPV proteins abrogate correct DSB repair. However, outstanding questions remain. Many studies of beta HPV and DSB repair are conducted in cells expressing the E6 gene in isolation. While HPV8 E6 alone did not significantly decrease CHK1 protein levels, the combination of HPV8 E6 and E7 reduced CHK1 protein abundance in vitro and in vivo . This suDSBs can be induced by physical rays and chemical reagents. The source of these lesions is important to consider when studying DSB repair. UV and ionizing radiation (IR) are two types of radiation commonly used to generate DSBs in cells. Since UV relies on the collapse of replication forks, DSBs induced by UV do not occur in all cells at the same time ,102. ThuChemicals can also be used to induce DSBs. For instance, radiation mimicking reagents such as bleomycin can cleave DNA via intercalation ,109. HydAll the physical rays or chemical reagents mentioned in the preceding paragraph cause genome-wide DSBs in a largely non-discriminative manner. This increases the challenge of dissecting the repair process at a single DSB site. To study the repair of a single DSB repair at a targeted locus, specified artificial endonuclease such as I-Sce1 can be used . HoweverIR and UV have been used to induce DSBs in cells with HPV5, 8, and 38 E6 ,84. RadiActivation of DNA repair is regulated by cellular signaling pathways ,116,117.Immunoblotting has been used to show that HPV5 and 8 E6 decrease major HR proteins including ATM, BRCA1, and BRCA2 . The appAs described above, DNA repair proteins should be recruited to the damaged site forming repair complexes, visible by microscopy as distinct foci. Immunofluorescence (IF) microscopy is often used together with DSB induction to examine the localization of repair factors to DSBs. In these analyses, phosphorylated H2AX (S139) or pH2AX is an established marker of DSBs that can be used to confirm the localization of repair factors to DSBs or define the overall rate of DSB repair by fixing cells at intervals after DSB induction ,123. IF IF microscopy has been used to detect repair kinetics of the HR pathway in cells expressing HPV5, 8, and 38 E6 . The appIF microscopy of fixed cells does not allow tracking of a single cell through the course of repair. Although technically more challenging, IF microscopy of living cells can address this barrier. Following DSB induction as described above, the recruitment and resolution of DSB repair factors can be recorded by time-lapse microscopy. This approach was used to reveal that NHEJ occurs in the G1 phase and NHEJ to HR pathway switch occurs in the S phase ,127. ThiWhile live-cell imaging has been used to examine other aspects of beta HPV biology , it has To measure the frequency of a specific DSB repair pathway, specialized reporters have been developed. The general design of these reporters is that they contain a fluorescent protein (most often GFP) that is inactivated by insertions in the open reading frame . I-SceI Reporter assays have been used to show that HPV5 and 8 E6 disrupt the HR pathway and that HPV8 E6 attenuates NHEJ ,99. Our IF microscopy allows the DNA repair process to be studied at a high resolution, but this benefit is offset by the time-consuming nature of image capturing and analysis to obtain robust data. The reporter constructs described above tend to result in fluorophore expression at levels too low (<5%) to be amenable to detection by IF microscopy. Instead, flow cytometry is more commonly used as a high throughput alternative with these assays. It can be used to detect GFP from fluorophore-based reporter constructs. Flow cytometry can also be used to determine the cell cycle position of the repair complex and offer valuable insight into the interplay between cell cycle position and DSB repair factor activation . With thFlow cytometry is used together with specified DSB reporters to measure the efficiency of different repair pathways . It alsoWhile the approaches above can be used to detect defects in DSB repair signaling, they do not measure the mutagenic impact of these defects (including increases in the number of changes in the types of mutations). Next-generation sequencing (NGS) is currently the best way to get this information. Whole-genome sequencing (WGS) is a type of NGS that offers an unbiased characterization of mutations by sequencing the entire genome. Whole exosome sequencing (WES) focuses this analysis by allowing mutations in non-protein-coding regions to be ignored. Targeted NGS can provide a robust analysis of a small region of interest allowing deep sequencing at an affordable price . TargeteNGS at a known region (CD4) around a single DSB induced by sgRNA/CAS9 shows that HPV8 E6 increases multiple types of mutations ,114. NGSWhile most of the approaches described above have already been used to investigate beta HPV biology, their ability to ask other questions fundamental to cell biology allows them to remain useful. However, we are unaware of two of the approaches being used in this field, namely live imaging of repair complexes and WGS . In addiEstablished and cutting-edge techniques have been used to show that beta HPV E6 proteins disrupt DSB repair and in some cases cause striking increases in mutagens during DSB repair. While these results support the “hit and run” hypothesis, they fall well short of providing definitive support for the idea. Ultimately, addressing outstanding questions  is criti


Similarly, live-cell imagingshowed a compartment-specific decrease in response to various drugs,such as CB-839, parthenolide (PTL), and piperlongumine (PLM). Theenzymatic activity assay revealed the mechanism underlying fluctuationsin GSH/GSSG levels in different subcellular compartments mediatedby these drugs in the GSH metabolic pathway, suggesting some potentialtherapeutic targets in AML cells.Glutathione (GSH),the constituent of the redox buffer system,is a scavenger of reactive oxygen species (ROS), and its ratio tooxidized glutathione (GSSG) is a key indicator of oxidative stressin the cell. Acute myeloid leukemia (AML) is a highly aggressive hematopoieticmalignancy characterized by aberrant levels of reduced and oxidizedGSH due to oxidative stress. Therefore, the real-time, dynamic, andhighly sensitive detection of GSH/GSSG in AML cells is of great interestfor the clinical diagnosis and treatment of leukemia. The applicationof genetically encoded sensors to monitor GSH/GSSG levels in AML cellsis not explored, and the underlying mechanism of how the drugs affectGSH/GSSG dynamics remains unclear. In this study, we developed subcellularcompartment-specific sensors to monitor GSH/GSSG combined with high-resolutionfluorescence microscopy that provides insights into basal GSH/GSSGlevels in the cytosol, mitochondria, nucleus, and endoplasmic reticulumof AML cells, in a decreasing order, revealing substantial heterogeneityof GSH/GSSG level dynamics in different subcellular compartments.Further, we investigated the response of GSH/GSSG ratio in AML cellscaused by Prussian blue and Fe AML cells show aberrantlevels of reduced as well as oxidized glutathione and perturbed expressionlevels of glutathione metabolism and homeostasis-linked enzymes.4 Glutathione, a ubiquitous tripeptide thiol, is widely recognizedas a reactive oxygen species (ROS) scavenger and a fundamental componentof the antioxidant system.5 The reducedglutathione (GSH) is converted to an oxidized moiety (GSSG) duringoxidative stress to maintain redox homeostasis.6 It modulates various cellular processes, such as cell signalingpathways, metabolism, defense, proliferation, stress response, anddetoxification of oxidants.7 The significanceof the GSH/GSSG ratio in tumor growth, progression, and drug resistancehas been highlighted.8 In different typesof cancers, GSH-mediated oxidative stress has been reported as a plausibletherapeutic target for most drugs. For example, in AML, there is anincreased expression of numerous glutathione pathway regulatory proteinsto combat oxidative stress. In addition, leukemic cells exhibit adecline in reduced GSH and an increase in oxidized glutathione (GSSG)levels, indicating that AML cells are hypersensitive to chemotherapeuticdrug-mediated inhibition of GSH metabolism.4 Moreover, the mechanism underlying the development of cancer cellresistance to therapeutic drugs is based on the overexpression ofglutamate–cysteine ligase  and glutathione synthetase(GSS) involved in GSH synthesis, leading to an increase in GSH/GSSG.8 Thus, it is important to measure the intracellularGSH/GSSG ratio in AML. GSH is found in various subcellular compartments,such as the mitochondria, cytosol, nucleus, and endoplasmic reticulum(ER), and its concentration varies significantly in different organelles.11 The monitoring of GSH content at the subcellular level reveals theredox state and the pathophysiology of cells, as the ratio of reducedand oxidized forms of glutathione (GSH/GSSG) indicates cellular oxidativestress in many malignancies. The abnormal GSH/GSSG level during oxidativestress in different subcellular compartments is evidence of distinctcellular activity. For example, in leukemic cells, the significantincrease in mitochondrial mass is due to the reduction of hydrogenperoxide by glutathione peroxidase (GPX) and the detoxification ofelectrophiles catalyzed by glutathione S-transferase (GSTP1) to maintainhomeostasis.12 Thus, it is crucial to measurethe real-time in vivo and compartment-specific GSH/GSSG-mediated redoxdifferences and fluctuations to comprehend the redox biology and pathophysiologyof leukemia cells. In most studies, the GSH/GSSG ratio is determinedusing chemical and disruptive methods at the whole cell or subcellularlevel using chemically synthesized trackers in AML cell lysates thatdisrupt cell integrity, which requires analytical separation methods,such as HPLC, LC/MS, and fluorimetry.16 However, the application of geneticallyencoded GSH sensors to overcome these limitations has barely beenexplored.18 Due to the “hard-to-transfect”nature of AML cells for exogenous genes,20 the introduction of the genetically encoded biosensor into AML cellswas not accomplished until recently by our group.21 Here, we exploited the application of endogenous geneticallyencoded biosensors comprising human glutaredoxin-1 (Grx1) linked tothe redox-sensitive green fluorescent protein, Grx1-roGFP2 , fusedwith subcellular localization guide peptides mitochondria localizationsequence (MLS), nuclear localization sequence (NLS), and endoplasmicreticulum localization sequence (ELS) with unprecedented sensitivityand temporal resolution to determine real-time and in vivo GSH/GSSGat the subcellular level.24 We successfully constructed