GA20oxs), GA3oxs, and GA2oxs, which correlate with bioactive GAs. The GA content and GA biosynthesis genes are affected by light, carbon availability, stresses, phytohormone crosstalk, and transcription factors (TFs) as well. However, GA is the main hormone associated with BR, ABA, SA, JA, cytokinin, and auxin, regulating a wide range of growth and developmental processes. DELLA proteins act as plant growth suppressors by inhibiting the elongation and proliferation of cells. GAs induce DELLA repressor protein degradation during the GA biosynthesis process to control several critical developmental processes by interacting with F-box, PIFS, ROS, SCLl3, and other proteins. Bioactive GA levels are inversely related to DELLA proteins, and a lack of DELLA function consequently activates GA responses. In this review, we summarized the diverse roles of GAs in plant development stages, with a focus on GA biosynthesis and signal transduction, to develop new insight and an understanding of the mechanisms underlying plant development.Plant growth and development are correlated with many aspects, including phytohormones, which have specific functions. However, the mechanism underlying the process has not been well elucidated. Gibberellins (GAs) play fundamental roles in almost every aspect of plant growth and development, including cell elongation, leaf expansion, leaf senescence, seed germination, and leafy head formation. The central genes involved in GA biosynthesis include GA20 oxidase genes ( Fusarium fujikuroi [In the early 20th century, active compounds named gibberellic acids (GAs) were found and isolated from ujikuroi . In thesujikuroi . GA is aujikuroi . Fundameujikuroi , floweriujikuroi , leaf exujikuroi , leaf foujikuroi , leaf anujikuroi , leaf siujikuroi , and leaujikuroi . Since t2+ metabolism [GA20oxs) and GA3 oxidases (GA3oxs), while GA2 oxidases (GA2oxs) act as GA-inactivating enzymes [GA20oxs (GA20ox1–GA20ox5) is crucial to determining the concentration of GA. An analysis of GA20ox transcripts revealed that their expression patterns differ, suggesting that they may play distinct roles in GA-regulated developmental processes [AtGA20ox1 and AtGA20ox2 are most strongly expressed during vegetative and early reproductive development in Arabidopsis. It was also found that GA20ox2 expression is tissue-specific or developmental stage-specific [GA20oxs, GA3oxs also have a direct role in determining GA biosynthesis and bioactive GAs. In the GA biosynthetic pathway, a GA3ox catalyzes the last step, which results in the synthesis of bioactive GAs [GA2ox catalyzes bioactive and/or immediate precursors of GAs to form inactive GAs. The gene encoding DkGA2ox1 is also well known as an important negative regulator of GA levels in plants or a dwarfism regulator gene, and it is a tissue-specific gene in Diospyros kaki [AaGA2ox1–AaGA2ox3 transcripts were detected in all organs of Artocarpus altilis, but were mostly displayed in stems and leaves [The GA biosynthesis pathway is significantly linked to GA biosynthetic enzymes , Ca2+ metabolism , and envtabolism . The mai enzymes ,16,17. Trocesses . Rieu etrocesses  revealedspecific . Similarros kaki . In contd leaves . Followid leaves ,23.3). Based on previous evidence, GA3 regulates organ formation and gravitropism by modulating the synthesis of the plant hormone auxin and transporters, and by increasing stress resistance [3 is correlated with the enhancement of indole acetic acid (IAA) and zeatin (ZR), and to reduced abscisic acid (ABA). At the same time, the expression levels of genes related to hormone metabolism and signaling were substantially changed through a phytohormone analysis [More than 135 GAs have been chronologically defined, but their names are not given based on their position in the biosynthetic pathway [sistance . In factsistance ,27,28,29analysis .53-aldehyde and GA12) were used as the substrates during GA biosynthesis [GA20ox catalyzes the conversion of GA53 to GA20 and GA12 to GA9 in three stages. Then, GA3ox catalyzes GA20 to GA1 and GA9 to GA4. In the last synthesis, GA2ox catalyzes GA5 to GA3 and GA20 to GA1 [Zea mays, it was found that the endogenous GA1 and GA8 levels in mutant plants were lower compared to the WT, while the concentration of endogenous GA44, GA19, GA20, GA1, and GA8 were higher compared to the WT in GA20ox1-overexpressing (OE) plants. Surprisingly, the transcript levels of ZmGA20ox1, ZmGA3ox2, and ZmGA2ox also increased in the GA20ox1-OE line. Altering GA levels significantly influences proportional changes in organ growth [As shown in ynthesis . GA20ox 0 to GA1 . Briefly0 to GA1 . GA meta0 to GA1 ,34. In dn growth .OsGA3ox1 on the lower side was higher (eight-fold) compared to the upper side in Oryza sativa [Previous studies found a correlation between the GA level and leaf distance from the base. As the length of the stem to the leaves increases, the bioactive GA level reduces. Normal cell elongation and differentiation in the stem are linked to leaf development with a higher bioactive GA content. GAs move from the leaves to the stem to provide sufficient bioactive GAs in the stem of rice . In watea sativa  , that regulate numerous enzymatic steps of GA metabolic pathways . The invongation , and SGD2 mutant . TherefoO. sativa, GA-deficient plants generally exhibit small grains, shorter panicles, a reduced leaf number, and a reduced internodal length [Based on cytological observations, a study found that a defective phenotype in a leaf was mainly caused by a decreased cell length caused by GA deficiency, which was controlled by the repression of GA biosynthesis, resulting in dwarfism . Howeverl length . Meanwhil length .3 application. A GA3 application results in an etiolated phenotype, including thinner leaves, a greater leaf area, and a reduced chlorophyll content [Lactuca sativa L. and Eruca sativa L., a GA3 application caused narrow and elongated leaves as well as lengthened internodes after 10–14 days of treatment [Brassica oleracea var. capitata, the head diameter and size of outer leaves increased in response to a GA3 application [Camellia sinensis, a GA3 application accelerated bud germination, but caused chlorophyll degradation resulting in photosynthesis reduction [3 and GA4+7 applied to the shoot apical meristems of cauliflower (B. oleracea var. botrytis) and broccoli  caused earlier curd formation and triggered a vegetative-to-reproductive transition [3 applications are more significant in GA-susceptible plants. For instance, vine elongation in dwarf pumpkins (GA-sensitive pumpkins) was more responsive to varying GA3 concentrations compared to the WT [3 applications have a different role in each species. In watermelons, a GA3 application only increased shoot biomass [3 application [3 application might cause an endogenous hormone imbalance via the regulation of hormone synthesis-related gene expression in Physalis heterophylla [GA deficiency can be solved by a GA content . In Lactreatment . In Braslication . In Cameeduction . Meanwhiansition . Based oo the WT . Apart flication . Howeverrophylla .Nicotiana tabacum [Stevia rebaudiana [GA biosynthesis inhibitors such as paclobutrazol (PAC), uniconazole, and daminozide are used to observe the opposite effect of a GA application. PAC induced the growth of smaller plants with a dark green phenotype and increased leaf thickness in  tabacum . Similarbaudiana . A densebaudiana . Moreovebaudiana .A drastic reduction in the GA level causes a huge disturbance at the metabolic level . MetabolLeaves are initiated at the flank of the shoot apical meristem (SAM). Cell division and expansion are critical processes in the formation of a complex, overlapping, and interconnected mature leaf . Cell diGA20ox and GA3ox, consequently inducing active GAs in Arabidopsis [GA20ox and GA3ox, resulting in reduced leaf expansion [GA20ox and GA3ox are dependent on the intensity and quality of light in Rosa sp. [GAs control leaf size by modulating cell division and increasing water absorption. Although cell size is not entirely related to leaf size or shape, the enhancement of protoplasmic content enlarges cell size, which is reflected in the surface area, diameter, and plant tissues . Light, bidopsis . In contxpansion ,64 and dRosa sp. .Solanum lycopersicum [OsGA2ox4-7. In contrast, phytochrome B (phyB) captures red light with the auxiliary action of phyA to mediate OsGA20ox2 and OsGA20ox4 repression, resulting in a lower GA1 level, which leads to the suppression of leaf sheath elongation in rice. The regulatory mechanism of phyB in O. sativa is different from dicots. In dicots, a reduction in active GAs is mediated by phyA by the suppression of PsGA3ox1 and the induction of PsGA2ox2. However, the phyA capacity could not decrease the active GA levels under B-light irradiation in peas [BIC1 and BIC2) is cryptochrome-interacting proteins. It functions in repressing cryptochrome 2 (CYR2) activity by participating in the GA regulation pathway by regulating the endogenous bioactive levels of GAs [OsBIC1 and OsCRY1s are involved in GA pathway regulation [Glycine max, the upregulation of GmGA2ox genes was mediated by GmCRY1, but repressed by low blue light (LBL), suggesting the involvement of GA homeostasis in LBL-induced cell elongation [Meanwhile, cryptochromes and phytochromes were important in reducing the active GA content in visible light in persicum . Cryptoc in peas . Moreoves of GAs . Then, Ggulation . In Glycongation .CRY1 is a light receptor that functions in repressing GA signaling by interacting with DELLA proteins  and GID1 to inhibit the GID1–GAI interaction competitively. TaCRY1a interacts with TaGID1 and Rht1 to minimize the interaction of TaGID1 and Rht1 in Triticum aestivum transgenic lines [https://version-11-5.string-db.org/, 10 February 2023) to identify the protein–protein interaction networks involved in GA biosynthesis, responses, perceptions, and signal transduction pathways in Arabidopsis. The k-means clustering was chosen as the clustering option for four cluster proteins: red, yellow, green, and blue. As shown in HY5 and ABA2, illustrating that each protein relates to and affects each other.The COP1/HY5 pathway mediates GA biosynthesis light regulation, while GAs modulate light signaling to prevent de-etiolation in the darkness. As mentioned previously, ic lines . DELLA pic lines . As showZmGA2ox3 and ZmGA2ox10 expression were increased by ethephon, which acts as a plant growth regulator, and at the same time, ZmPIN1a, ZmPIN4, and ZmGA3ox1 transcript levels were reduced, resulting in decreased auxin and GA accumulation in Z. mays [Pharbitis nil, GAs repressed endogenous JA and JA biosynthesis by collaborating with an inhibitor of JA biosynthesis, salicylhydroxamic acid [Most consistently, GA-regulated genes contain auxin and BR-related genes ,48. The  Z. mays . A previ Z. mays . However Z. mays . BR sign Z. mays , while J Z. mays . Accordi Z. mays . In Pharmic acid . In Arabmic acid .CsGA20ox1 was positively correlated with cytochrome P450 90A1 (CsCPD) and Dwarf4 (CsDWF4) in response to BR biosynthesis [HB40, induced by GA and, in turn, decreased the levels of endogenous bioactive GAs by inhibiting GA biosynthesis while increasing the GA deactivation gene. HB40 directly activates the transcription of JUNGBRUNNEN1 (JUB1), a key TF that restricts growth by impairing GA biosynthesis and signaling. HB40 also induces genes that encode GA2oxs. Nuclear growth-repressing DELLA proteins induced HB40 in plant growth [Nelumbo nucifera in the promotion of leaf sheaths [ynthesis . GA and t growth . The act sheaths .SlDREB) from Solanum lycopersicum was mainly expressed in the leaf suppressed by GAs, but induced by ABA. In a SlDREB-OE plant, the expression of the key genes ent-copalyl diphosphate synthase (SlCPS), SlGA20ox1, SlGA20ox2, and SlGA20ox4, which are involved in GA biosynthesis, were downregulated, causing lower endogenous bioactive GA and resulting in a decline in leaf expansion and internode elongation. In GA20ox1-OE lines, the bioactive GA level increased, leading to the production of more and larger cells and resulting in larger leaves [GA20ox4 [SlGA20ox1 and SlGA2ox4 [As previously mentioned, TFs regulate leaf size and might positively and negatively correlate with GAs . A dehydr leaves . Meanwhir