asensor system to monitor the basal level of glutathione in four subcellularcompartments of AML: the cytosol, nucleus, mitochondria, and ER. Ourstudy demonstrates the utility of genetically encoded redox biosensorsin dissecting redox mechanisms, as well as the potential targets determinedvia enzymatic activity assays associated with the effect of chemotherapeuticdrugs on GSH/GSSG levels in different compartments of AML cells, whichcan present a compelling strategy for better and selective targetingof leukemic cells.Acute myeloid leukemia (AML)is a very aggressive malignancy of immature myeloblasts that is morecommon in adults and has a poor prognosis with a high relapse rate.g for 1 h at 37 °C. Subsequently, the cells were culturedand maintained in a culture medium containing 3 μg/mL puromycinfor 1 week, and cells expressing Cyto-Grx1-roGFP2, MLS-Grx1-roGFP2,NLS-Grx1-roGFP2, and ELS-Grx1-roGFP2.iL in the cytosol, mitochondria,nucleus, and endoplasmic reticulum, respectively, were sorted viafluorescence-activated cell sorting .TheHL60 stable cell lines expressing sensor proteins  were obtainedvia lentivirus infection and subsequent selection with 3 μg/mLpuromycin . The 293T cells grown at a confluencyof 50–60% were cotransfected with three lentiviral packagingvectors  accompanied by pLVX-sensor vectorsusing Lipofectamine 3000 (Invitrogen), according to the manufacturer’sprotocol, and after 72 h, the supernatant containing the recombinantlentivirus was harvested. We seeded HL60 cells into six-well cultureplates for lentiviral infection along with 4 μg/mL Polybrene and then centrifuged them at 10005 cells/mL) andtreated respectively at 50% confluency with DOX (0–10 μg/mL),Ara-C (0–10 μM), Fe3O4 NPs (0–100μg/mL), and PBNPs (0–100 μg/mL) for 24 h. In addition,cells were incubated with a combination of NPs and chemotherapeuticdrugs as follows: Fe3O4 NPs and PBNPs togetherwith 1 μM Ara-C for 24 h, respectively. The final concentrationof DMSO and absolute ethanol in the cells was <1%. The controlswere always treated with the same amount of solvent.DOX and Ara-C were dissolved in absolute ethanol andDMSO, respectively.The HL60 cells were seeded . The AML cells expressing theprobes Cyto-Grx1-roGFP2, MLS-Grx1-roGFP2, and NLS-Grx1-roGFP2 in thecytosol, mitochondria, and nucleus, respectively, were imaged every5 min for a total of 90 min time series after the addition of a singlebolus of 1 mM NMM, 1 μM CB-839, 20 μM PTL, and 20 μMPLM, respectively, carefully to avoid the disturbance and movementof the cells. We acquired the images for the Grx1-roGFP2 at 405 and488 nm excitation wavelengths and the 525 and 530 nm emission wavelengths.The collected imaging data were processed using Imaris software. Allmaterials, reagents, instruments, and other experimental methods usedin this study are described in detail in the Supporting Information.Live-cellimaging experiments after treatment with multiple drugs, such as NMM,CB-839, PTL, and PLM, were performed using a Zeiss LSM 980 with a63× objective lens Airyscan 2 (Zeiss Axio Observer) and a motorizedstage to capture multiple view fields controlled by ZEN software.Live-cell imaging was carried out in a temperature-, humidity-, andCO26 In particular,the Grx1-roGFP2 and Grx1-roGFP2.iL can have a specific fluorescentresponse to the GSH/GSSG ratio when excited with 408/488 nm laser.23 However, as mentioned previously, due to the “hard-to-transfect”nature20 of AML cells, exogenous gene expressionhas barely been explored in AML cells. In the current study, the geneticallyencoded fluorescent probes, Grx1-roGFP2/Grx1-roGFP2.iL, were employedto design and construct an AML sensor system based on lentiviral transductionto investigate GSH/GSSG fluctuations within various subcellular compartmentssuch as cytosol, nucleus, mitochondria, and ER. The recombinant lentiviruswas used to infect the HL60 type of AML cells to generate four stablecell lines expressing GSH-specific fluorescent indicators in the cytosol,nucleus, mitochondria, or ER. Live-cell imaging confirmed the exactlocalization and expression of Grx1-roGFP2/Grx1-roGFP2.iL in differentsubcellular compartments , indicatingthe correct distribution of MLS-Grx1-roGFP2 in the mitochondria. Thenuclear localization of NLS-Grx1-roGFP2 was also verified throughcolocalization with Hoechst 33342 , which exhibits the successful nuclear localization ofNLS-Grx1-roGFP2. The homogeneous colocalization of ELS-Grx1-roGFP2.iLand ELS-Tracker Red , confirming the accurate ELS-Grx1-roGFP2.iL expressionin the ER. Eventually, the live-cell imaging data confirmed the successfulconstruction of four stable AML cell lines expressing fluorescentglutathione probes in the cytosol, mitochondria, nucleus, and ER,respectively.The in vivo GSH/GSSG levels in varioussubcellular compartments have been measured using genetically encodedbiosensors or chemical probes in several types of human cell lineslike HeLa, 293T, Jurkat, and A549 cell.artments 1. The ceartments 1A1–A3. Martments 1B1–B3,aartments S1A, indiartments S1A, indi27 exhibited the lowest GSH/GSSG levels determinedusing ELS-localized Grx1-roGFP2.iL  was recorded forCyto-Grx1-roGFP2 stably expressed in the cytosol of HL60 cells 30min after ALA (0–2 mM) and 60 min after NMM (0–2 mM)(2O2 (0–500 μM) -treated AML cells in the  cytosol,  mitochondria,and  nucleus. The quantificationanalysis of live-cell imaging exhibited that the normalized fluorescencesignal intensity ratio  of probes Cyto-Grx1-roGFP2, MLS-Grx1-roGFP2 , and NLS-Grx1-roGFP2  decreased as plotted over time inresponse to NMM, whereas Figure S2G representsthe viability of NMM-treated HL60 cells after 24 h. It is known thatthe GSH enhancer (ALA) increased the GSH levels in A549 cells, whereasthe inhibitor (NMM) caused a decrease in GSH levels.26 Likewise, in the current study, the sensor in AML cellsresponded to the fluctuations in the GSH/GSSG levels regulated byALA and NMM. These results showed that the sensors are functionalin AML cells which agree with studies carried out in other mammaliancells.35The ratiometric calibration was performedin the Cyto-Grx1-roGFP2-expressingstable AML cell line using flow cytometry to characterize the sensitivityand specificity of Grx1-roGFP2 to GSH/GSSG levels. The HL60 cellswere also treated with H(0–2 mM)1F, 2 min–500 μM) 1G, and 2–500 μM) 1H treatm4 Here, the response ratio demonstrated that Cyto-Grx1-roGFP2 andMLS-Grx1-roGFP2 were responsive to cytarabine ranging from 0 to 10μM in a concentration-dependent manner, revealing that cytarabinedecreased the GSH/GSSG levels in the cytosol and mitochondria in accordanceto the correlation between GSH/GSSG levels and the fluorescence signalratio of Grx1-roGFP2  were, respectively, treated with cytarabine(Ara-C) and DOX at different concentrations for 24 h. Previously,the total GSH/GSSG levels were determined in human AML cells in responseto Ara-C using DTNB colorimetric methods.1-roGFP2 2A,B, con1-roGFP2 2C,D. The37 Notably, by measuring the basal GSH/GSSG level of different subcellularcompartments, we are able to investigate the effects of chemotherapeuticdrugs on GSH/GSSG levels at the subcellular level in AML cells, whichwould help to elucidate the drug’s mechanism of action in AMLtherapy. Furthermore, the sensor also responded to the changes causedby the nanoparticles ) to the GSH/GSSG level in AML cells.As reported previously, the Fe3O4 NPs inducedthe depletion of GSH and increased the ROS level in different cancercells, e.g., HepG2 and A549 selectively kill cancer cells via theP53 pathway43 and PBNPs acted as multienzymemimetics and ROS scavengers to protect the cells against oxidativestress, which could be the reason for the increase in GSH/GSSG levels.44At the same time, inthe ER, it was increased slightly after theconcentration was maximum. Still, the same depletion pattern of GSH/GSSGlevels was observed in the ER as compared to the control group, revealingthat the cytosol, mitochondria, and nucleus were the most sensitivesubcellular compartments while the ER was comparably less sensitiveto the DOX 2E–H. WhiThe GSH/GSSG level-targeting efficiency of variouschemotherapeutic drugs in different subcellular compartments of AMLsuspension cells was evaluated using live-cell imaging. The drugsCB-839, PTL, and PLM were used to treat the AML stable cell linesto target the GSH/GSSG levels, as observed via live-cell imaging.The imaging data suggested that the treatment of AML stable cell linesexpressing the probes Cyto-Grx1-roGFP2 3A, MLS-GSimilarly, the treatment of AML stable cell linesexpressing differentsubcellular targeted probes, Cyto-Grx1-roGFP2 4A, MLS-GLikewise, treating the AML stable cell lines expressingCyto-Grx1-roGFP25A, MLS-GFigure S4). The five keyenzymes  involved in GSH biosynthesiswere found in specific subcellular compartments.9 They maintain the homeostasis of reduced and oxidized GSHlevels to combat cell oxidative stress. To further explore the mechanismbehind the change in GSH/GSSG levels and investigate how drugs affectthe GSH metabolism, as observed in live-cell imaging of differentsubcellular compartments, molecular docking of enzymes was performedwith CB-839, PTL, and PLM. It has been reported that CB-839 disruptsglutamine metabolism and significantly impairs antioxidant GSH synthesis,resulting in increased mitochondrial ROS and apoptotic cell death.38The GSH metabolic pathway showed avaried distribution of the GSH and enzymes involved in its synthesisin different subcellular compartments, such as the cytosol, mitochondria,nucleus, and ER for the measurement of GSH/GSSG levels in the four subcellular compartmentsof AML suspension cells. The established sensor system in AML cellsenables GSH/GSSG gradient and basal level profiling at the subcellularlevel, conferring substantial heterogeneity and investigating thedrug-sensitive compartments in AML cells via live-cell fluorescencemicroscopy. With the GSH/GSSG-specific biosensors, we profiled theresponse spectrum of different subcellular compartments in AML cellsunder treatment by various chemotherapeutic drugs. Moreover, the live-cellimaging conferred a compartment-specific decrease in GSH/GSSG levelsin response to various drugs, CB-839, PTL, and PLM. The mechanismunderlying the fluctuations in GSH/GSSG was revealed through an in-depthstudy of the enzymatic activity influenced by these drugs in the GSHmetabolic pathway. We found some potential therapeutic targets inthe GSH metabolic pathway of AML cells, as investigated through dockinginteraction and enzymatic activity assays. Therefore, this study couldhelp elucidate the mechanism of GSH-mediated redox regulation in AMLfor the development of novel and more efficient AML treatment strategies.


Ryanopathies are associated with the impaired function of the RyR, leading to heart diseases such as congestive heart failure (CHF), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular dysplasia type 2 (ARVD2), and calcium release deficiency syndrome (CRDS). The aim of the current review is to provide a short insight into the pathological mechanisms of ryanopathies and discuss the pharmacological approaches targeting RyR2.Cardiac diseases are the leading causes of death, with a growing number of cases worldwide, posing a challenge for both healthcare and research. Therefore, the most relevant aim of cardiac research is to unravel the molecular pathomechanisms and identify new therapeutic targets. Cardiac ryanodine receptor (RyR2), the Ca This mechanism is called Ca2+-induced Ca2+ release (CICR), which is the crucial step of cardiac excitation–contraction coupling (ECC). The Ca2+ current serves as an essential trigger for Ca2+ release, but most of the Ca2+ required for contraction comes from the SR. In the beginning of diastole, Ca2+ release is terminated and RyR2s remain closed during the whole course of diastole. [Ca2+]i is restored by the sarcoplasmic reticulum Ca2+ ATP-ase (SERCA), the plasma membrane Ca2+ pump (PMCA), and the Na+-Ca2+ exchanger (NCX) [RyR2 is a ligand-gated Caer (NCX) .2+]i; therefore, it is determined by the SR Ca2+ content, the magnitude of the LTCC current, and the sensitivity of RyR2 to Ca2+ (ECC gain). Ca2+ fluxes through the sarcolemma, and SR are linked through a dynamic balance, which protects the cardiomyocyte against cytoplasmic Ca2+ overload while maintaining Ca2+ release amplitudes. The basis of this autoregulation is that (1) RyR2 is activated by increasing SR [Ca2+], (2) LTCC inactivation is Ca2+-dependent, and (3) NCX-mediated Ca2+ clearance rate is enhanced by elevated [Ca2+]i i transients drive larger Ca2+ efflux through the NCX, which eventually decreased the SR Ca2+ content to a lower steady state level. This experimental setup was designed to simulate the situation in certain heart diseases such as congestive heart failure (CHF) or catecholaminergic polymorphic ventricular tachycardia (CPVT) when RyR2 is substantially active during diastole; however, the results suggested that the autoregulation of Ca2+ release is broken in these conditions. Ca2+ leaked out of the SR and the resulting Ca2+ overload, which induced an inward current through the NCX, causing two problems: (1) it generated premature depolarization , associated with triggered arrythmias, and (2) depleted the SR Ca2+ content to severely low levels (below the capacity of autoregulation) that depressed Ca2+ release amplitude i . The autmplitude ,6. Apparthe RyR2 ,7. Next,The primary causes of CHF include myocardial infarction and chronic hypertension, which initiate the morphological and functional remodeling of the cardiac tissue, resulting in cardiac hypertrophy. Cardiac remodeling is an adaptive response to increased wall stress and altered neurohormonal stimuli after myocardial infarction or hypertension, which compensates for the impaired pump function. However, in the long term, ongoing maladaptive remodeling gradually demolishes the structural integrity of the tissue, leading to decompensation and heart failure with reduced ejection fraction (HFrEF) ,10,11,122+ concentrations ([Ca2+]i), generated by enhanced Ca2+ release  reaches a threshold and triggers Ca2+ spillover by opening RyR2s, is called store overload-induced Ca2+ release (SOICR), manifested as propagating Ca2+ waves  that accounts for the manifestation of CPVT (gain-of-function) i following caffeine activation  and also account for the development of SOICR , resulting in higher NCX-mediated Ca2+ efflux, which would decrease SR Ca2+ content and therefore the probability of SOICR as well -dependent, as the drug was not potent at ≥10 µM cytoplasmic [Ca2+], indicating that it would not inhibit the amplitude of Ca2+ transients. Most importantly, phenytoin selectively inhibited RyR2s isolated from failing human hearts, as it did not affect RyR2s from healthy individuals -7-methoxy-2,2,4,5-tetrahydro-1,4-benzothiazepine, also known as JTV519) was developed by Kirin Pharmaceutical Laboratory and first tested by Kaneko in 1994, who reported that it suppressed myocardial injury in a myofibrillar overcontraction model . The autre model . In anotre model ,135. An re model . In linere model ,138.In order to determine whether restoring FKBP12.6 binding to RyR2 improves cardiac function in CHF, MI was induced in wild-type and FKBP−/− mice. K201-treated wild-type mice showed a significant increase in ejection fraction, while K201 failed to cause improvement in FKBP−/− mice compared to placebo controls. Coimmunoprecipitation experiments demonstrated an increased amount of FKBP12.6 bound to RyR2 in K201-treated animals. These data suggest that FKBP12.6 is essential for the beneficial effects of K201 in CHF, and K201 acts by enhancing FKBP12.6 affinity to RyR2 .+, Ca2+, and K+ current inhibiting actions that motivated further research to develop newer derivatives  that lack these off-target effects. One compound from this group, S107, was reported to have K201-like effects [2+ leak and reduced ventricular arrhythmias, infarct size, and left ventricular remodeling in a rat ischemia–reperfusion model [K201 has non-specific Na effects ,140,141.on model . Clinica2+ release offers an effective therapy in both CHF and CPVT; however, currently, there is not any clinically available RyR2-specific inhibitor. There is a substantial effort to find RyR2-selective inhibitors, but not all RyR2 blockers would theoretically make an appropriate antiarrhythmic medicine. Drug repurposing looks to be the primary strategy in RyR2-targeted drug development and modification of these lead molecules have promising prospects. These efforts probably will lead to many newer RyR2-selective drugs with strong therapeutic potential soon.Fixing dysregulated Ca


As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.Calreticulin is an endoplasmic Ca Cycling of Ca2+ in cardiomyocytes drives muscle excitation–contraction (E-C) coupling. The sarcoplasmic reticulum (SR), a functionally specialized form of the endoplasmic reticulum (ER) and a component of the cellular reticular network, is the source of Ca2+ for muscle contraction and relaxation [2+ regulatory proteins that control and regulate the E-C coupling of the cardiac muscle. Calsequestrin, the ryanodine receptor/Ca2+ channel (RyR), junctin, junctate, sarcalumenin, and histidine-rich protein are examples of proteins that are unique to the SR and play an important role in SR Ca2+ handling. Ca2+ release from the SR via the RyR initiates muscle contraction [2+ is decreased in the cytoplasm by the action of SR-associated Ca2+-ATPase (SERCA), a plasma membrane Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase [2+ binding protein of the ER, and its impact on cardiac physiology/pathology, and we provide evidence that ER functions are essential components of cardiomyocyte biology.Cardiovascular disease is one of the major health burdens in developed countries. Key causes of heart failure are ischemic heart disease and myocardial infarction, which damage the heart muscle, thereby compromising heart function. Calaxation . This me+-ATPase . As impo+-ATPase . The pot2+-binding chaperone present in a number of diverse species [2+ in the ER lumen with high capacity and participates in the folding of newly synthesized proteins and glycoproteins. Calreticulin, together with calnexin  and PDIA3 , constitute a so-called “calreticulin/calnexin cycle”, which is responsible for the folding and quality control of newly synthesized glycoproteins [2+ to carry out mechanical functions in the heart. Calreticulin protein contains functionally specialized domains including an N-terminal globular domain and an extended arm proline-rich domain (P-domain), which are responsible for the chaperone function of the protein. In addition, the C-terminal highly acid C-domain is responsible for Ca2+ binding and buffering [Calreticulin is an ER-resident Ca species . The proproteins ,7,8,9,102+ handling by calreticulin is responsible for the embryonic lethality in calreticulin-deficient mice [2+/calcineurin-dependent transcriptional events during cardiac development [Cardiac development is a well-controlled molecular and morphogenetic event, and even small perturbations in this process can have devastating consequences in the form of congenital heart disease . In miceent mice ,14. Thiselopment .2+ and calreticulin-dependent checkpoint in cardiac myofibrillogenesis [2+-dependent transcriptional activities and impaired myofibrillogenesis due to decreased activity of the muscle-specific transcription factor MEF2C [2+ signaling during early cardiac development [The ultrastructure of the myofibrils is disorganized in the developing heart in the absence of calreticulin, and the transcriptional function of MEF2C shows impaired nuclear localization, further supporting the involvement of a critical Caogenesis . A role ogenesis . In the or MEF2C . Inhibitor MEF2C . In calrelopment . Calretielopment .2+ concentration, leading to inflammation and upregulation of a RelA protein, which is part of the NF-κB family [2+ binding function of calreticulin involved, or is its role in ER-dependent stress responses, including unfolded protein response (UPR), responsible? Likely, both are involved. Many miRNAs are targets of the nuclease activity of the ER stress sensor IRE1α, driving apoptotic events that are due to ER stress [2+ channel ORAI1, which is responsible