leaves . Contras[GA20ox4 . In addiSlGA2ox4 . GA alsoSlGA2ox4  and HD-ZSlGA2ox4 , resultiSlGA2ox4 . In concThe leaf angle refers to the angle formed by the stem and leaf blade’s adaxial side, which forms the plant architecture into a compact or broad structure. It affects the competition and interception of light among plants, resulting in increases or decreases in plant yield and productivity. The leaf angle is an important agronomic trait due to its effect on sunlight capture efficiency and nitrogen reservoirs . SeveralRumex palustris by an increase in ABA catabolism, but inhibition of ABA biosynthesis. The correlation of ABA and GA occurs during submergence conditions where ABA mediates GA production for elongation [ABA and GA have different roles in regulating leaf angles. ABA acts as a negative regulator of petiole elongation . As prevongation . In a prongation .OsOFP22 interacts with 3-aa loop extension (TALE) homeodomain proteins and BR signaling components to regulate BR/GA responses and biosynthesis [SLR1, and DELLA proteins [GID1 and DELLA, DELLA is rapidly degraded through the ubiquitin–proteasome pathway, causing GA signaling repression. On the other hand, in the G-protein-dependent pathway, D1/RGA1, a subunit of the G-protein (Ga), plays a crucial role in the leaf angle. GA binds to an extracellular receptor, signals are transduced to effector proteins by Ga proteins, and finally, the suppressive activity of SLR1 was eliminated [OsMIR396d was also found to be involved in GA and BR signaling and the regulation of internal GA biosynthesis; consequently, GA signaling pathway gene (OsGID2) and GA biosynthesis gene  transcript levels decreased. In contrast, BR signaling was enhanced in OsMIR396d-OE plants, implying that OsmiR396d could control the leaf angle through GA and BR signaling [Furthermore, it has also been revealed that specific GA-related genes interact with BR genes to regulate the leaf angle ,83. OsOFynthesis . Two GA proteins . When GAiminated . OsMIR39ignaling .Brassica rapa L. ssp. Pekinensis). Chinese cabbage has four growth stages: seedling, rosette, folding, and heading. The leaves grow upward and inward after the rosette stage, and the leafy head forms when the leaf whorls intertwine [The leafy head is a unique organ in vegetables and is used as an identifying trait in the Brassicaceae family, which includes plants such as Chinese cabbage  in B. rapa. BrKS1 encodes an ent-kaurene synthase, the key enzyme involved in GA biosynthesis. Ent-kaurene synthase functions as a catalyst in the cyclization or rearrangement of the substrate ent-copalyl diphosphate (ent-CPP) to produce ent-kaurene, a precursor of GAs [BrCPS1, which encodes ent-CPP synthase 1, was also discovered to be involved in GA biosynthesis. A mutation in the fourth and seventh exons of BrCPS1 was responsible for nhm phenotypes. The GA content of mutant plants was lower than for the WT, and the tendency to form leafy heads was found after a GA3 application in nhm lines [BrKAO2 as the candidate gene, which encodes ent-kaurenoic acid oxidase 2 in the GA biosynthetic pathway. Two non-synonymous mutations in the second BrKAO2 exon caused mutations named nhm3-1 and nhm3-2. BrKAO2 was expressed at all stages of leaf development [Previous studies have revealed that GA affects leafy head formation in Chinese cabbage ,93. Hower of GAs . Similarelopment . A list B. rapa for leafy head formation, a total of ten GA-related genes  were increased [B. rapa [LsKN1 played an important role in leafy head development by binding to the promoter of LsAS1 to alter the dorsoventral pattern. However, the increased LsKN1 expression was prominent, but deficient for heading compared to GA biosynthesis genes [LsKN1 promoted auxin biosynthesis and repressed the biosynthesis of GA in lettuce (L. serriola). LsKN1 triggered the expression of two critical genes in the GA biosynthesis pathway (LsGA3ox1 and LsGA20ox1) by binding to their promoter regions [LsKN1, the transcription coactivator ANGUSTIFOLIA (BrAN) from B. rapa also regulated leafy head formation by mediating the leaf width and regulating the arrangement of cortical microtubules and the shape of pavement cells [Although the expression of auxin-regulated genes was dominant in ncreased . Moreove[B. rapa . In anotis genes . LsKN1 p regions . Similarnt cells .BrAN3 level was different in the rosette and heading stage. BrAN3 RNAi lines showed changes in phytohormone signaling pathways, including auxin, ethylene, GA, JA, ABA, BR, CK, and SA. Two DEGs (Bra039460 and Bra003520) related to GA signaling pathways were downregulated in BrAN3 silencing plants, contributing to leafy head formation and proving to be positively correlated with BrAN3 [BrAN3 in rosette leaves affects leafy head formation. This assumption was reinforced by Alemán-Báez et al. [B. oleracea and provide the majority of the energy required to produce the leafy head. BolC02g009950.2J and Bol035882, similar to OFP8, influenced the leaf ratio by regulating cell elongation and by repressing GA20ox1 at the rosette and heading stages. Other candidate genes that may control leafy head formation are BrpSAW1, BrpESR1, and BrpGL1, which are also related to trichomes, petioles, serration, and cell division in B. rapa [Interestingly, the th BrAN3 . Howeverz et al. , who fou B. rapa .B. rapa and Chrysanthemum morifolium by reducing or increasing endogenous bioactive GAs, indicating that GA biosynthesis is inactivated/activated by Pro-Ca [A. graveolens, 12 lignin synthesis gene expression profiles changed with an increase in the GA concentration and GA biosynthesis genes, including AgKO, AgKAO, AgGA20ox1, AgGA20ox2, AgGA3ox1, AgGA2ox1, and AgGA2ox3. Contrastingly, the AgKS and AgGA2ox2 expression was reduced with an increase and change in the GA biosynthesis genes and bioactive GAs as well as the lignin synthesis gene, demonstrating that lignin might be correlated with leafy head formation [GA20ox1 with the reduced inactivation activity of GA2ox1 to encode enzymes resulted in the final level of bioactive GAs, including GA1 and GA4. GA1 and GA4 levels are prominent factors leading to cellulose biosynthesis, resulting in reduced lignin content in Sorghum bicolor [GA20ox1 were not correlated with the levels of bioactive GA in Arabidopsis [Moreover, prohexadione-calcium (Pro-Ca) contributes to leafy head development in y Pro-Ca ,111. In ormation ,112. The bicolor . In factbidopsis . In summLeaf senescence is the final stage of leaf development, significantly influencing plant survival and productivity . SenescePlant hormones are a major player affecting leaf senescence during the initial development, progression, and terminal phase of senescence. GA, cytokinin, and auxin delay leaf senescence ,117, whiRBOHs), senescence-associated genes (SAGs), and chlorophyll catabolic genes (CCGs) increased due to the application of exogenous ABA. The increased level of endogenous ABA caused a reduction in the GA3 level, resulting in accelerated leaf senescence, possibly by the simultaneous suppression of gene expression associated with ABA catabolism and GA biosynthesis [3-inhibited senescence, a GA3 treatment inhibited RBOHs, SAGs, CCGs, and GA deactivation genes, whereas the GA biosynthesis genes were induced in GA3-treated cabbages [Based on a previous study, GA biosynthesis genes, GA deactivation genes, respiratory burst oxidase homolog genes (ynthesis . Contrascabbages .4+7) and light supplementation could prevent rapid leaf senescence caused by dark and cold conditions in Lilium sp. A drastic reduction in photosynthetic pigments and antioxidant activity, proteolysis, and lipid peroxidation enhancement can be prevented by both light and additional GA4+7 [In contrast to GA, dark and cold conditions induced rapid leaf senescence ,123,124.al GA4+7 . Furtherga1-3) and a Q-DELLA/ga1-3 mutant whose DELLA repressor was removed to evaluate the relationship between DELLA and GA in Arabidopsis leaf senescence. The transcript levels of senescence-specific genes (SAG12 and SAG29) in the leaves of the WT were much lower compared to those of the Q-DELLA/ga1-3 mutant during senescence, and vice versa with the ga1-3 mutant, illustrating that leaf senescence is influenced by the removal of the DELLA repressor [The relationship between DELLA and GA in leaf senescence has been identified. Although DELLA is a GA repressor, DELLA contributes to reducing leaf senescence in plants. A study investigated a GA-deficient mutant (epressor .SAG113 and AAO3) expression related to chlorophyll degradation. The RGA and GAI interaction with NAP impaired the transcriptional activities of NAP on SAG113 and AAO3 expression, which was related to chlorophyll degradation, illustrating that NAP plays a considerable role in GA signaling [BrNAC08 regulates leaf senescence by modulating structural genes  in B. rapa. NAC binding sites (NACBS) were found in the promoter regions of those structural genes and were specific [BrNAC041 at the transcriptional and translational levels, but contributes to GA biosynthesis to regulate leaf senescence. The transcription patterns of BrNAC041 were opposite from those of BrCYP707A3, BrKAO2, and BrGA20ox2 and acted as transcriptional repressors to regulate the gene functions of BrCYP707A3, BrKAO2, and BrGA20ox2. BrNAC41 binds to the NACBS of BrCYP707A3, BrKAO2, and BrGA20ox2 promoters and was validated using an electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP)-qPCR assay [A close relationship exists between GA signaling and TFs. However, upstream TFs that control GA biosynthesis in association with GA-mediated leaf senescence remain unclear. A member of NAC TF, NAP/ANAC029 , was found to be involved in leaf senescence in Arabidopsis. NAP positively regulated age-dependent and dark-induced leaf senescence by inducing senescence-related gene  and chlorophyll content, reduced electrolyte leakage and the expression level of a series of SAGs and CCGs, and increased GA biosynthetic gene transcript levels [WRKY75 also contributes to leaf senescence. An involvement of AtWRKY75 was found in regulating age-triggered leaf senescence through a GA pathway in Arabidopsis. WRKY75 regulates leaf senescence through the GA-mediated signaling pathway, illustrating that WRKY75 is a part of the GA-mediated senescence regulatory network. GA-treated plants showed an enhancement of photosynthetic pigments and SAG12, demonstrating that the leaf senescence caused by GA was delayed in wrky75 mutants, but was accelerated in WRKY75-OE plants [WRKY45 was a positive regulator of age-dependent leaf senescence. WRKY45 mutations led to increased leaf longevity, whereas WRKY45-OE plants remarkably increased age-triggered leaf senescence. Senescence-associated gene  transcript levels were significantly increased in WRKY45-OE plants. WRKY45 binds to the promoter of senescence-associated genes and interacts with RGA-LIKE1 (RGL1), a GA signaling pathway repressor [Although recently discovered, WRKY TF is becoming an important TF in leaf senescence. t levels . PreviouE plants . In the epressor .BrTCP21, was involved in GA-delayed leaf senescence. A GA3 application maintained a higher expression of BrTCP21 and suppressed the expression of senescence-associated genes by binding to the promoter region of BrGA20ox3 to activate its transcription, thus delaying leaf senescence. In senescing leaves, the BrTCP21 transcript level was reduced and gradually decreased following leaf senescence [A TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF, nescence . HoweverGA20oxs, GA3oxs, and GA2oxs, and these genes are correlated with bioactive GAs. The roles of GAs in regulating leaf size, leaf angle, leafy head formation, and leaf senescence have been revealed to correlate with other phytohormones, such as auxin, BR, SA, ABA, and JA. However, the mechanism of GAs in regulating leaf development is a complicated process that is still not fully understood, especially for leafy head formation. As mentioned previously, numerous factors in GA biosynthesis, namely light, Ca2+ metabolism, water availability, environmental stresses, various phytohormone interactions, varied species, and growth stages, remain to be determined [Studies about phytohormones have become increasingly important and helpful in finding a way to increase the yield and productivity of crops. In this review, we summarized the roles of GAs in plant organs and developmental stages. Bioactive GAs and GA biosynthesis gene expression varied among plant development stages and species. The main genes involved in GA biosynthesis are termined ,84,118.


Most solid tumors contain hypoxic and nutrient-deprived microenvironments. The cancer cells in these microenvironments have been reported to exhibit radioresistance. We have previously reported that nutrient starvation increases the expression and/or activity of ATM and DNA-PKcs, which are involved in the repair of DNA double-strand breaks induced by ionizing radiation. In the present study, to elucidate the molecular mechanisms underlying these phenomena, we investigated the roles of AMPK and FOXO3a, which play key roles in the cellular response to nutrient starvation. Nutrient starvation increased clonogenic cell survival after irradiation and increased the activity and/or expression of AMPKα, FOXO3a, ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 in MDA-MB-231 cells. Knockdown of AMPKα using siRNA suppressed the activity and/or expression of FOXO3a, ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 under nutrient starvation. Knockdown of FOXO3a using siRNA suppressed the activity and/or expression of AMPKα, ATM, DNA-PKcs, FOXO3a, Src, EGFR, PDK1, and SOD2 under nutrient starvation. Nutrient starvation decreased the incidence of apoptosis after 8 Gy irradiation. Knockdown of FOXO3a increased the incidence of apoptosis after irradiation under nutrient starvation. AMPK and FOXO3a appear to be key molecules that induce radioresistance under nutrient starvation and may serve as targets for radiosensitization. Most solid tumors contain cells in hypoxia, and these hypoxic cells show resistance to ionizing radiation . HypoxicAMPK is a cellular energy sensor capable of detecting shifts in the AMP/ATP ratio, activated when cellular energy is low. Cellular energy is low under hypoxia and under nutrient starvation, and AMPK is activated in these conditions. AMPK is a heterotrimeric protein complex composed of α, β, and γ subunits, and the phosphorylation at Thr 172 of AMPKα induces AMPK activation . RecentlC. elegans) DAF-16, which activates genes involved in longevity, lipogenesis, heat shock survival, and oxidative stress responses ,53,5]. HSeveral studies have reported that FOXO3a is involved in the response to DNA damage in a non-transcriptional manner ,32,33. TIn response to DNA double-strand breaks, ATM and DNA-PKcs are rapidly activated through phosphorylation at serine 1981 and serine 2056, respectively. Then, ATM and DNA-PKcs activate many kinases involved in DNA damage responses ,37. ATM While the AMPK/FOXO3a pathway is involved in DNA double-strand break repair and radiosensitivity through the increased activity and expression of ATM and DNA-PKcs under nutrient starvation, AMPK has other functions related to DNA double-strand break repair. AMPK is reported to directly phosphorylate 53BP1 at Ser1317 and promote 53BP1 recruitment in the process of NHEJ, which facilitates the end joining of distal DNA ends .Ionizing radiation generates ROS, which show high reactivity with various cellular molecules, including DNA, and cause cytotoxicity, mutagenicity, and apoptosis ,43. SOD2In summary, our findings suggest that the AMPK/FOXO3a pathway increases the activity and/or expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 and induces radioresistance under nutrient starvation . AMPK anHuman MDA-MB-231 breast cancer cells  were cultured in DMEM  containing 10% fetal bovine serum (FBS)  and penicillin–streptomycin . For nutrient starvation, the cells were cultured in glucose-free DMEM  without FBS. For the control condition, the cells were cultured in DMEM with 1.0 g/L glucose and 10% FBS. In this manuscript, “nutrient starvation” refers to culturing in a glucose-free medium without FBS.X-ray irradiation was performed using an X-ray generator, Model M-150WE , at 130 kV-8 mA with a 0.5 mm aluminum filter at a dose rate of 0.56 Gy/min.Clonogenic cell survival was assessed using a colony formation assay. Cells were cultured under nutrient starvation or the control condition for 12 h before irradiation. Four hours after irradiation, cells were trypsinized to produce a single-cell suspension, seeded into 60 mm dishes at various cell densities, and cultured with the control medium. After 12 days of culture, the colonies were stained with crystal violet, and the number of colonies containing more than 50 cells was counted.Cells were cultured under nutrient starvation or the control condition for 12 h before 8 Gy irradiation. Four hours after irradiation, the medium was replaced with the control medium. Twenty hours later, cells were collected with trypsin and washed twice with phosphate-buffered saline. Apoptotic cells were stained using the annexin V-FITC Apoptosis Detection Kit  following the manufacturer’s instructions. The cells were analyzed using the BD FACS Canto II system . Over 10,000 cells were analyzed for each sample.Western blot analysis was carried out as previously reported . The folRNA silencing was achieved with a siRNA specific for AMPKα1/2  or FOXO3a  at a final concentration of 10 nM. The siRNAs were delivered to the cells using RNAiMAX reagent  following the manufacturer’s instructions. A non-targeting siRNA  was used as a control for non-sequence-specific effects in each transfection. Forty-eight hours after adding siRNA, the medium was replaced with the nutrient-starved or control medium.t-test. Any p-values less than 0.05 were considered statistically significant.All data are expressed as means and standard deviations from at least three independent experiments. The statistical significance of the difference in survival fraction and the incidence of apoptotic cells between the two groups was analyzed with Welch’s


However, their aberrant concentration can result in various serious diseases. Therefore, it is essential to monitor biological species in cellular organelles such as the cell membrane, mitochondria, lysosome, endoplasmic reticulum, Golgi apparatus, and nucleus. Among various fluorescent probes for species detection within the organelles, ratiometric fluorescent probes have drawn special attention as a potential way to get beyond the drawbacks of intensity-based probes. This method depends on measuring the intensity change of two emission bands , which produces an efficient internal referencing that increases the detection's sensitivity. This review article discusses the literature publications (from 2015 to 2022) on organelle-targeting ratiometric fluorescent probes, the general strategies, the detecting mechanisms, the broad scope, and the challenges currently faced by fluorescent probes.Biological species, including reactive oxygen species (ROS), reactive sulfur species (RSS), reactive nitrogen species (RNS), F −, Pd2+, Cu2+, Hg2+, and others, are crucial for the healthy functioning of cells in living organisms.Biological species, including reactive oxygen species (ROS), reactive sulfur species (RSS), reactive nitrogen species (RNS), F However, their chemical imbalance can cause cellular malfunction.3 Therefore, smart research work on organelles and biological species is essential, which is challenging.In this modern era, everyone is interconnected. Similarly, in living beings, cells, organelles and biological species are interconnected and act cooperatively for the normal functioning of living systems.4,5 Bioimaging technique has been developed for examining biomolecules in living cells and tissues.6 Choi et al. studied fluorescent probes for organelles with respect to recent advances and bio-applications.7Over the years, significant studies and research work have been done on organelles and biological species present within the organelles. For example, organelle-targeting medicine has been developed for curing various diaeases.8 Notably, many single emission probes are unable to withstand environmental interference. Through a built-in correction of two emissions at different wavelengths, ratiometric fluorescence probes could eliminate background interference.9 Additionally, ratiometric fluorescence imaging has an even better resolution due to the two clearly defined emission peaks.10 Therefore, the development of ratiometric fluorescent probes is a winning strategy for the sensitive detection of small molecules, thanks to its reduced environmental effect.Nowadays, fluorescent probes for targeting organelles have gained more attention due to their excellent photo-physical characteristics, high sensitivity, rapid response, low cost, non-invasiveness, and real-time imaging.et al. reported ratiometric optical nanoprobes for molecular detection and imaging.10 In an excellent review, the design principles and applications of fluorescent probes in the ratiometric detection of anions, cations, and biological molecules has been beautifully demonstrated.11 Another review article highlighted various chemo/probe-based semiconductor quantum dots (QDs).12Although there are several outstanding reviews in the literature based on ratiometric probes with sensing applications, each one has concentrated on a single aspect, such as the optical processes, a particular class of fluorophores, or a specific subset of target analytes. In 2018, Huang et al. summarized recent advancements in the metal–organic framework (MOF)-based ratiometric fluorescent probes.13 Recently, Bigdeli et al. published a review article on fluorescent nanoprobes for visual detection.8In 2020, Wu We illustrate the design principles, fundamental detection mechanisms, and applications of organelle-targeting ratiometric fluorescent probes. The current limitations and prospective future directions, which would spur additional research interest and bring up fresh opportunities for biological analysis, are also covered.214In designing fluorescent probes, photo-physical parameters such as photo-induced electron transfer (PET), internal charge transfer (ICT), monomer–excimer formation, Förster resonance energy transfer (FRET), and excited state intramolecular proton transfer (ESIPT) are frequently used. FRET is a relatively excellent strategy for designing ratiometric fluorescent probes and increasing the Stokes shift.8 Generally, ratiometric changes in the emission spectrum are of the following type:Generally, the design of ratiometric probes involves the combination of two fluorophores, one reference fluorophore  and another dynamic fluorophore .15,16(I) Static + dynamic change: herein, upon the addition of an analyte, the emission intensity of one fluorophore is almost kept unchanged, whereas other fluorophores may undergo an increase/decrease/shift in emission intensity .15,1617(II) Dynamic + dynamic change: herein, both the fluorophores change (increase/decrease/or shift in emission intensity), but in opposite directions .1718 In addition, the polarity-dependent approach helps create various biological probes, which are helpful for imaging multiple organelles.19Generally, organelle-targeting ratiometric fluorescent probes contain a fluorophore, recognition units, and targeting moieties . The org2.120 The common mitochondrial-targeting units include the quaternized pyridine moiety,21 triphenylphosphonium (TPP),22,23 indole,24 cyanine,25,26 pyridinium,27,28 and rhodamine29  a moderate size (conjugated band numbers (CBN < 40)), (2) a cationic character, and (3) an appropriate lipophilicity (+6 > log Poct > 0).42 The ER-targeting fluorescent probes mainly track cellular concentrations of stress-responsive substances like NO, H2S, H2O2, and HOCl.43,44The commonly used ER-targeting moieties include glibenclamide,de group .40,41 ER2.4l-cysteine as an effective Golgi apparatus targeting ligand. They created various probes using this technique . Some of them have been made available for purchase.50,51 From a molecular docking experiment, Ma et al. demonstrated electrostatic binding between a positively charged probe and negatively charged nucleus RNA major groove .52 Nucleus-targeting unit functionalization is an excellent strategy for delivering fluorescent functional probes into the nucleus of living cells  to target the nucleus.ng cells .53,542.67Currently, the