for Ca2+ entry from the extracellular space due to store-operated Ca2+ entry, there is an increase in several miRNAs with target degradation that is dependent on intracellular Ca2+ levels [miRNA plays a significant role in cardiovascular differentiation, function, and disease and has been the target of intense scrutiny ,20,21. IB family . In calrR stress . In fibr+ levels .2+ and the Ca2+ binding function of calreticulin in cardiac development comes from studies of GRP94, another ER Ca2+ binding chaperone and resident protein. GRP94 and calreticulin have many similar features. Firstly, GRP94 has a highly acidic C-terminal domain that binds approximately 20 moles of Ca2+ per mole of protein [2+ binding function of calreticulin and proper ER Ca2+ homeostasis are essential for cardiac development.Evidence of the importance of ER Ca protein . Second, protein . Lastly, protein . This pr2+ binding C-domain [2+ binding residues to a large cluster of positively charged amino acids in the C-domain [2+ binding C-domain) is embryonic lethal in a way similar to that seen for silencing of the calreticulin gene [2+ binding to calreticulin that affects cellular Ca2+ signaling is sufficient to induce embryonic lethality in mice [2+-dependent events are critical during cardiogenesis and play different roles in fully differentiated cardiomyocytes  or deletion of calreticulin (reduced ER Ca2+ capacity) in the adult heart impairs systolic and diastolic functions. Calreticulin deficiency in adult cardiomyocytes has also resulted in a 50% reduction in spXBP1 mRNA compared to a 30% increase in spXBP1 when calreticulin is overexpressed (The Cre:LoxP tamoxifen-inducible system combined with the myosin heavy-chain-promoter-driven expression of Cre recombinase ,46,47 ha2+ capacity/homeostasis is essential for cardiac development and leads to heart disease when dysregulated.ER Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis.Calreticulin supports Ca2+ binding protein in the ER/SR of the developing heart and is downregulated after birth when calsequestrin, a muscle-specific Ca2+ binding/storage protein, is upregulated to supply Ca2+ that supports E-C coupling.Calreticulin is a major Ca2+ capacity produce cardiomyocytes with enhanced efficiency, triggering detrimental mechanical stretching of cardiac fibroblasts and leading to cardiac pathology.The increased expression of calreticulin and an increased ER/SR Ca2+ pool in adult cardiomyocytes must be controlled as any increase or decrease in calreticulin results in heart pathology.The calreticulin-dependent CaResolving the specific functions of ER versus SR in muscle cells remains a challenge.The role of calreticulin mutants needs to be explored for a better understanding of their role in cardiac pathophysiology.2+ homeostasis?Do other ER-associated chaperones play a role in cardiomyocyte Ca2+ homeostasis will allow for the development of more targeted approaches to combat heart disease.Further understanding of the role of ER/SR lumenal Ca


Stimulation with denatonium benzoate, a bitter taste receptor agonist which activates largely nuclear and mitochondrial Ca2+ responses, resulted in a dramatically increased cytosolic Ca2+ response in cells treated with lactisole. This cytosolic Ca2+ signaling activated NO production in the presence of lactisole. Thus, lactisole may be useful coupled with bitter compounds as a therapeutic nasal rinse or spray to enhance beneficial antibacterial NO production in patients suffering from chronic inflammatory diseases such as chronic rhinosinusitis.Human airway sweet (T1R2 + T1R3), umami (T1R1 + T1R3), and bitter taste receptors (T2Rs) are critical components of the innate immune system, acting as sensors to monitor pathogenic growth. T2Rs detect bacterial products or bitter compounds to drive nitric oxide (NO) production in both healthy and diseased epithelial cell models. The NO enhances ciliary beating and also directly kills pathogens. Both sweet and umami receptors have been characterized to repress bitter taste receptor signaling in healthy and disease models. We hypothesized that the sweet/umami T1R3 antagonist lactisole may be used to alleviate bitter taste receptor repression in airway basal epithelial cells and enhance NO production. Here, we show that lactisole activates cAMP generation, though this occurs through a pathway independent of T1R3. This cAMP most likely signals through EPAC to increase ER Ca T1R3 has also been proposed to signal independently of T1R1 or T1R2, either as a homodimer or with another GPCR partner [In humans, the taste perception of either sweet or savory/umami chemicals occurs through type 1 taste receptors (T1Rs). The perception of sweet taste signals the presence of beneficial energy-rich sugars, and this pathway is thought to be signaled by the interaction of sweet tasting molecules with a G protein-coupled receptor (GPCR) heterodimer of T1R2 and T1R3 [ partner ,6,7,8,9. partner ,6,7,8,9  partner . Pharmac partner ,6,7,8,9.2+ pathways activated by the detection of sweet [Experiments where T1R nutrient sensing is inhibited often employ lactisole, a molecule isolated from coffee beans . Lactisoof sweet  or umamiof sweet . Consequof sweet ,11,12,132+ to stimulate nitric oxide (NO) production [2+ within the SCC to trigger a release of Ca2+, acetylcholine, and other signaling molecules through gap junctions into surrounding multi-ciliated epithelial cells [Apart from their function in taste signaling pathways, T1Rs are also expressed in the human upper airway, where they function with bitter type 2 taste receptors (T2Rs) to detect pathogenic growth. T2Rs are localized to the cilia of multi-ciliated epithelial cells where they detect “bitter” bacterial byproducts, such as quorum sensing molecules. Once activated, T2Rs signal through intracellular Caoduction ,15,16 anoduction , increasoduction ,19 along2+ signaling pathways through reducing total ER Ca2+ levels [Viruses such as influenza  or SARS-+ levels . Thus, w2+ content and denatonium-induced cytosolic Ca2+ elevations, ultimately signaling an increase in NO production. We propose here that these off-target effects of lactisole may be therapeutically useful for boosting airway NO production. However, our data also suggest that studies using lactisole to experimentally or therapeutically inhibit T1R sugar or amino acid detection must take into account the potential effects of T1R-independent cAMP signaling and/or changes in ER Ca2+ signaling that may also occur.Here, we show that lactisole increases intracellular cAMP levels independent of T1R1 or T1R3 expression. Additionally, lactisole both increases ER CaA list of all reagents used in the current study are shown in 2+, cells were incubated with 5 µM of Fluo-8 AM for 1 h in Hank’s Balanced Salt buffered with 20 mM HEPES free acid. Fura-2 AM (5 µM) was utilized for assessment of baseline intracellular Ca2+ levels. Images of cells loaded with Fura-2 were captured using Fura-2 filters . To assess NO production, cells were loaded with 10 µM DAF-FM diacetate for 45 min. For intracellular or nuclear cAMP, nuclear, mitochondrial, or non-nuclear Ca2+ measurements, cells were transfected with the appropriate biosensors 48 h prior to experiments. Images taken with either FITC or TRITC utilized 49002-ET or 49004-ET Chroma respectively. All images were taken on an Olympus IX-83 microscope (20 × 0.75 NA objective) with excitation and emission filter wheels from Sutter Instruments , an Orca Flash 4.0 sCMOS camera  and used MetaFluor  software. Light sources included a xenon lamp for Fura-2 and a X-Cite 120 boost LED source  for all other imaging.To assess intracellular CaResidual human surgical material was used a source for isolating primary sinonasal cells. Samples were obtained conforming to the University of Pennsylvania guidelines for use along with the Declaration of Helsinki and the U.S. Department of Health and Human Services code of federal regulation (Title 45 CFR 46.116). Institutional review board approval (#800614) was obtained, and informed consent was collected from each patient. Patients were adults, >18 years old, and underwent surgery to address a sinonasal disease or for other causes.2 in PneumaCult-Ex Plus medium  supplemented with penicillin (100 U/mL) and streptomycin (100 ug/mL). After 2 h had passed, the media were then transferred to a cell culture dish and the resulting cultures were expanded utilizing PneumaCult-Ex Plus medium. To obtain fully differentiated cultures, cells were seeded in air–liquid interface (ALI) cultures and exposed to air for at least 21 days prior to use in experiments.Unless otherwise noted, the primary nasal epithelial cells were isolated from the residual surgical material via enzymatic digestion as previous described . In shorPrimary bronchial epithelial cells were purchased through Lonza (CC-2540S) and propagated and differentiated using the same methods as described for primary nasal epithelial cells.Subconfluent cultures of Beas-2B cells  were transfected via lipofectamine 3000 for 48 h with either 10 nM of TAS1R1.6 or TAS1R3.2 or control non-targeting RNAi duplex . Validation of knockdown was achieved via qPCR analysis.RNA was collected by resuspending cultures in TRIzol . Suspensions were used immediately or stored at 70 °C for later use. The Direct-zol RNA kit  was used to purify the preserved RNA. cDNA libraries were created using RT-PCR using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Resulting cDNA libraries were then subjected to qPCR using Taqman qPCR probes and analyzed via the QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific). Microscoft