available probes for the membrane share a common approach: the conjugation of an environment-sensitive fluorophore to generate membrane-specific signals and a membrane-anchoring moiety to minimize the diffusion of the probe .7355–57 and some excellent reviews.9,58 In this section, we explain the fluorescence-based sensing mechanisms briefly. The HOMO localizes on the donor moieties in the ICT-based fluorescent probe. The LUMO is centered on acceptor moieties, thus creating a solid dipole with a charge transfer phenomenon upon excitation. The preferential interaction of the analyte at either the donor or acceptor results in a change in dipole strengths and, consequently, spectral shifts  (The various fluorescence-based sensing mechanisms include photo-induced electron transfer (PET), internal charge transfer (ICT), monomer–excimer formation, Förster resonance energy transfer (FRET), and excited state intra-molecular proton transfer (ESIPT). These mechanisms are available in detail in our recent publicationsl shifts . To effel shifts , upon exl shifts . The propectrum) . FRET-bapectrum) .459–62Mitochondria are organelles that play a vital role in cell physiology, including oxidative respiration, ATP production, and signal transduction. Mitochondria are widely known as the main generators of various ROS, RSS and RNS. Thus, probes that can specifically target mitochondria play a key role in monitoring multiple functions of mitochondria and mitochondrion-related illnesses.4.12O2 and HOCl are potent oxidants with antibacterial capabilities.63,64 However, the aberrant synthesis of ROS species in vivo has been linked to several illnesses, including lung damage, atherosclerosis, osteoarthritis, and rheumatoid arthritis.27 Therefore, real-time and on-site detection of ROS is an exciting research topic.The reactive oxygen species Het al. constructed probe 1 with far-red emission by combining a pyrene unit  with a benzo[e]indolium unit  linked by an ethylene bridge . Upon the addition of ClO−, emission at 632 nm gradually fades away with a concomitant increase in fluorescence intensity at 455 nm (blue emission). Probe 1 with excellent mitochondrial targeting features, such as high selectivity (detection limit = 182 nM), fast response time, significant Stokes–Stokes shift (107 nm), photostability, and live cell membrane permeability, displayed potential for detecting ClO− in mitochondria.Hu e bridge , Table 12O2, functions as a signaling molecule to control various cellular processes, such as cell division, proliferation, and migration.66,67 However, high H2O2 concentrations can harm proteins and nucleic acids, which are strongly related to many disorders such as malignancies, diabetes, and Alzheimer's disease.68,69 Therefore, monitoring of H2O2 concentration is essential. He et al. designed benzothiazole dye  based probe 2 containing an aromatic boronic ester moiety (H2O2 recognition group) , photostability, and Pearson's colonization coefficient (0.94), displayed potential for detecting H2O2 in mitochondria.An important ROS, endogenous Hn group) , Table 1et al. designed probe 3 based on the FRET platform for detecting OCl−  with the concentration of OCl− was linear in the range of 0 to 5 μM. The limit of detection was 10.2 nM. From the fluorescence imaging experiment, the applied probe 3 successfully examined endogenous OCl− in Murine RAW 264.7 cells.Shen ing OCl− , Table 14.22S), and hydrogen polysulfides (H2Sn), are produced in large quantities in mitochondria and are associated with critical mitochondrial-related pathological and physiological processes.7 Cys can act as an antioxidant in mitochondria by removing various ROS from mitochondria to stop oxidative damage.72 Additionally, Cys is necessary for the mitochondrial process of protein turnover.73–75 Therefore, it is essential to identify and measure Cys in real-time within cells, particularly in the mitochondria, to understand the pathological and physiological processes properly.Reactive sulfur species (RSS), such as cysteine (Cys), hydrogen sulfide  and a benzyl group (for easy distribution in the mitochondria) showed strong red fluorescence at λem = 605 nm (λex = 490 nm). In the presence of cysteine, probe 4 underwent ratiometric fluorescence change with the formation of new emission maxima at 540 nm and the simultaneous decrease in emission intensity at 605 nm. The limit of detection was 33.7 nM for cysteine. Applied probe 4 examined endogenous cysteine levels in HeLa cells through bioimaging, demonstrating potential applications in natural areas.Yang chondria , Table 1et al. developed probe 5 for detecting cysteine over other biothiols , linear in the range between 0.5 and 40 μM. Interestingly, the merocyanine fluorophore and probe 5 exhibited a large two-photon cross-section (Φσmax) of 72.6 GM (λex = 760 nm) and 65.2 GM (λex = 740 nm), respectively, favorable in producing bright and high contrast images of living samples. In addition to detecting cysteine in live cells and mitochondria, probe 5 exhibited promising application for monitoring cysteine concentration in living tissues (down to 150 μm depth) using two-photon fluorescence microscopy.Considering the advantage of two-photon fluorescence microscopy and ratiometric detection, Niu iothiols , Table 12S plays essential roles in mitochondria, such as scavengers for reactive oxygen species,77 and is associated with various mitochondrial-related pathological and physiological processes.78,79 Therefore, monitoring of H2S level in mitochondria is crucial. Liu et al. proposed probe 6 integrated with cyanine (mitochondria-targeting group) and naphthalimide group (responsive to H2S) for ratiometric detection of H2S in mitochondria  showed good linearity in the range of 1–9 μM. The detection limit was 1.31 μM for Na2S. In the presence of Na2S, reduction of the azide group to the amino group (electron donating) occurs, activating the ICT process and turn-on fluorescence of the naphthalimide moiety. Probe 6 found applications for fluorescence imaging and ratiometric detection of H2S in live cells.Hchondria , Table 1et al. engineered probe 7 integrated with triphenylphosphonium (mitochondria-targeting group), 2-fluoro-5-nitrobenzoic unit, and 1,8-naphthalimide fluorophore (well-known ICT fluorophore)80 for the detection of H2Sn  solution, fluorescence titration of probe 7 with Na2S2 revealed a decrease in emission intensity at 485 nm. A new emission maximum at 550 nm emerged and increased (Stokes-shift = 109 nm). Probe 7 successfully demonstrated its application in imaging intracellular H2Sn with good selectivity and sensitivity.Han  of H2Sn , Table 12 is an essential endogenous signaling molecule that performs significant roles in many physiological processes. However, increased SO2 concentration is associated with severe lung cancer, nervous system diseases, and respiratory problems.82–85 Therefore, detecting SO2 and its derivatives in living systems becomes a high priority. Notably, a platform with fluorophores attached to aromatic heterocycles through C–C bonds was frequently used as a Michael addition receptor. The Michael-addition principle allows nucleophiles, such as bisulfite, to attack the C–C double bond.86,87 In this section, most probes for bisulfite detection follow the nucleophilic addition reaction mechanism.SOet al. developed near-infrared (NIR) fluorescent probe 8 by combining a coumarin carboxylic acid group with a piperazine substituted benzopyrylium salt (mitochondrion-targeting group) . The nucleophilic addition reaction of SO32− at the double bond of the benzopyrylium unit resulted in the interruption of the conjugated π-electron cloud, and the fluorescence at 635 nm decreased, accompanied by the simultaneous increase in the new emission peak at 455 nm. From the co-localization experiment, probe 8 efficiently targeted mitochondria , and further investigations in HeLa cells and the nude mice experiment showed the application of probe 8 in biological systems for detecting SO32−  , Table 1ng SO32− .et al. exploited probe 9, prepared by the condensation reaction between indolium or pyridinium and 3-formyl-9-methyl carbazole unit  varied from 0.0383 to 3.8769 (101-fold enhancement). The in vitro imaging experiment proved the application of probe 9 for the quantification of SO2 derivatives in the mitochondria.Liu ole unit .89 In th90 In the presence of HSO3−, the emission spectrum of probe 10 revealed a blue shift of 162 nm, and the emission intensity ratio (I463/I625) was 56  or by drugs.The same group presented another probe, probe 10, containing a carbazole and an alkyl sulfonated benzoindole  as the basic skeleton , Table 1) was 56 . The cha32− or HSO3−, Wang et al. developed probe 11 to detect SO2 derivatives in aqueous medium and the mitochondria of living cells  vs. the concentration of HSO3− was linear in the range of 1–9 μM. The sensing mechanism could be attributed to the interruption of the π-conjugation of probe 11 upon the addition of HSO3− at the responsive site of probe 11, leading to a ratiometric change in the absorption and fluorescence spectrum. The fluorescence co-localization experiment revealed the unique distribution of probe 11 within the mitochondria of live MCF-cells . Furthermore, the applied probe 11 examined mitochondrial SO2 derivatives quantitatively through a fluorescence imaging experiment.Based on the 1,4-addition reaction between the polymethine chain of hemicyanine and SOng cells , Table 1et al. applied the strategy of combining two classical dyes to construct long-wavelength probe 12, prepared from benzopyrylium and chromenoquinoline dyes  and an increase in blue fluorescence at 514 nm (λex = 405 nm) upon the addition of HSO3−. The detection limit was 103 nM and 17 nM for the red and green channels, respectively. The nucleophilic addition reaction of HSO3− at the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bond of probe 12 resulted in the interruption of π-conjugation/inhibition of the ICT process from the chromenoquinoline to the benzopyrylium group. Thus, a significant blue shift (99 nm) in the emission spectrum was observed. Furthermore, probe 12 displayed application for detecting SO2 derivatives in the solid state.Wang ine dyes , Table 1et al. developed probe 13 based on pyrazoline  and hemicyanine dyes   changed from 0.45 to 445 (989 times). Intriguingly, probe 13 demonstrated an application for the ratiometric imaging of mitochondrial SO2 derivatives in living cells.Wang g group) , Table 1et al. designed probe 14 based on the conjugated platform of dansyl, piperazine, and benzothiazolium, in which the benzothiazole moiety acts as a recognition unit and mitochondrion-targeting group  of probe 14 showed a change from 0.3 to 1.5 (5-fold), and the limit of detection was 69 nM. Furthermore, probe 14 demonstrated a successful application for detecting HSO3− in the mitochondria of living cells through fluorescence imaging , and mitochondrion-targeting group) with carbazole-3-aldehyde .α,β-Unsaturated compounds are prone to a nucleophilic addition reaction by HSOaldehyde , Table 1et al. constructed probe 16 composed of the coumarin-hemicyanine skeleton, in which the FRET process occurs from coumarin to hemicyanine and the ICT process occurs from the styryl to the indolium group (mitochondrion-targeting) . The staining experiment revealed probe 16 as a specific mitochondrion-targeting probe and can detect endogenous SO32− in living cells such as HepG2 and L02 cells.Zhang rgeting) , Table 1et al. developed probes 17 and 18 by using diethyl 2,2′-(phenylazanediyl)diacetate (electron-donor) and 1H-benzo[e]indolium  , and high selectivity and sensitivity . The sensing mechanism for both probes was a nucleophilic addition reaction. However, the path of the sensing mechanism was different. Interestingly, both the probes displayed the capability of quantifiable tracking and fluorescence imaging of HSO3−/SO32− in the mitochondria of living cells.Zhao g group) , Table 1et al. constructed probe 19 by the condensation reaction between trimethylbenzoindolium (mitochondrion-targeting moiety) and p-diphthalaldehyde  and then turn-on emission (at high concentrations). The sensing mechanism was a twice nucleophilic addition reaction. The cell imaging revealed that the probe 19 could specifically detect HSO3− in the mitochondria of living cells (HepG2 cells).Zheng aldehyde , Table 14.399 As a result, it is essential and beneficial to monitor fluoride ions in mitochondria.The fluoride ion is one of the most significant anions crucial to many biological and medicinal processes. According to literature reports, a high fluoride ion concentration can damage mitochondria through oxidative stress and reduce the mitochondrial respiratory chain's efficiency, resulting in mitochondrial malfunction and the development of neurodegenerative disorders.et al. applied ICT-modulated strategies for constructing probe 20 based on a diethylaminocoumarin derivative (a well-known ICT fluorophore) in which a pyridinium salt behaves as a mitochondrion-targeting group  attributed to the ICT process between the pyridinium cation and 7-diethylamino-coumarin unit. Upon the addition of F−, a new emission band at 539 nm emerged and increased at the expense of a decrease in emission at 639 nm. The sensing mechanism was F−-induced cleavage of the Si–O bond between the phenyl and triisopropylsilyl groups. Furthermore, the applied probe 20 successfully examined mitochondrial F− in living cells.Shen ng group , Table 14.4101,102 Therefore, developing an efficient tool for palladium ion detection in a biological system is necessary.Due to its high stability and reliability characteristics, palladium has been extensively employed in various applications, including catalysis, synthesis of organic compounds, medicine, dental crowns, fuel cells, and electronics production. However, palladium ion enrichment in living organisms can cause severe health-related diseases.et al. developed probe 21 to detect Pd2+ in living cells based on the FRET process and rhodamine ring-opening mechanisms . Upon adding Pb2+, a decrease in emission at 472 nm and an increase in the new emission band at 594 nm were observed. Moreover, the fluorescence imaging experiment revealed the application of probe 21 for the ratiometric visualization of Pd2+ in the mitochondria of living cells.Wang chanisms , Table 14.5Most organelle-targeting methods discussed above focus on lipophilic cationic fluorescent probes that selectively target mitochondria. However, problems with cationic probes, such as the effect on membrane potential and cellular toxicity, are yet to be overcome. Furthermore, to explain the probe's potential to target mitochondria, mostly co-localization experiments have been performed using commercially available mitochondria-specific dyes such as MitoTracker Green FM, MitoTracker Red, and MitoTracker Orange. However, there can be several other contributing factors and principles behind the probe's potential to target mitochondria, and these factors need a clear discussion.In addition, mitochondria contain hundreds of biomolecules, such as anions, cations, enzymes, mitochondrial DNA, RNA, lipids, and so forth. Using fluorescent probes, it is still challenging to selectively label bioactive compounds at low concentrations (often nanomolar levels). No doubt, ratiometric fluorescent probes hold promise for removing various background interferences. However, nonetheless, just a few mitochondrion-targeting ratiometric fluorescent probes have been developed to date.5103 As key indicators of lysosome function and oxidative stress, reactive oxygen species (ROS) such as HClO and H2O2 and reactive sulphur species (RSS) such as H2S have been the focus of numerous probe designs. Since the lysosome lumen is acidic, developing probes that can detect HOCl in an acidic medium is challenging.4,30Lysosomes, an essential subcellular organelle, which can function as a digestive compartment in eukaryotic cells and include a variety of enzymes and proteins, are critical regulators in metabolic processes under acidic pH circumstances.5.12O2 is a significant reactive oxygen species (ROS) with distinct destructive oxidation characteristics. Lysosomes can produce hydrogen peroxide (H2O2) to combat pathogens.104–106 Therefore, it's crucial to create a reliable method for measuring H2O2 in inflammatory tissues to assess the physiological and pathological link between lysosomal H2O2 and inflammation.Het al. constructed probe 22 based on naphthalimide, benzylboric acid (H2O2 responsive group), and pyridine group (lysosome-targeting group) for monitoring H2O2 in living tissue and in inflamed tissue , Zhou d tissue , Table 1107,108 With this in mind, Shen et al. synthesized probe 23 based on the imidazopyridine moiety (donor) and rhodamine moiety (acceptor)  , Table 1et al. provided the coumarin and rhodamine based FRET platform 24 for HOCl detection in living cells . However, in the presence of HOCl, the emission spectrum of probe 24 showed a decrease in intensity at 480 nm and an increase in new maxima at 580 nm (which belonged to rhodamine), assigned to a ring-opening and FRET (FRET efficiency = 93.75%) based detecting mechanism.Using a similar mechanism to the one described above, which converts the rhodamine spiro-form into the ring-open form when HOCl is present, Yuan ng cells , Table 1et al. constructed probe 25 from phenothiazine coumarin and a morpholine unit for hypochlorite detection  and zebrafish  to the benzopyronin dye  and from 704 nm to 512 nm , respectively. The sensing mechanism was the 1,6-conjugate addition reaction. Probe 26 specifically targeted lysosomes and displayed application for monitoring intracellular bisulfite levels.Tamima onin dye , Table 1115 These biothiols are significant indications of lysosomal function because they are produced by lysosomal proteolysis.116Cysteine, homocysteine, hydrogen sulfide, and glutathione are examples of cellular thiols frequently studied employing lysosome-targeting probes.et al. devoted benzo[b]xanthene-derived probe 27 for the two-photon ratiometric fluorescence-based detection of cysteine .Tamima cysteine , Table 1et al. applied a cleavable FRET-based strategy for designing a lysosome-targeting probe 28, based on a coumarin–NBD (nitrobenzofurazan) cassette . Furthermore, probe 28 preferentially targeted lysosomes and displayed potential application for detecting H2S in lysosome.Zhang cassette , Table 15.3119 At the organelle level, improper copper homeostasis can cause several severe illnesses.120–122 As a result, it is still essential to monitor the concentration of copper levels in cells, particularly in lysosomes.Many essential physiological processes involve the role of copper. Copper plays important roles in a variety of fundamental physiological processes.et al. applied FRET-based strategies for constructing ratiometric probe 29 , and ratiometric fluorescence response towards Cu2+. From fluorescence imaging experiments, live cells (L929 cells) incubated with probe 29 showed blue and weak green fluorescence. However, cells pre-treated with Cu2+ and then incubated with probe 29 showed solid green and diminished blue fluorescence. In addition, probe 29 was applied for fluorescence imaging of Cu2+ in the lysosome of living cells.Liu probe 29 , Table 12+-induced ring opening of rhodamine,124 Wu et al. synthesized probe 30 for Cu2+ detection (125 In CH3CN–H2O (8 : 2) solution, probe 30 behaved as a selective and sensitive probe for Cu2+ over other tested ions. The sensing mechanism was Cu2+-induced ring opening and the FRET process from pyrrole to rhodamine.Inspired by Czarnik's report on Cuetection , Table 15.4etc.126 Therefore, it is crucial to have an effective method for detecting mercury ions.Mercury is a hazardous and pervasive heavy metal that can result in numerous serious health issues, including renal failure, damage to the central nervous system, et al. developed an NBD-based probe 31 by introducing a morpholine moiety to the NBD fluorophore for Hg2+ detection , is crucial for protein synthesis, folding, distribution, and calcium ion storage. Literature reports revealed that ER stress, which is linked to significant diseases, can cause autophagy and even cell death. Therefore, studying ER is an exciting field of research.6.12O2, an important reactive oxygen species, plays a crucial role in various pathological and physiological processes. During ER stress, the increased concentration of H2O2 can damage cellular proteins and may result in cancer, metabolic diseases, cardiovascular diseases, neurodegenerative diseases, etc.130,131 Therefore, developing efficient analytical methods for H2O2 in ER is crucial.H2O2 in ER. However, quantitative detection of endogenous H2O2 remains challenging. To solve this issue, Gao et al. synthesized probe 32 based on α-ketoamide and naphthalimide groups , both under normal conditions (0.692 μM H2O2) and under ER stress (1.26 μM H2O2).In the past, researchers developed various probes for detecting exogenous He groups , Table 1− specific sensors.132–134 On the other hand, the sulfonamide group has been known to target ER.135 Taken together, Hou et al. constructed biosensor 33 for ClO− detection in ER  vs. ClO− concentration was linear between 0 and 120 μM concentrations. The detection limit was 0.59 μM for ClO−. The applied biosensor 33 examined exo/endogenous ClO− in the ER of living cells.In the past, the diaminomalenonitrile-based Schiff base has been used by several researchers for developing ClOon in ER , Table 1136 Ma et al. constructed biosensor 34 using a 4-aminonaphthalimide moiety (fluorescent group), (2-aminoethyl) thiourea unit (HClO recognition site), and p-toluenesulfonamide unit (ER-targeting group)  solution, free sensor 34 showed emission at 533 nm (green emission), attributed to the ICT process. After adding HOCl, biosensor 34 revealed ratiometric fluorescence change with blue-shift (49 nm) of the emission maxima from 533 nm to 484 nm, which was assigned to inhibition of the ICT process. Furthermore, biosensor 34 demonstrated an application for HOCl detection in the ER of PC-12 cells.Inspired by the excellent optical properties and ICT process of 4-aminonaphthalimide moiety,g group) , Table 1137 such as peroxynitrite (ONOO−),138 hypochlorite (OCl−)139 and hydrogen peroxide.140,141 Pak et al. designed NHC-borane-based fluorescent sensor 35, constructed from a naphthoimidazolium precursor (HOCl responsive unit) for specific detection of HOCl over other ROS (18 Sensor 35 followed an electrophilic oxidation mechanism that involved B–H bond cleavage. Sensor 35 successfully targeted ER and demonstrated an application for monitoring HOCl in living cells (Raw 264.7 cells) and rat hippocampal slices by a two-photon fluorescence microscopy experiment.In the past, aryl boronic acid has been widely utilized in designing fluorescent sensors for biological species,ther ROS , Table 16.22S during ER stress and ER functions,142,143 Shu et al. pioneered ratiometric NIR-fluorescent biosensor 36 based on dicyano-isophorone  and an O-carboxybenzaldehyde unit (H2S recognition site) for H2S detection in ER  and zebrafish. Interestingly, sensor 36 successfully detected endogenous H2S produced during ER stress incited by Tunicamycin.Taking into account the essential role of Hon in ER , Table 12 and its derivatives are gaining increasing attention. According to literature reports, SO2 is vital in cardiovascular processes145 and can regulate hippocampal neuron apoptosis.146 Thus, SO2 detection is essential. Li et al. developed a FRET-based platform 37, constructed from benzoindole-based hemicyanine (acceptor) and naphthalimide derivatives (donor), for the detection of SO2 derivatives in ER (147 Sensor 37 exhibited emission maxima in buffer solution at 610 nm (λex = 440 nm), attributed to the FRET process. The sensing mechanism was a nucleophilic addition reaction and inhibition of the FRET process. Furthermore, sensor 37 successfully targeted ER and demonstrated an application for imaging exogenous and endogenous SO2 derivatives in living cells.Nowadays, physiological functions of SOes in ER , Table 16.3148 Furthermore, lysosomes in damaged tissue contained high concentrations of copper ions.149 However, it is unclear whether or not the copper ions in these organelles are the cause of the ER's harmful actions. Thus, to understand copper-related diseases, a reliable technique for imaging copper at the level of organelles is crucial.Mitochondria and lysosomes implicate the cellular homeostasis of copper.et