Excel and GraphPad PRISM v8 were used to both analyze and plot the resulting data.Unless otherwise noted, genotyping methods followed as previously described . Samplest-tests were utilized. Graphs or plots with >2 comparisons utilized the ANOVA test, whereas the Tukey–Kramer post-test was used for comparing multiple samples to each other, the Bonferroni post-test was used for selective pair comparisons, Sidak’s post-test for paired comparisons, or Dunnett’s post-test for multiple comparisons relative to one control. In all comparisons, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001, “n.s.” . All data presented are the mean ± SEM of at least 3 experiments.For graphs with just two comparisons, 2+ signaling [Both sweet and umami taste signaling pathways are thought to share a common component, the GPCR T1R3. In humans, lactisole inhibits the perceived taste of both sweet  and umamignaling ,27,28. Pignaling  and the ignaling . Therefo2+ detection dye Fluo-8 AM, lactisole alone had no effect on intracellular Ca2+  in 0-Ca2+ HBSS with 2 mM EGTA. By inhibiting ER Ca2+ uptake, the total ER Ca2+ stores slowly leaked and caused Fluo-8 to fluoresce before continuing to disperse out of the cell and chelating to extracellular EGTA, preventing re-uptake. Beas-2B cells treated with 20 mM lactisole for 1 h revealed a 275% increase in ER Ca2+ release as observed through comparisons of peak Ca2+ elevations and a 200% increase in ER Ca2+ stores as calculated by the area under the curve  from the ER. As seen in 2+ content and the rate of Ca2+ efflux from the ER. With such a dramatic increase in ER Ca2+ stores, we wanted to investigate if the ER itself increased in size.Even though lactisole did not stimulate Cahe curve a. While + channels prominent on the ER, to visualize the ER’s morphology. Here, we show that in live cells loaded with ER-Tracker, there is a 25% reduction in fluorescence signal in cells pretreated with 20 mM lactisole for 1 h relative to untreated cells  signaling via a Gq. Moreover, we only observed a denatonium-induced Ca2+ release in airway cells line RPMI-2650 ((2+ in response to denatonium regardless of lactisole pretreatment (2+ in response to thujone (3 mM), however this signaling was unaffected by lactisole pretreatment  increased PTC-induced Ca2+ release by 300% , a bitter compound associated with T2R38 . In humaof T2R38 . We genopression . While B by 300% . Utiliziartments . Thus, l2+ signaling pathways was not unique to T2R agonists. Histamine-induced Ca2+ release was also increased with lactisole pretreatment  or Exchange Protein Directly Activated by cAMP (EPAC). Both have been shown to affect Caor AKAR4 ,34, we dor AKAR4 . Beas-2Bevations d. Intereevations d. These 2+ efflux and influx pathways via modulation of SERCA and RYR Receptors to create a Ca2+-induced Ca2+ release pathway [2+ content.Though PKA activity was not likely elevated by lactisole, we wanted to test if EPAC activity was affected. Using the ratiometric cAMP Epac-S-H74  biosenso pathway ,38,39,402+ dynamics in airway epithelial cells, we first utilized qPCR to determine the expression levels of inositol 1,4,5-triphosphate (IP3) receptors, ryanodine receptors, and sarcoendoplasmic reticulum calcium ATPase (SERCA) pump expression. Here, we show that in Beas-2Bs, primary basal, and primary differentiated airway epithelial cells express all forms of IP3 receptors (ITPR1-3) and SERCA pumps (ATP2A1-3) however they lack expression of ryanodine receptor 2 (RYR2)  . Additio2B cells , support2+ content as described above in 2+ efflux from the ER as observed through increases in the peak Ca2+ elevations and the relative magnitude of the intial linear slope in Fluo8 fluorescence relative to untreated cells, however there was no impact on total ER Ca2+ content  increased the peak Ca2+ release per cell by 65%  blocked lactisole’s ability to increase denatonium-stimulated Ca2+ elevations  glucose conditions . Primary basal epithelial cells produced NO when in the presence of lactisole  and/or sugars (T1R2/3 or T1R3 homodimers) to regulate cell physiology that affects diverse processes such as taste and insulin secretion. Many studies have described the function of lactisole as an inhibitor of T1R3 and have used lactisole as a tool to pinpoint role of T1Rs. Here we showed a novel, uncharacterized function for lactisole that seemingly impacts all downstream Ca2+ modulates NO production [2+-signaled NO production in HBSS containing amino acids. Therefore, both amino acids and lactisole may be useful to employ in conjunction with bitter compounds to drive NO production to help combat infections.We also showed that these off-target effects of lactisole might be therapeutically leveraged in airway epithelial cells. Intracellular Caoduction . NO is aoduction . We prevoduction . Moreove


Plants perceive bacterial Nod factors or fungal signals to induce the Ca2+ oscillation in the nucleus of root hair cells, and subsequently activate calmodulin (CaM) and Ca2+/CaM-dependent protein kinase (CCaMK). Ca2+ and CaM-bound CCaMK phosphorylate transcription factors then initiate down-stream signaling events. In addition, distinct Ca2+ signatures are activated at different symbiotic stages: microbial colonization and infection; nodule formation; and mycorrhizal development. Ca2+ acts as a key signal that regulates a complex interplay of downstream responses in many biological processes. This short review focuses on advances in Ca2+ signaling-regulated symbiotic events. It is meant to be an introduction to readers in and outside the field of bacterial and fungal symbioses. We summarize the molecular mechanisms underlying Ca2+/CaM-mediated signaling in fine-tuning both local and systemic symbiotic events.Cytoplasmic calcium (Ca For example, the plant plasma membrane receptor flagellin-sensitive 2 (FLS2) recognizes the conserved bacterial PAMP, flg22, to induce transient Ca2+ influxes , calcineurin B-like protein (CBL)-CBL-interacting protein kinases (CIPK) and Ca2+calcium-dependent protein kinases (CDPKs or CPKs)  . InteresMedicago-rhizobial symbiotic relationship, the symbiosis signaling pathway is initiated when the plant receptor complex LysM receptor kinase 3 (LYK3)-Nod factor perception (NFP) recognizes lipo-chitooligosaccharide signals  from rhizobial bacteria  and DMI1  from nucleus to ribosome for polypeptides or protein biosynthesis. Another possibility is that the NPCs regulate the localization of CASTOR, POLLUX/DMI1, CNGC15 to the nuclear membranes, although the biological mechanism involved deserves further study.The nuclear pore complex (NPC) in llations . Nod faceratures . In L. jnization . The nuc2+ channels and components of the nuclear pore complex , other cation channels are also required for nuclear Ca2+ oscillations. Medicago truncatula DMI1 and its two homologs CASTOR and POLLUX in L. japonicus were once thought to be potassium (K+) channels  failed to form rhizobia-induced nodulation. This finding links defects in Ca2+ binding to Ca2+ channel regulation, which ultimately affects the legume-microbe symbiosis  to form a complex protein in nuclear membranes which was required for the activation of nuclear Ca2+ spiking , displayed spontaneous nuclear Ca2+ spikes and constitutive activation of nodulation . A hypothesis proposes that the inner-layer-localized MCA8 mediates the recapture of nuclear Ca2+ spikes, while the outer-layer- and the ER-localized MCA8 may reload the Ca2+ store at the ER or nuclear envelope from the cytoplasm ] and heterotrimeric G-proteins including Gα, Gβ, and Gγ subunits are also involved in root nodule symbiosis . The GTPase, belongs to the Rho/Rop family, directly regulates reactive oxygen species (ROS) production through activating the respiratory burst oxidase homolog B (RBOHB) . The ROSesponses , which sesponses . Interesymbiosis : the exped roots ; and gened roots . Furthered roots . Anothered roots . Active ed roots . More st2+ and CaM. Hence, it was named Ca2+/CaM-dependent protein kinase  which were discovered in animal cells, the CCaMK reported from plants contained a C-terminal visinin-like domain, including three EF-hand motifs, which functioned as a Ca2+-sensitive molecular switch  and one CaM-binding domain with autoinhibitory function , Ca2+/CaM also binds to CCaMK, and the protein becomes activated  increased the CaM-binding capability of the mutant, which underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of Ca2+ and CaM. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results suggest that altering the CaM-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of the CCaMK  participate in symbiotic regulation. The answers to the above questions should provide new insights into nodulation and arbuscular mycorrhizal colonization. This knowledge would empower us to develop strategies to improve and manipulate plant-microbe symbioses and, thus, increase crop yield and agricultural productivity.Although exciting advances in CaPY, FL, CG and BP were involved in writing this review. All authors contributed to the article and approved the submitted version.USDA NIFA (Hatch Project 1015621 to BWP) and past support of the National Science Foundation (grants 1021344 and 1557813 to BWP) are acknowledged. This publication was partly supported by Provincial Natural Science Foundation of Hunan (2021JJ40241) and the National Technology System for Grape Industry (CARS-29-ZP-9). This publication was supported in part by the USDA National Institute of Food and Agriculture, Hatch project 1015621 to CG.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.