al. presented a naphthalimide and hydrazide based biosensor 38 for copper ion detection in living cells , and solution color changed from blue to yellowish-green. The detecting mechanism was assigned to the copper-mediated hydrolytic reaction of sensor 38, forming aminonaphthalimide. Additionally, the biocompatible sensor 38 specifically targeted the ER of living cells and displayed potential for qualitative and quantitative detection of Cu+/Cu2+ under physiological conditions.Park ng cells , Table 16.4151 The ER pH is the same as that of the cytoplasm under normal physiological conditions.152 Recent research has shown that ER stress potently stimulates autophagy, strongly linked to many diseases , causing the ER pH to drop.153,154 Therefore, it is crucial to quantitatively evaluate the pH change in ER to explain the biological functions of ER fully.The physiological functions of the ER, such as targeting during secretion, protein sorting, and retrieving resident chaperones, are regulated by the pH of the ER, which serves as a crucial parameter.155 With this in mind, Dong et al. pioneered probe 39 based on the naphthalimide–coumarin platform and employed hydroxyl and morpholine groups as the pH-responsive sites  and two emission maxima at 446 nm and 527 nm between pH 5.08 and 7.73, and further single emission maxima at 446 nm at pH 7.73 (basic pH). The sensing mechanism was assigned to the FRET–PET–ICT process (p-toluenesulfonamide group (ER-targeting group), probe 39 successfully targeted ER. It demonstrated an application for quantitatively detecting the pH changes in the dexamethanose-treated cell and during ER stress.Fluorescent probes with dual responsive sites have shown improved sensitivity to pH and enhanced emission wavelength.ve sites , Table 1 process . Further6.5156,157 Tian et al. developed ratiometric fluorescent probe 40 based on p-toluenesulfonamide (ER-targeting group) for CES2 detection  and tumor tissue (Carboxylesterase 2 (CES2) has critical roles in ER, such as metabolic, drug detoxification, molecular target for the prodrug design, and ER stress-associated diseases.etection , Table 1r tissue . Notably6.62S, H2O2, and HOCl. However, the mechanism behind ER selectivity still needs to be clarified. Many biological species present in ER still need to be detected due to a lack of efficient probes. In addition, most of the probes discussed above emit visible light. Thus, their in vivo applicability is restricted.Over the past few years, scientists engineered several ER-targeting fluorescent probes for the selective detection of various substances like NO, H7159–161 Thus, to study in depth various functions of the Golgi apparatus, it is essential to develop efficient Golgi apparatus-targeting probes.The Golgi apparatus is an organelle with a phospholipid membrane composed of cisterna. It transforms proteins from the rough ER, then separates them into vesicles for transport to other cell regions. According to recent findings, CO is crucial for the Golgi apparatus.7.12O2) plays a critical role in this process. To address this issue, Wang et al. developed a two-photon fluorescent probe 41, which targets the Golgi apparatus with the aid of a phenylsulfonamide group. The response mechanism was a reaction-based process  is essential for the study and treatment of drug-induced liver injury (DILI) associated with oxidative stress. Targeting the Golgi apparatus has emerged as a new approach for DILI research and treatment. Feng et al. developed a new probe, 42, by conjugating a sulfanilamide moiety to a coumarin–hemi-cyanine conjugated system , probe 43 showed a significant 95 nm red shift with a decrease and increased emission maxima at 425 nm and 560 nm, respectively. The detecting mechanism was attributed to the cleavage of the allycarbamate group of probe 43 to form 43a mediated by a Pd0 Tsuji–Trost reaction. Furthermore, probe 43 found application for CO imaging in cells and zebrafish and to visualize CO levels during cellular oxidative stress stimulated by lipopolysaccharide  is directly linked to several ailments, such as genotoxicity, respiratory disease, and neurological abnormalities. However, the protective barrier of the cell nucleus membrane makes it challenging for fluorescent probes to investigate the correlation of FA and SO2 in the nucleolus regions. Ma et al. took this challenge and constructed probe 45 based on the benzopyryllium-dansyl FRET platform (52 The probe 45 was used for SO2 reversible sensing and recovered by FA. The sensing mechanism of SO2 was the interruption of the FRET process between dansyl (donor) and benzopyryllium (acceptor). The FA addition restored the FRET process because of the reversible Michael addition reaction. Probe 45 found application for quantitatively monitoring endogenous SO2/FA in the nucleus region of live cells and living animals.Organisms' excessive generation of formaldehyde (FA) and sulfur dioxide . However, due to nucleus localization, 46p could be an excellent green-fluorescent probe for the nucleus.Nitric oxide (NO) is a significant signal molecule involved in a variety of physiological and pathological processes. Thus, to understand these processes, real-time detection of short-living NO in the biological medium is crucial. Based on their previous reports on NO detection,duct 46p , Table 18.3etc. Thus, to better understand their molecular process and function, an efficient tool that can target the nucleus is in demand. Sun et al. developed D–π–A type probe 47 based on triphenylamine (donor) and N-methyl benzothiazole (acceptor and nucleus-targeting group)  , Table 1et al. reported dual-organelle targeting (LD and nucleus) fluorescent probe 48 for monitoring cellular microenvironment polarity change . However, there needs to be concrete proof of this. Wang y change , Table 1et al. developed a ratiometric fluorescent probe 49 based on a naphthalimide dye and a Hoechst (nucleus-targeting unit)  , Table 18.4172 Nowadays, newly developed nucleus-targeting probes are more specific to a particular analyte but are still rare. Furthermore, each developed method needs a clear explanation of organelle-specific/targeting mechanism, cellular toxicity, targeting ability in the altered environment, probe stability, response time, etc.For chemical biology purposes, nucleus-targeting systems have been thoroughly investigated. However, commercially available nucleus-targeting fluorescent probes are limited to staining DNAs.9173 and viral infection.174,175 The cytomembrane also contributes to amyloid formation in neurodegenerative diseases.176,177Cytomembrane is an essential target for the study of dynamics and morphology due to the recent discovery of membrane microdomains (rafts) in cancer9.1et al. developed probes 50 and 51 based on coumarin derivatives . Furthermore, probe 52 monitored the acidification of the vesicles throughout the endocytic pathway. The main limitation associated with probe 52 is that we cannot use it for long-term tracking due to the instability of the basic form.The cell’s various biomolecules, pathogens, and fluids are regulated by intracellular vesicles that the plasma membrane produces.vesicles , Table 19.3Despite several efforts in designing membrane-targeting ratiometric fluorescent probes, literature reports are rare. To date, no commercially available membrane-targeting fluorescent probes are available. In the future, researchers must consider several parameters for designing efficient membrane-targeting fluorescent probes, such as probe stability, selectivity, sensitivity, response time, orientation/location in the membrane, and detailed mechanism of interaction with the membrane.10Developing a powerful molecular tool that can target other organelles simultaneously is necessary to study the relationship between different organelles.10.12 is a critical gas messenger that plays a vital role in many cellular processes, including apoptosis in lysosomes and mitochondria. Therefore, to know the relationship between lysosome and mitochondria in regulating SO2-related cellular activities, Kong et al. pioneered probe 53 (183 Free probe 53 showed emission at 600 nm (red fluorescence) owing to the FRET process between the naphthalimide unit and the semi-cyanine unit. Upon the addition of SO2, the emission intensity at 600 nm decreased. In comparison, a new emission maximum at 530 nm appeared and increased, assigned to Michael's addition reaction of SO2 in the semi-cyanine unit and inhibition of the FRET process. In living cells, probe 53 found an application for simultaneously detecting endogenous SO2 in lysosome and mitochondria by one and two-photon modes.From a recent study, SOprobe 53 , Table 110.2et al. developed probe 54 based on hemicyanine and rhodamine dyes for selectively monitoring mitochondria and lipid droplets under different pH values through a dual-emission channel (19 The pH (from 2.52 to 10.50) sensing behavior of probe 54 revealed a notable decrease in the emission band at 580 nm, accompanied by the increase in a new emission peak at 450 nm, attributed to two different structural forms under acidic and basic medium. Under acidic conditions, the ring-open form of probe 54 targeted mitochondria and displayed solid red emission. In contrast, the ring-closed form of probe 54 targeted LDs and gave blue emission. Furthermore, applied probe 54 monitored the pH fluctuation in living cells in the presence of different exotic chemicals.Understanding the fundamental connection between fluctuating mitochondrial pH and lipid droplet (LD) generation is crucial for understanding cell physiology. Bai  channel , Table 110.3Dual organelle-targeting probes hold great promise for elucidating the relationship between organelles and deepening our understanding of the biological processes behind the biological species. However, building molecular probes with two or more sensitive moieties is challenging. Furthermore, dual organelle-targeting probes are rare in the literature.11etc. Additionally, we highlighted the methods utilized to construct these probes and sensing mechanisms for their response to particular species.This review article highlighted recent advances in organelle-targeting ratiometric fluorescent probes reported since 2015. In particular, we discussed synthetic fluorescent probes with potential applications in biological systems such as biological species detection, fluorescence imaging, studying physiological and pathological processes, Nowadays, significant research has been put into developing organelle-targeting fluorescent probes to create fluorescent tools with improved resolution and sensitivity and a better understanding of the molecular mechanisms behind various biological processes. In conclusion, high selectivity, high reactivity, fast response time, low detection limit, good solubility, and organelle-targeting ability are the multiple advantages of the fluorescent probe. Currently, ratiometric probes that meet all the above properties are rare in the literature.184 Thus, the development of ratiometric probes with two-photon properties is greatly needed. Second, for in vivo application, near-infra-red (NIR) probes provide several advantages, such as deep tissue penetration, reduced photon scattering, and reduced photodamage to the living organism.185 Thus, ratiometric probes with emissions in the NIR region are in demand. Third, Reversible probes hold great promise for revealing the dynamic states of relevant analytes in various processes.52 Thus, developing ratiometric probes for biological species detection based on reversible reactions is essential. Fourth, probes targeting organelles such as the nucleus, membrane, lipid droplet (LD), melanosome, and Golgi apparatus are rare in the literature (Researchers will always be very interested in the advancement of ratiometric probes and their commercial applications. Despite the abundance of current outcomes, the following areas require additional work in the future. First, compared to traditional one-photon fluorescence probes, two-photon fluorescent probes provide several advantages, such as deep visualization, reduced photo-toxicity, minimal light scattering, and highly bright and contrast images.terature . Fifth, terature . Sixth, https://scholar.google.com/ and https://www.sci-hub.se/. Microsoft Office Word 2007 was used for writing the article, while Microsoft Office PowerPoint 2007 was used for graphical presentation. ChemBioDraw was used for chemical structure drawing.All the data were collected freely from websites such as Dr Manoj Kumar Goshisht and Dr Neetu Tripathi contributed equally to writing the original draft and to reviewing and editing the manuscript. Dr Goutam Kumar Patra and Dr Manohar Chaskar contributed to reviewing and editing the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The endpoints measured included mitochondrial reactive oxygen species and cytostructural changes assessed via confocal laser scanning microscopy; cell morphology evaluated via scanning electron microscopy; biochemical reactions assessed via protein arrays; transcriptome assessed via gene arrays, and cell surface activation markers evaluated via flow cytometry. The results showed that, compared to untreated cultures, NiO increased markers for dendritic cell activation, trafficking, and antigen presentation; oxidative stress and cytoskeletal changes, and gene and cytokine expression of neutrophil and other leukocyte chemoattractants. The chemokines and cytokines CCL3, CCL7, CXCL5, IL-6, and IL-8 were identified as potential biomarkers of respiratory sensitization.Dust, both industrial and household, contains particulates that can reach the most distal aspects of the lung. Silica and nickel compounds are two such particulates and have known profiles of poor health outcomes. While silica is well-characterized, nickel compounds still need to be fully understood for their potential to cause long-term immune responses in the lungs. To assess these hazards and decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. To understand the implications of these two compounds reaching the distal aspect of the lungs, the alveoli, an architecturally relevant alveolar model consisting of epithelial cells, macrophages, and dendritic cells in a maintained submerged system, was utilized for high throughput testing. Exposures include crystalline silica (SiO The lungs are a complex network of cell types involving cellular crosstalk, communication, and varying motions . Because of this heterogeneity of cellular structure, one of the most critical aspects of pulmonary in vitro study is the ability to adequately maintain relevant cellular architecture in selected models .With the primary function of the lungs being gas exchange, it is critically important to test and assess the potential for poor health outcomes associated with inhaled air. Inhaled air can consist of chemicals and particulates that, depending on various physicochemical properties, can deposit on cells and affect cellular responses throughout the respiratory system . Of the Sensitization, whether in the skin or lungs, involves two consecutive steps: (i) Induction, where an exposure leads to a cascade of innate and adaptive cells activating and maturating to provide a specific elevated immune response on secondary exposure; and (ii) Elicitation, where an exacerbated immune response occurs on second exposure, leading to a variety of inflammation, as seen in acute and chronic asthma, as well as anaphylaxis . TypicalCurrently, most research into respiratory sensitization has focused on low molecular weight (LMW) and high molecular weight (HMW) chemicals . Most knIdentifying and understanding the mechanisms associated with respiratory sensitization has primarily focused on rodent studies or gathered from population-level studies in humans ,10,11. T2) is ubiquitous in the earth’s crust and is known to lead to adverse pulmonary health through silicosis, where trapped silica lead to inflammation; scarring; lung cancer; chronic obstructive pulmonary disease (COPD), and kidney disease [2 is created from sawing, drilling, crushing, grinding, and cutting [2 is not known to lead to respiratory sensitization despite decades of research on human populations after exposure; however, it is known to be a respiratory irritant that leads to oxidative stress for all cell types within the lungs on exposure [Crystalline silica  and immune cells, specifically alveolar macrophages (AMs) in the luminal space and dendritic cells (DCs), which are scattered among the basement membrane .2) and a suspected sensitizing respiratory particulate (NiO). Like the previous study, the results suggest that multiple techniques and endpoints can show objective distinctions in immune responses after different particulate exposures.Previously, an easy, reliable, and verified cell culture model that can be adopted by any lab capable of performing molecular toxicology studies was used to study a known chemical respiratory sensitizer, isophorone diisocyanate (IPDI), and a known cell activator, phorbal 12-myristate 13-acetate (PMA) and ionomycin . Here, t®. The characteristics of toxicology and immunology assessments, as well as basal properties, have been studied. This includes evaluating transepithelial electrical resistance (TEER) [The model setup is based on in vivo alveolar cellular architecture, which contains epithelial cells (ECs), alveolar macrophages (AMs), and dendritic cells (DCs). e (TEER) ,30,31,322 and NiO along with the quantitative physicochemical properties of each material listed in the table. SiO2 had an average size of approximately 3 μm, with NiO having an average size of approximately 80 nm. The surface charge of SiO2 averaged −56.8 and NiO −9.05, with the hydrodynamic diameter at 1.807 μm for SiO2 and 0.963 μm for NiO.2- and NiO-treated cultures, respectively  was assessed using the Olympus CellSens software V4.2. Resultant MFI calculations were compared across untreated  vs. SiO2-treated  vs. NiO-treated  cultures. While both treated cultures showed increased nuclear binding activity, no statistical significance was seen. Only NiO treatment induced significant increases in ROS. ROS from all exposures were as follows: untreated, 19.02; SiO2-treated, 24.49; NiO-treated, 60.94. F-actin, a measure of proliferation, increased significantly in both SiO2- and NiO-treated cultures compared to untreated cultures . NiO treatment showed significant increases in ROS and F-actin compared to untreated, while SiO2-treated cultures only showed significant increases in F-actin.Confocal laser scanning microscopy (CLSM) was performed to measure reactive oxygen species (ROS), nuclear binding activity, and cytoskeletal structure. 2- and NiO-treated cultures revealed several inflammatory genes associated with inflammatory responses and associated explicitly with cell activation and recruitment  were upregulated in NiO treatment relative to untreated and SiO2-treated cultures, indicating possible sensitizing potential.Transcriptome related to innate and adaptive cytokines was performed on delta Ct values and normalized to the reference gene ubiquitin C (UBC) by subtracting gene(s) of interest from the reference gene. Using delta Ct values where higher values represent increased expression, heatmaps were created to compare transcriptomics across the array of cytokine-related genes. A comparison between SiO2 and untreated cultures: BMP6, CCL1, CCL2, CCL3, CCL17, CCL18, CCL19, CCL20, CNTF, CXCL1, CXCL2, CXCL5, CXCL9, CXCL10, CXCL13, IL-1RN, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12β, IL-15, IL-16, IL-17α, IL-17F, IL-22, MSTN, OSM, TGFβ2, THPO, TNF, TNFRSF11, TNFSF10, TNFSF11, VEGFa, ADIPOQ, NODAL.2 and untreated cultures include: C5, CSF3, CD40LG, CXCL16, CXCL10, IFNa2, IL-1b, IL-23, XCL1, BMP4, IL-27, and CCL21. DCs in the basolateral chamber showed that the following genes were upregulated in NiO-treated cultures compared to SiO2-treated and untreated cultures: CCL3, CCL20, CCL24, CSF2, IL-5, IL-11, IL-12b, IL-17F, OSM, TNFSF10, TNFSF11, BMP4, and CX3CL1. Genes downregulated in DCs in NiO-treated cultures include ADIPOQ, BMP7, CD70, CXCL3, IL-4, IL-15, IL-21, IL-22, LIF, and CXCL12.Downregulated genes from ECs and AMs for NiO compared to SiOdavid.ncifcrf.gov database, and KEGG pathways were investigated to examine potential biological consequences. To better understand which biological pathways may be perturbed, genes were subsequently loaded to the ignaling .Genes in DCs following NiO treatment corresponded to the following pathways: chemokine signaling, cytosolic DNA-sensing, rheumatoid arthritis, Toll-like receptor signaling, Jak-STAT signaling, inflammatory bowel disease, RIG-I-like receptor signaling, type I diabetes mellitus, asthma, PI3K-Akt signaling, and T cell receptor signaling .2 also showed significant increases in RANTES from the cell lysate compared to NiO and untreated cultures. The cell lysates of DCs showed significant increases in IL-6 and MIP-1a in NiO- and SiO2-treated cultures compared to untreated cultures. Significant decreases were seen in IL-5 from NiO- and SiO2-treated cultures compared to untreated cultures. Supernatants from DCs showed only a significant increase in IL-8 for NiO compared to both SiO2 and untreated cultures, indicating a prolonged recruitment of neutrophils . CD40 expression was increased in both SiO2- (2.16%) and NiO-treated cultures (2.53%) compared to untreated cultures (0.66%), but not significantly so. CD80 expression was significantly increased in both SiO2- and NiO-treated cultures compared to untreated cultures . CCR7 expression was increased in both SiO2- (2.98%) and NiO-treated cultures (1.51%) when compared to untreated cultures (0.46%), but the results did not reach statistical significance.Flow cytometry was performed to identify specific DC markers related to activation and antigen presentation  and migration (CCR7) . MHCII eThe local milieu of the lungs is designed to be anti-inflammatory to prevent excessive inflammation and exacerbated immune responses to every exogenous material inhaled. Specifically, alveolar macrophages (AMs) phagocytose and continually patrol the lumen of the alveolar spaces where they engulf and dispose of foreign materials. In the steady state, AMs are suppressive by secreting immunosuppressive cytokines to surrounding cells ,34,35. DWhile there are no current biosignatures of respiratory sensitization common to all known respiratory sensitizers, there are general principles of sensitization that appear to hold for most known sensitizers at the respiratory junction: neutrophil influx and general cell activation of recruited cells . The cytDuring respiratory sensitizing reactions to chemical sensitization, our previous study found that specific cytokine-related genes, including CXCL5, IL-6, IL-8, and CCL7, were expressed in a perturbed manner ,41,42. SUnlike skin sensitization, the lungs lack a validated model that accurately identifies known or potential sensitizers . The curAs an alternative to animal testing and to circumvent many of the issues associated with failure to translate to humans, human-derived cells can and should be a current method of investigation ,14,15. S2) and a suspected respiratory sensitizer (NiO). Endpoint measurements included: (1) Cell morphology measured by microscopy; (2) Transcriptomics measured by real-time polymerase chain reactions (rt-PCR); (3) Cytokine profiling and expression measured by a Luminex multiplex assay; (4) Expression of cell surface markers measured via flow cytometry; and (5) Biological pathway analyses probed via the Database for Annotation Visualization and Integrated Discovery (DAVID). Respiratory sensitization typically requires an initial exposure, induction, subsequent re-exposure, and elicitation, for typical symptomatic responses. However, specific biochemical  responses are required for innate cells to recruit and activate immune-specific adaptive cells . Because of this requirement, it is hypothetically possible to identify respiratory sensitizers before the elicitation phase by examining innate cells at the exposure site. This would allow for the development of a rapid assay capable of predicting sensitizing potential before exposure, preventing the implementation of novel materials that may lead to poor health outcomes. Some cytokines, such as IL-8, peak 24 h post-exposure [This study showed an alveolar cell culture model mimicking in vivo architecture to differentiate responses induced by a known respiratory irritant (SiOexposure . Additioexposure . To