Interplay between the ER and mitochondria plays a crucial role in regulating energy metabolism and cell fate control under stress. The mitochondria-associated membranes (MAMs) denote physical contact sites between ER and mitochondria that mediate bidirectional communications between the two organelles. Although Ca2+ transport from ER to mitochondria is vital for mitochondrial homeostasis and energy metabolism, unrestrained Ca2+ transfer may result in mitochondrial Ca2+ overload, mitochondrial damage and cell death. Here we summarize the roles of MAMs in cell physiology and its impact in pathological conditions with a focus on cardiovascular disease. The possibility of manipulating ER-mitochondria contacts as potential therapeutic approaches is also discussed.The endoplasmic reticulum (ER) and mitochondria are interconnected intracellular organelles with vital roles in the regulation of cell signaling and function. While the ER participates in a number of biological processes including lipid biosynthesis, Ca MAMs refer to membranous contact sites between ER and mitochondria, which mediate bidirectional communications between the two organelles.2+ transmission from ER to mitochondria, molecule translocation, protein-protein interaction or lipid metabolism.MAMs control cell homeostasis and cell death. The death signals governed by MAMs may be presented as excess CaChanges in physiological events modulated by ER and mitochondria communication are common triggers for apoptosis, autophagy, necroptosis, pyroptosis and ferroptosis.2+ ions either boost metabolism or evokes apoptosis. Ca2+ flux regulated by MAMs dictates the fate of cellular events.CaDysregulation of MAMs functions is correlated with etiology of cardiovascular diseases.Targeting MAMs and associated components exhibits promises in therapeutic intervention of cardiovascular pathologies.Identification of the functional significance of various components on MAMs to distinct the forms of cell death.2+ ions transported through MAMs in cell death control?Determination of second messages or metabolites other than CaDevelopment of small molecule drugs or gene therapy strategies directly targeting MAMs to improve cell physiology in disease conditions or prevent cell death.2+ transport and storage, synthesis, transfer, modification and processing of lipids and proteins [2+ transfer proteins and lipids. It is not surprising that such membranous association facilitates the interaction between ER and mitochondria. The ER may respond to various physiological or pathophysiological stimuli to transfer ions, lipids and protein signals into mitochondria, resulting in a fine-tuning of mitochondrial physiology. On the other side of the coin, the mitochondria may also deliver ions, lipid or protein signals to the ER [The endoplasmic reticulum (ER) and mitochondria are tightly intertwined intracellular organelles in eukaryotic cells with a concerted role in the maintenance of cellular homeostasis. The ER is crucial to Caproteins –3. On thproteins –7. Ampleproteins , 8–10. Mproteins , 12. As o the ER –15.2+ transmission from ER to mitochondria, molecule translocation, protein-protein interaction and lipid metabolism. Mitochondrial permeability transition pores (mPTPs), a high-conductance channel composed of adenine nucleotide translocase (ANT) in the inner mitochondrial membrane (IMM), the voltage-dependent anion channel (VDAC) resided in the OMM and cyclophilin D in the matrix, is the main driver of cell death [As mentioned earlier, MAMs control not only cell homeostasis but also cell death events. The cell death signals governed by MAMs may be presented in various forms, including Call death . Since Mll death –19. In pll death . Therefo2+ signaling, lipid metabolism, mitochondrial dynamic alteration and apoptosis [MAMs are deemed an independent specialized cell compartment where multiple biological events take place, including Capoptosis . The compoptosis . WithoutMAMs perform biological functions through a number of protein complexes with not only tethering property but also unique functionality . Recogni3Rs) are vital Ca2+ outflow channels located on the ER surface mediating Ca2+ transfer from the ER lumen into cytoplasm. Elevated IP3Rs levels or activity are common in pathological conditions including hypertension, cardiac hypertrophy, ischemic dilated cardiomyopathy and heart failure [2+ flux. It was shown that VDACs promotes the bridging or connectivity between ER and mitochondria to facilitate Ca2+ flux into mitochondria. Silencing or inhibition of VDAC1 exhibits a loosened ER-mitochondria contact [3Rs with VDACs to sustain MAMs architecture, with higher levels of GRP75 favoring a higher degree of IP3R-VDAC interaction. This is in line with the fact that silencing GRP75 disrupts the connection between IP3Rs and VDAC  and B-cell receptor-associated protein 31 (BAP31) not only carries tethering capacity, but also transfers apoptotic signals between the two organelles , which presides over the unfolded protein response and vesicle trafficking, is recognized as a binding partner for vesicle-associated membrane protein-associated protein-B (VAPB) - an ER-resident protein regulating cell development and tumorigenesis  or ER through Ca2+ reuptake pump sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). SERCAs manifest in several subtypes, of which the ubiquitous SERCA2b possesses the highest affinity for cytoplasmic Ca2+ [2+ ions in response to electrical excitation or pharmaceutical stimulation of the ryanodine receptor (RyR). Ca2+ can move freely through the OMM with the assistance of VDACs, whereas the IMM is not permeable and Ca2+ can only influx through the mitochondrial Ca2+ uniporter (MCU) with a relatively weak affinity to cytosolic Ca2+ [2+ levels around SR/ER are considerably higher than global cytosolic Ca2+. Given the vicinity of mitochondria to SR/ER Ca2+ stores, MCU would exhibit a much higher affinity to SR/ER Ca2+ [2+ uptake efficiency [Camic Ca2+ . SR/ER rlic Ca2+ . However/ER Ca2+ . Moreoveficiency . Throughficiency .2+ transfer from the ER to the mitochondria is mainly regulated by the IP3Rs-GRP75-VDACs complexes. When ER-resident Ca2+ release channels (IP3Rs) open, a high Ca2+ microdomain can be formed within the ER vicinity. This prompts Ca2+ uptake by VDACs localized on the OMM [3Rs-GRP75-VDACs channel complex [2+ is directly transmitted from the ER to cytoplasm and cross OMM, subsequently into mitochondrial matrix through MCU [As shown in Fig.  the OMM . The thi complex . In thisough MCU .Fig. 2Re2+ in mitochondrial matrix has various functions. Firstly, it can improve the efficiency of tricarboxylic acid (TCA) cycle and the electron transport chain (ETC), thus facilitating ATP production [2+ ions that enter mitochondria boost energy metabolism by favoring the enzymatic activity of key rate limiting dehydrogenases of the TCA cycle, including isocitrate dehydrogenase (IDH), pyruvate dehydrogenase (PDH), oxoglutarate dehydrogenase (OGDH or α-KGDH), and glycerol-3-phosphate dehydrogenase (GPD1/G3PDH) [2+ may control metabolism by modulating glucose transporter activity [2+ serves as a double-edged sword. Ca2+ ions may boost cell metabolism or evoke cell death. Thus Ca2+ flux as the main Ca2+ conveyance dictates in occurrence of these cellular events, thus supporting a role for MAMs in cell fate control [2+ overload result in cell death. Ca2+ interacts with cyclophilin D and ANT to form mPTP. Excess mitochondrial Ca2+ uptake evokes mPTP opening, mitochondrial swelling and rupture of the OMM. OMM rupture is responsible for release of pro-apoptotic factors such as cytochrome C (Cyt c) and apoptosis inducing factor (AIF) [2+ overload facilitates disassembly of respiratory chain complex II through binding to cardiolipin in the IMM, thus prompting release of various subunits. Such changes trigger substantial reactive oxygen species (ROS) production and eventually apoptosis [Existence of Caoduction . Ca2+ io1/G3PDH) . In addiactivity . However control . High leor (AIF) –57. Morepoptosis . Ample epoptosis .2+ is necessary to induce apoptosis, it is not surprising that factors affecting ER-to-mitochondria Ca2+ transfer positively or negatively influence this process of programmed cell death. Multiple factors alter Ca2+ levels in mitochondria via direct or indirect action on the IP3R-GRP75-VDACs complex  can inhibit Akt and promote Ca2+ mobilization in MAMs, ultimately sensitizing cells to apoptotic stimuli [3R phosphorylation via counteracting Akt through recruitment of protein phosphatase 2 A (PP2A) at the MAMs, which then induces cell death by mediating Ca2+ transfer into mitochondria [L), two Bcl-2 family members on MAMs, connect with central modulatory domain of IP3Rs to suppress Ca2+ release or indirectly inhibit IP3Rs through regulating its phosphorylation. Besides, both Bcl-2 and Bcl-XL can conjugate with VDAC1 to suppress mitochondrial Ca2+ intake [3R-mediated Ca2+ release but also binds with VDACs to modulate mitochondrial Ca2+ uptake. To the contrary, Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer (Bak), pro-apoptotic proteins from Bcl-2 family, regulate ER Ca2+ levels by binding to and displacing Bcl-2 from IP3R1, where the anti-apoptotic protein regulates phosphorylation status of IP3R1 and Ca2+ efflux from ER [3Rs degradation may be suppressed by Sigma-1 receptor (Sig-1R), a vital component of MAMs. Sig-1R can be dislodged from binding Ig protein (Bip) and binds with IP3Rs under conditions of mitochondrial stress, thereby inhibiting degradation of IP3Rs to boost Ca2+ transmission into mitochondria [3Rs and alleviates mitochondrial Ca2+ overload, thus dampening the sensitivity to Ca2+-dependent apoptosis [Given that mitochondrial Capoptosis . The mam stimuli . Furtherchondria . In addi+ intake . Moreove from ER . IP3Rs dchondria , 66. Morpoptosis .2+ uptake in the ER, cluster in MAMs where multiple proteins modulate its function orchestrating apoptosis  is a sorting protein located on ER surface engaged in the modulation of lipid metabolism, ER homeostasis and ER-mitochondria tethering. Besides, PACS2 participates in apoptotic signaling in MAMs. High levels of ER stress evoke dephosphorylation of the full-length BH3-interacting domain death agonist (Bid) and its connection with PACS2, fostering Bid-bound PACS2 to translocate from ER to mitochondria where Bid is cleaved by caspase-8 on mitochondria producing truncated form of Bid (tBid) to bind with Bax/Bak, leading to increased permeability of OMM, release of cytochrome c and initiation of apoptosis  damage in cardiomyocytes [3R-GRP75-VDAC complex, and this interaction strengthens along with cell death following H/R injury [3R function, abolished mitochondrial Ca2+ overload and alleviated cell death as well as infarct area in reperfused hearts [MAMs possess a key role in managing mPTP opening, a main mediator of cardiomyocyte death during reperfusion damage . Cycloph stimuli , 73, 97. stimuli . The Cypmyocytes . MoreoveR injury . Inhibitd hearts . In acco2+ influx and apoptosis via widening the proximity between SR and mitochondria. Qiao and colleagues noted overtly elevated PTPIP51 in mouse hearts following I/R challenge, while knockout of PTPIP51 markedly ameliorated I/R-induced cardiomyocyte dysfunction and infarct size [2+ to protect against I/R injury although excess autophagy in reperfusion triggered further myocardial injury [2+, mitochondrial fission and mitochondrial ROS in cardiomyocytes, maneuvers which regulate SERCA function may be closely associated with myocardial susceptibility to I/R challenge. For instance, transmembrane protein related to thioredoxin 1 (TMX-1) and FUNDC1 were shown to attenuate the sensitivity of the heart to reperfusion damage through modulation of SERCA function [PTPIP51, a mitochondria-resident protein, facilitates mitochondrial Carct size . Neverthl injury . Hence, function , 104.2+ overload and respiratory chain activity in reperfusion relieves oxidative stress, Mfn1/2 knockout might be effective in alleviating cardiac dysfunction in short