bestDendritic cells (DCs), the primary antigen-presenting cells, are critical to immune responses throughout the body and are essential to eliciting long-term immune responses. On activation, these cells will readily take up and process exogenous material, increase the surface expression of MHCII, where the foreign antigen is presented, and migrate to local draining lymph nodes to train and activate T and B cells ,74,75,76In ECs and AMs, perturbations in the transcriptome are related to biological pathways, which can affect immune cell recruitment, proliferation, differentiation, and survival; increases in ROS production and cytoskeletal component changes; cellular migration, and activation of fibroblasts. Biologically, these pathways affect acute and chronic inflammatory responses, the ability of lymphoid cells to home relevant tissues of interest, and cell signaling. Downregulated transcriptomic profiles in ECs and AMs lead to perturbed biological pathways, which can cause decreases in cell cycling and cell-effector functionality.In the lungs, it has been shown that excessive increases in oxidative stress can hinder AM functionality, leading to pathologic inflammation . While m2 and NiO, respectively). Further changes were seen when comparing the suspected sensitizer NiO to the irritant SiO2 and untreated cultures. Significant increases in cytokine production, perturbations in the transcriptome, and surface marker expression related to inflammation, allergy, and sensitization were all noted after exposure to NiO. Taken together with previous studies, this study helps further the idea that various endpoint readouts  can help establish a high throughput assay capable of assessing the sensitizing potential of new and existing substances.In dendritic cells, it has been shown that a known sensitizer will cause upregulation of the major histocompatibility complex (MHC) class II, co-stimulatory molecules , and inflammatory cytokines . ResultsThe lungs are highly complex and constantly changing. Current models do not account for important processes such as cell turnover, activation, and communication between cells. Additionally, there are barriers such as fluids, such as surfactant and mucus, that capture and eradicate foreign substances but are not present in current models. To improve accuracy, studies should use fluidic devices and cell migration assays and include T and B cells to assess the activation of immune cells. While in vitro models have limitations compared to live organs or organisms, they offer the ability to study specific mechanisms and test new materials and contaminants. This can lead to significant advancements in understanding and treating lung-related issues.Experimental premise. To better understand the utility of this model, a model comparing a suspected respiratory sensitizing particulate  and a known respiratory irritant  were utilized. SiO2 and NiO were made in-house with physicochemical properties shown in Reagents. A commercially available engineered nickel oxide particle was purchased from Nanoshel, LLC . Similarly, a commercially available engineered silicon dioxide particle was purchased from Sigma-Aldrich .Physicochemical characterization of the materials. Hydrodynamic diameter, polydispersity, and zeta potential measurements were taken using a Zetasizer Nano ZS . All measurements were performed in triplicate with sample parameters for absorbance and refractive indices set to 0.01 nm and 1.580, respectively.Nanoparticle preparation for cell culture studies. Samples were diluted to 0.002 wt% in triplicate. Dilutions were performed in phenol-free cell culture media.Cell culture. A549 epithelial cells and U937 monocytes were grown in complete RPMI (cRPMI) 1640  and supplemented with 10% FBS and 1% penicillin-streptomycin. JAWSII cells were cultured in complete Alpha minimum essential medium (cAMEM) with nucleosides  and supplemented with 5 ng/mL murine GM-CSF ; 20% fetal bovine serum, and 1% penicillin-streptomycin. All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere until ready for use.® membranes  at 28 × 104 cells/cm2. Cells were allowed to adhere for 2–3 days until a confluent monolayer was formed. Media were removed, and inserts were inverted and placed into sterile glass dishes. JAWSII cells were resuspended in 500 μL of cAMEM and plated on the basal surface of the membrane at 7 × 104 cells/cm2 and allowed to adhere for hours. After excess media were removed, inserts were reverted into the well plate, and 1 mL of cAMEM was added to the basolateral chamber. U937 macrophages were added at a 1:9 ratio of U937:A549 in cRPMI, and the apical chamber was replenished to 500 μL [2 atmosphere to allow cells to rest for 24 h before exposure.Cells were plated as previously described . A549 epo 500 μL . The modMacrophage differentiation. U937 monocytic cells were incubated with 100 ng/mL phorbol 12-myristate-13-acetate (PMA) for 24 to 48 h, as previously described [2 atmosphere for 72 h before use. The adherent cells were dissociated using trypsin, resuspended in cRPMI, counted, and plated according to use.escribed . The celChemical exposure. All exposure materials were added to the apical chamber of the Transwell® membrane. SiO2 was added at 50 ppm and was a positive control for cellular irritation. NiO was added at 50 ppm as a test compound for suspected sensitization. The post-exposure period was 24 h to assess early markers of respiratory sensitizing potential.Cell imaging. Imaging occurred as previously described [® plates were removed from the incubator, and cells from both chambers were washed twice with 1X phosphate-buffered saline (PBS) solution. Glutaraldehyde, at a concentration of 1:10 in 1X PBS, was added to both chambers for 10 min and followed by three washes of 1X PBS at 10 min intervals. PBS was replaced with 4% osmium tetroxide in PBS for 2.5 h at 4 °C. Three consecutive wash steps were repeated, followed by a series of dehydration steps that occurred twice, each at 10 min intervals: 50% ethanol (EtOH); 70% EtOH; 90% EtOH; 100% EtOH. The well inserts were removed and submerged in 100% EtOH in sequence. The membranes were carefully excised with a razor blade, placed into sterile buckets, and dried in a critical point dryer . Imaging was performed on a focused ion beam scanning electron microscope  at 5 kV with a spot size of 5.0 and a working distance of 10 mm using an Everhart–Thornley detector.escribed . BrieflyTM live cell stain , MitoTracker™ Red CM-H2Xros (Thermo Fisher Scientific Inc.), and ActinGreenTM 488 ReadyProbes reagent (Thermo Fisher Scientific Inc.) for analysis of the nucleus, reactive oxygen species (ROS), and F-actin cytoskeleton, respectively. Images were captured using a confocal laser scanning microscope . Quantifying fluorescence was performed with CellSens software V4.2 on a Wacom Cintiq 22HD workstation (Olympus Corp.).Cells were stained with NucBlueTranscriptomics. Polymerase chain reaction (PCR) plates for a panel of innate and adaptive cytokine were purchased (AB Applied Biosystems TaqMan® Array 96-well plates) for mouse and human . The Transwell® compartments were evaluated separately by collecting cells and supernatant from both the apical and basal sides. Macrophage and epithelial cells were analyzed from the former, while the latter was used for dendritic cells. RNA collection, cDNA formation, and plating protocols followed manufacturer instructions. Plates were assessed on a QuantStudio 6 Flex RealTime PCR system (ThermoFisher Scientific), delta Ct values were calculated —Ct (housekeeping gene)), and heatmaps were created. Only ∆Ct values greater than 0.5 were considered for statistical analyses.https://www.genome.jp/kegg/) pathway analyses.Analysis of PCR data was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID v6.8) . BrieflyCytokine and chemokine multiplex analysis. Using the manufacturer’s instruction, cytokines and chemokines were measured using Milliplex MAP cytokine/chemokine magnetic bead panels for both mouse and human . Fluorescence was analyzed using the Bio-Plex Luminex 100 XYP  with the Bio-Plex Manager 4.1 software. Subsequently, a 5-parameter curve-fitting algorithm was applied for standard curve calculations.Flow cytometry. Fluorochrome-conjugated antibodies to CD40 (3/23), I-A/I-E (M5/114.15.2), CCR7 (4B12), and CD80 (B7-1) were purchased from BioLegend . TruStain FcX™ (anti-mouse CD16/32) was used to block against non-specific Fc binding, and 7-amino-actinomycin D (7-AAD) was used to measure live/dead cells. A live/dead cell gating was obtained, and the analysis was performed on a FACSVerse  with a subsequent analysis performed utilizing FlowJo v10. Cells were prepared as follows: after 24 h, the media were removed from the basal chamber, and 0.25% trypsin-EDTA (Fisher Scientific) was added for 5 min in a 37 °C humidified incubator at a 5% CO2 atmosphere. Equal parts of complete media were added to each well and mixed to resuspend cells. The samples were then spun in a temperature-controlled incubator at 4 °C, and the supernatant was removed. After removing the supernatant, cells were washed and spun before cell staining. Staining was performed as previously described [3 in PBS), blocked with anti-mouse CD16/32 (93), and placed on ice for 10 min. Consecutively, the cells were stained on ice for 45 min using anti-CD40, I-A/I-E, CD80, and CCR7 at 1:200 dilutions in FACS buffer. Lastly, the cells were washed three times in a FACS buffer and resuspended to a final volume of 0.2 mL before a FACS analysis.escribed . BrieflyDynamic light scattering (DLS) analysis. Particles were suspended in phenol-free cell culture media. Hydrodynamic diameter, dispersity, and zeta potential measurements were taken using a Zetasizer Nano ZS . All measurements were performed in triplicate with sample parameters for absorbance and refractive index set to 0.01 nm and 1.580, respectively.Statistical Analyses. Unless otherwise noted, all samples were performed in triplicate with three replicates for each methodology for nine samples in each experimental setup. Data were analyzed using analysis of variance (ANOVA) followed by a t-test using Microsoft Excel v16.72 and GraphPad Prism 9.4.1. Significance is noted in the figure caption where applicable, with data presented as mean with ± standard deviation.While it is possible that any chemical or particulate can lead to sensitization within the respiratory system leading to lifelong allergies, hypersensitivity, and other complications, the incidence rate is still low within the more significant population. However, to prevent poor health outcomes, especially in areas of lower development, assessing for respiratory sensitization is a continued focus in immunotoxicology. To provide preventative, protective, or curative responses, it is critical to understand the processes that promote long-term immune reactions before, during, and/or after toxicant exposure.The model utilized in this study can rapidly adjust cell types to mimic the area of the lung  to be studied. The simplicity of design, low cost of setup, ability to switch to an air–liquid interface if needed, and the ability to modify the endpoints measured are all strengths in using the model herein. While this model is static and does not include adaptive immune cells, subsequent studies are needed. They are underway to assess the ability of the cells in this model to activate and recruit T and B cells after exposure to known respiratory sensitizers and novel materials.Although submerged conditions are still commonly used in most studies, air–liquid interface (ALI) cultures have proven increasingly successful in recent years . HoweverWhen selecting and optimizing a co-culture system, the type of cell used is crucial. Although some human dendritic cell lines are available, they are not well-established in the literature and can be difficult to obtain. Obtaining human PBMCs that have differentiated into dendritic cells is also challenging, expensive, and can vary significantly between individuals. Therefore, researchers often use cell lines to ensure response consistency and simplify the validation process. Studies have demonstrated that murine dendritic cells exhibit similar responses to human dendritic cells, with JAWSII cells being a good example ,89,90. I