term. However, long term Mfn2 deletion was shown to result in inhibition of autophagy and mitochondrial fusion, en route to cardiac dysfunction [Mitochondrial dynamics, a major modulator of cellular metabolism, also plays a vital role in I/R through modulating mPTP opening , 48. As function . Fig. 3 Heart failure is a complicated and progressive disorder mainly caused by initial cardiac insufficiency and relies on a vicious cycle between neurohormonal activity and progressive cardiac remodeling –110. Mai2+ by SR Ca2+ leakage via RyR2 channel in MAMs was found in post-myocardial infarcted mice models, contributing to altered mitochondrial activity and a deleterious elevation in mitochondrial Ca2+ concentration [2+ output and mitochondrial ROS, causing RyR2 leakage, intracellular Ca2+ raise and eventually apoptosis. Based on this view, blocking calcium-sensing receptor (CaSR) diminishes intercellular Ca2+ transmission, mitochondrial Ca2+ overload and apoptosis. Mitochondria Ca2+ influx was also shown to be interfered by FUNDC1, an OMM protein governing mitophagy. Patients diagnosed with heart failure present decreased FUNDC1 expression accompanied with poor SR-mitochondria contacts [3R3 though interacting with FBXL2 (receptor subunit of human SCF (SKP1/cullin/F-box protein) ubiquitin ligase complex) to sustain mitochondrial function and Ca2+ homeostasis. We further noted that FUNDC1 deficiency exacerbated cardiac remodeling and reduced systolic function in mice fed high-fat diet [2+ transfer and ATP production under Sig-1R stimuli [2+ transport, contractility and rhythmicity. Furthermore, with a high Sig-1R affinity, fluvoxamine ameliorated heart failure and cardiac dysfunction in rodent model of transverse aortic constriction [2+ dysfunction in heart failure to trigger of mPTP opening, transgenic mice were established with induced, cardiac-specific overexpressed β2a subunit of L-type Ca2+ channel. These mice developed Ca2+ overload, spontaneous myocardial necroptosis, and heart failure. Intriguingly, deletion of cyclophilin D, a key regulator of mPTP and necroptosis, rescued this phenotype, suggesting necroptosis as a crucial component in the pathogenesis of heart failure [Overloading of mitochondrial Cantration . There mcontacts , 113. Hecontacts . In addifat diet . Anotherfat diet . Hypertr stimuli . Cardiomtriction , 118. Fu failure , 120.Maladaptive cardiac hypertrophy leads to heart failure and various functional deterioration, including aberrant mitochondrial dynamics. Substantial evidence has defined downregulated Mfn2 level in both in vivo and in vitro models of heart failure, for example, spontaneously hypertensive rats and hypertrophy induced by transverse aortic constriction . Multipl2+ transfer from ER to mitochondria. Deficiency of UCP2 decreased mitochondrial Ca2+ level and Ca2+-dependent enzymatic activity in PASMCs, evoking mitochondrial damage including decreased mitochondrial biogenesis and mitophagy in endothelium [PAH is vascular remodeling induced by phenotypical changes in pulmonary artery smooth muscle cells (PASMCs). PASMCs present a quiescent physiological contractile phenotype, which is converted to a hyperproliferative phenotype to favor small pulmonary vessel occlusion in PAH . A numbeothelium , 127. Inothelium .2+ transmitted from the ER to the mitochondria modulates PDH phosphorylation through activation of PDH phosphatase and inhibition of PDH kinase, thereby boosting PDH activity and promoting glucose oxidation [Moreover, phenotype alterations in PASMCs are linked with metabolic patterns transition, with altered ATP generation from mitochondrial oxidation to mainly rely on cytoplasmic glycolysis as the energy source . Inhibitxidation , 132. MoMitochondrial dynamics are also associated with phenotypic transition in PASMCs. Notably, PASMCs isolated from PAH patients showed mitochondrial fragmentation associated with elevated fission protein Drp1 and reduced fusion protein Mfn2 in MAMs  also plays a role in atherosclerosis and hypertension . The rolMore data are available on the role of mitochondrial dynamics in pathogenesis of systemic vascular diseases. During VSMCs shift from physiological quiescent contractile phenotype to the hyperproliferative phenotype which is resistant to apoptosis, reduction in Mfn2 expression at MAMs might be associated with the disturbance of mitochondrial network in proliferative cells. Moreover, platelet-derived growth factor (PDGF), as a known inducer of phenotypic changes in VSMCs, was shown to stimulate mitochondrial fission, VSMCs proliferation and migration , 141. BeActing as the most direct interacting bridge between the ER and mitochondria, MAMs are critical for integrating activities carried out by these two organelles, especially transmission and coordination of energy metabolism and regulation of apoptotic signals under chronic stress. Dysregulation of MAMs function is correlated with pathogenesis of cardiovascular diseases. Available evidence points out for possible biomedical applications of targeting MAMs and associated proteins as therapeutic strategies for the management of cardiovascular diseases.As an interface between energy metabolism, proteostasis and cell fate control, the modulatory mechanisms and molecules participating in the maintenance of MAMs integrity and function are vital to identification of new therapeutic targets for prevention or treatment of heart diseases. Novel and more effective treatments are exigently required for cardiovascular diseases. MAMs-associated proteins and regulatory molecules may influence the progression of cardiovascular diseases directly, which is of great importance to improve patient survival. Further studies should focus on the identification of novel MAMs proteins and modulators, as well as the therapeutic potentials of these proteins and modulators in cardiovascular diseases.check list


We hypothesized that intramuscular administration of autologous total immunoglobulin G (IgG) could induce an immunomodulatory effect in human subjects. In our previous studies, we showed that intramuscular administration of autologous total IgG could induce significant clinical improvements and increases of the serum levels of interleukin-10 (IL-10) and interferon-gamma (IFN-γ) in patients with atopic dermatitis.To investigate the mechanism of immunomodulation induced by intramuscular administration of autologous total IgG, we evaluated changes in T cells before and after intramuscular administrations of autologous total IgG in this study.Thirteen healthy adults received 8 intramuscular injections of 50 mg autologous total IgG for 4 weeks (from week 0 to week 4). The percentages of IL-10- or IFN-γ-producing peripheral blood T cells, as well as serum levels of IL-10, IFN-γ, and immunoglobulins, were measured at baseline (week 0) and at weeks 4, 8, and 12.+ T cells was significantly increased at weeks 8 and 12 compared to baseline (P < .05), while the percentage of IFN-γ-producing CD3+ T cells was significantly increased at week 12 compared to baseline (P < .05). There were no significant differences in the serum levels of IL-10, IFN-γ, and immunoglobulins before and after intramuscular administration of autologous total IgG (P > .05). No serious adverse events were observed.The percentage of IL-10-producing CD4Intramuscular administration of autologous total IgG induced immunomodulatory effects on T cells in healthy human subjects. This simple intervention could be a safe, effective, and economical T cell immunomodulation method for human subjects (NCT03695757). Previous studies have showed that a stimulation of interleukin-10 (IL-10)-producing CD4+ regulatory T cell is critical for immunomodulatory and anti-inflammatory effects of polyvalent IgG.–9 Natural IgG antibodies to the antigen binding sites (idiotypes) of pathogenic antibodies (IgG autoantibodies or IgE antibodies) have been suggested as the main therapeutic components of polyvalent IgG exerting immunomodulatory effects by neutralizing pathogenic antibodies.–12The current evidences suggest that polyvalent IgG modulates the function of immune cells, including dendritic cells, neutrophils, monocytes, macrophages, T cells, and B cells.–15 We hypothesized that active induction of anti-idiotypic immune response to pathogenic antibodies by intramuscular administration of autologous total immunoglobulin containing pathogenic antibodies could induce an immunomodulatory effect and provide a clinical improvement in patients with autoimmune and allergic diseases.The idiotype-anti-idiotypic immune response has been suggested to contribute to the development and maintenance of immune homeostasis (immune tolerance) in healthy human subjects.,18 Autologous blood therapy or autologous serum therapy  involves repeated administrations of a small amount (1–5 mL) of autologous blood or serum to the same subject by intramuscular injections, immediately after venous blood sampling.–21 These have been practiced as common alternative and complementary medical modalities to treat various immune diseases for more than 100 years since the first report in 1913.–22 Randomized, double-blind, placebo-controlled studies have demonstrated favorable clinical efficacies of autologous blood therapy and autologous serum therapy in patients with atopic dermatitis and chronic urticaria, respectively.,20 However, the therapeutic component of blood producing the clinical efficacy of autologous blood therapy and the underlying therapeutic mechanism have not been identified. We hypothesize that the blood component responsible for the therapeutic efficacy of autologous blood therapy is an autologous total immunoglobulin, including pathogenic antibodies, and that the therapeutic mechanism is an anti-idiotypic immunomodulation induced by the intramuscular administration of autologous total immunoglobulin.The concept of this study, an induction of immunomodulatory effect by intramuscular administration of autologous total IgG in healthy human subjects, originated from our understanding of the therapeutic mechanism of autologous blood therapy.,23–25 In that study, intramuscular administration of 50 mg autologous total IgG twice a week for 4 weeks significantly decreased the clinical severity scores of atopic dermatitis and serum total IgE levels, and significantly increased serum levels of IL-10 and interferon-gamma (IFN-γ) at weeks 4, 8, and 12 compared to baseline (week 0) without serious adverse events.,23–25 In a subsequent randomized, double-blind, placebo-controlled study, we demonstrated that 8 weekly intramuscular administrations of 50 mg autologous total IgG over a 7-week period could induce significant clinical improvements and increases of serum levels of IL-10 and IFN-γ at weeks 4, 8, 12, and 16 compared to baseline in 51 adolescent and adult patients with moderate-to-severe atopic dermatitis without serious adverse events. These results showed that intramuscular administration of autologous total IgG could provide a systemic immunomodulatory effect in patients with atopic dermatitis.To prove the concept, we evaluated the clinical efficacy, safety, and immunomodulatory effect of intramuscular administration of autologous total IgG in 20 adult patients with severe atopic dermatitis as an open-labeled prospective single-arm pilot clinical trial.To further evaluate the mechanism of immunomodulation induced by intramuscular administration of autologous total IgG, we evaluated changes in T cell before and after intramuscular administration of autologous total IgG in 13 healthy human subjects.22.1This study was conducted at a single academic center  as an open-labeled prospective single-arm pilot clinical trial. Study subjects were recruited from November 2018 to May 2019. This study was conducted in compliance with the guidelines for Good Clinical Practice and the Declaration of Helsinki with the approval from the institutional review board of Ajou University Hospital. All study participants provided written informed consent. This trial is registered with ClinicalTrials.gov (NCT 03695757).2.2 participated in this study  without underlying disease, and compatible with the criteria for autologous blood donation (hemoglobin level ≥11.0 g/dL and body weight ≥40 kg)9 years w2.3,28 The solution containing the purified autologous total IgG was tested for bacterial contamination using an endotoxin assay kit . The autologous total IgG solution was transferred into sterile glass vials in 50-mg aliquots and stored at −20°C.Autologous venous blood (400 mL) was collected from each participant using a double blood bag containing anticoagulant  at the initial visit (week −4)  and then were followed until week 12  supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 2 mM l-glutamine, and Cell Stimulation Cocktail (plus protein transport inhibitors) containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin  for 20 hours at 37°C with 5% CO2. Next, cells were incubated with PerCP-Cyanine5.5-labeled anti-CD3 , FITC-labeled anti-CD4 , PE-labeled anti-CD4 , PE-Cyanine7-labeled anti-CD8 , APC-labeled anti-CD25 , and PE-Cyanine7-labeled anti-CD19  antibodies for 30 minutes at 4 °C. For intracellular staining, cells were incubated with fixation/permeabilization buffer (eBioscience) for 30 minutes at 4 °C, followed by incubation with PE-labeled anti-IL-10  and FITC-labeled anti-IFN-γ  antibodies. Flow cytometric analysis was performed using a BD FACSAria III flow cytometer (BD Biosciences). Data were analyzed using the FACSDiva software version 7.0 (BD Biosciences).Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation using Cell Preparation Tubes  at baseline (week 0) and weeks 4, 8, and 12. Flow cytometric analysis was performed as previously described.2.6Serum levels of IL-10 and IFN-γ were measured using enzyme-linked immunosorbent assay (ELISA) monoclonal antibody kits and standards (BD Biosciences) according to the manufacturer's instructions.2.7Serum concentrations of IgG, IgA, and IgM were measured by a turbidimetric immunoassay using a COBAS INTEGRA analyzer . The serum total IgE level was measured using the ImmunoCAP assay .2.8Dermatophagoides farinae, HollisterStier, Spokane, WA), and bovine thymus extract , as extractable nuclear antigens, were measured using ELISA as previously described.–33 Serum levels of IgG antibodies to those antigens were expressed in arbitrary units using serially diluted high-titer serum samples as standards.Serum levels of IgG antibodies to pneumococcal capsular polysaccharide antigens , influenza antigens , house dust mite antigens  counts were assessed using an automated hematology analyzer . Routine chemistry tests, including liver and kidney function tests were performed using a Cobas c702 analyzer .2.10P value was obtained by Bonferroni correction. The Bonferroni corrected P value  <.05 was considered statistically significant.Data are presented as the mean ± standard error of the mean. Statistical significance of the differences in values before and after intramuscular administration of autologous total IgG was analyzed using the Wilcoxon signed-rank test. Bonferroni correction was applied to compensate Type I statistical error developed due to multiple comparisons. A corrected 33.1+ T cells was significantly increased at weeks 4 and 12 compared to baseline (P < .05)     3.3P > .05)  Table .3.4P > .05)  Fig. .3.5P > .05)  counts at weeks 4, 8, and 12 compared to those at baseline  during the study period . The difference in the baseline functional states of regulatory T cells might result in discrepancy of changes in serum IL-10 and IFN-γ levels between patients with atopic dermatitis and healthy human subjects. However, we cannot exclude a possibility that the dose and duration of intramuscular administration of autologous total IgG required to increase serum IL-10 and IFN-γ levels in healthy human subjects are higher and longer compared to patients with atopic dermatitis. Further studies are needed to explain the reason for the discrepancy of immunomodulatory effects induced by 8 intramuscular administrations of 50 mg autologous total IgG for 4 weeks between patients with atopic dermatitis and healthy human subjects.In this study, intramuscular administration of autologous total IgG significantly increased the percentage of IL-10-producing CD4+ T cells increased significantly at week 8 and reached a peak at week 12 after intramuscular administrations of autologous total IgG in healthy human subjects. These results are consistent with the pattern of changes in serum levels of IL-10, which are also significantly increased at week 4, with the peak level reached at week 12 after the injections of the same amount of autologous total IgG for the same time period in adult patients with severe atopic dermatitis in our previous study. These results collectively suggest that T cell activation induced by intramuscular administration of autologous total IgG has characteristics of delayed onset and prolonged effect in human subjects. These immunomodulatory characteristics could be further supported by long-term clinical improvements and decrease in serum total IgE levels lasting for more than 9 months observed in 2 out of 3 adult patients with severe atopic dermatitis who were followed up for more than 2 years after 8 intramuscular administrations of 50 mg autologous total IgG for 4 weeks.In this study, the percentage of IL-10-producing CD4+ T cells from healthy human subjects increased intracellular expression of IL-10 in CD4+ CD25high T cells, suggesting a direct activation of regulatory T cells by polyvalent IgG. Interestingly, the expansion of IL-10-producing CD4+ T cells induced by intramuscular administration of autologous total IgG in healthy human subjects observed in this study was similar to the expansion of IL-10-producing CD4+ T cells induced by intravenous administration of heterologous polyvalent human IgG in patients with autoimmune diseases, including immune thrombocytopenia and eosinophilic granulomatosis with polyangiitis.,37 The doses of intravenously administered heterologous polyvalent human IgG that ameliorated autoimmune or inflammatory diseases and expanded regulatory T cells were high (1–2 g/kg body weight every 3 or 4 weeks).–38 However, the dose of intramuscularly administered autologous total IgG required for clinical improvements in adult patients with severe atopic dermatitis,23–25 and for the expansion of IL-10-producing CD4+ T cells in healthy adult human subjects observed in this study, was relatively low . The reason for requirement of high dose of polyvalent human IgG to treat autoimmune or allergic diseases was attributed to low concentration and low affinity of natural anti-idiotype antibodies to pathogenic autoantibodies contained in polyvalent human IgG Clinical trials investigating high-dose intravenous polyvalent IgG therapy in adult patients with severe atopic dermatitis showed little clinical benefit. The nature of immune response provoked by a specific antigen was determined by the dose and route of administration of the antigen.,40 These results collectively suggest that the differences in dose (high or low), route of administration (intravenous or intramuscular), and origin of IgG (heterologous or autologous) could result in different kinds of immunomodulatory effects (passive anti-idiotypic immune modulation or active anti-idiotypic immune modulation). We speculate that intramuscularly administered high concentration of purified autologous total IgG can induce immunomodulatory effects in healthy human subject due to differences in antigen dosage, purity, and type of antigen-presenting cells involved, compared to the naturally present autologous total IgG in blood circulation. Further studies are necessary to evaluate the detailed mechanisms of systemic immunomodulation induced by intravenous or intramuscular administration of heterologous or autologous human IgG.It has been reported that the addition of polyvalent human IgG to purified CD4,41 A simple affinity purification is required for the preparation of autologous total IgG from autologous plasma, with a minimal risk of transmission of infectious agents due to this procedure. The cost for preparation of autologous total IgG from autologous plasma is apparently lower than the manufacturing cost of heterologous polyvalent human IgG. Therefore, our study suggests that the intramuscular administration of autologous total IgG could be a simple, safe, and economical immunomodulation method, with a potential to replace the intravenous polyvalent human IgG therapy, for patients with various immune diseases.The intravenous polyvalent human IgG therapy is expensive due to the safety issues and manufacturing process of immunoglobulin preparation from the plasma pool of multiple blood donors; furthermore, there is a serious concern about the shortage of supply of blood donation in the present and in the future. However, we could not detect any significant changes in serum levels of IgG antibodies to the F(ab’)2 fragments of autologous total IgG in those patients before and after intramuscular administration of autologous total IgG.The major limitation of our study is the absence of mechanistic insight into the T cell immunomodulation induced by intramuscular administration of autologous total IgG. We initially hypothesized that the intramuscular administration of autologous total IgG could induce a systemic immunomodulatory effect through the anti-idiotypic immunomodulation. Although we showed the induction of significant immunomodulatory effects on T cells, we could not provide any direct evidence of the development of anti-idiotype T cells or anti-idiotype antibody responses in this study. In the previous study, we attempted to measure the changes in serum levels of IgG anti-idiotype antibodies to autologous total IgG by ELISA in 3 adult patients with severe atopic dermatitis.This study has several limitations as a clinical trial. This was an uncontrolled clinical trial, without a placebo control group, and included relatively small number of participants due to small amount of research grant. The intervention duration of the study was also relatively short (4 weeks). Further clinical trials with a randomized placebo-controlled study design, a larger number of subjects, and longer intervention duration are needed to confirm the immunomodulatory effects of intramuscular administration of autologous total IgG in healthy human subjects.In conclusion, intramuscular administration of autologous total IgG induced immunomodulatory effects on T cells in healthy human subjects. This simple intervention could be a safe, effective, and economical T cell immunomodulation method for human subjects. Further studies are required to evaluate the detailed mechanism of this immunomodulatory effect.D-HN determined the study concept, participated in the study design, analysis and interpretation of the data, funding, and drafting of the manuscript, and has the final responsibility for the submission of the manuscript for publication. BK, S-JY, S-MC, and M-EK contributed to the acquiring of data. BK performed experiments and data extraction, and interpretation of flow cytometry assay. S-JY and S-MC contributed to the measurement and analysis of the serological changes. BK and M-EK contributed to the statistical analysis and interpretation of the data. S-JY and M-EK conducted the administrative, technical or material support, and management of this study. All authors contributed in the writing to the manuscript and approved the final version of the manuscript.Conceptualization: Dong-Ho Nahm.Data curation: Byul Kwon, Seung-Jung Yang, Su-Mi Cho, Myoung-Eun Kim, Dong-Ho Nahm.Formal analysis: Byul Kwon, Seung-Jung Yang, Su-Mi Cho, Myoung-Eun Kim, Dong-Ho Nahm.Investigation: Dong-Ho Nahm.Methodology: Byul Kwon, Dong-Ho Nahm.Project administration: Byul Kwon, Dong-Ho Nahm.Visualization: Byul Kwon, Myoung-Eun Kim, Dong-Ho Nahm.Writing – original draft: Byul Kwon, Seung-Jung Yang, Su-Mi Cho, Myoung-Eun Kim, Dong-Ho Nahm.Writing – review & editing: Byul Kwon, Seung-Jung Yang, Su-Mi Cho, Myoung-Eun Kim, Dong-Ho Nahm.