The apoptosis assay by Annexin V-FITC/PI staining showed that all tested compounds induced cell death mainly via apoptosis. Cell cycle analysis assessed using RNase/PI staining showed that organotin(IV) dithiocarbamate compounds induced cell cycle arrest at different phases. In conclusion, the tested organotin(IV) dithiocarbamate compounds demonstrated potent cytotoxicity against Jurkat E6.1 cells via apoptosis and cell cycle arrest at low IC50 value. However, further studies on the mechanisms of action are required to probe the possible potential of these compounds on leukemia cells before they can be developed into anti-leukemic agents.The discovery of cisplatin has influenced scientists to study the anticancer properties of other metal complexes. Organotin(IV) dithiocarbamate compounds are gaining attention as anticancer agents due to their potent cytotoxic properties on cancer cells. In this study, a series of organotin compounds were assessed for their toxic effects on the Jurkat E6.1 cell line. WST-1 assay was used to determine the cytotoxic effect of the compounds and showed that six out of seven organotin(IV) dithiocarbamate compounds exhibited potent cytotoxic effects toward T-lymphoblastic leukemia cells, Jurkat E6.1 with the concentration of IC Leukemia is known as bone marrow and blood cancer. Four common types of leukemia are acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). ALL with a prevalence of 25% of cancers in children under the age of 15 years, is the most common cancer in childhood. This ALL has a 93.5% five-year survival rate when treated with the newest protocol and top chemotherapy [Clinically, various anticancer drugs are used to treat ALL patients . HoweverThe toxicity research on metal-based substances become a hot topic after the discovery of cisplatin as a potent chemotherapy drug towards various cancer cells ,8. OrganThe common types of cell death that occur in the body are apoptosis and necrosis . ApoptosTherefore, seven organotin(IV) dithiocarbamate compounds, which are diphenyltin(IV) diisopropyl dithiocarbamate (ODTC 1), diphenyltin(IV) diallyl dithiocarbamate (ODTC 2), triphenyltin(IV) diisopropyl dithiocarbamate (ODTC 3), triphenyltin(IV) diallyl dithiocarbamate (ODTC 4), triphenyltin(IV) diethyl dithiocarbamate (ODTC 5), dimethyltin(IV) diisopropyl dithiocarbamate (ODTC 6) and dimethyltin(IV) diethyl dithiocarbamate (ODTC 7)  were invThe percentage of viability for cells treated with organotin(IV) dithiocarbamate is shown in 50 values obtained from organotin(IV) dithiocarbamate compounds and Vincristine on Jurkat E6.1 and WIL2-NS cell line are summarized in 50 value below 1.0 µM (0.67–0.94 µM). The diphenyltin(IV) dithiocarbamate compounds produced a less toxic effect on the cells with IC50 values ranging from 1.05–1.45 µM. The dimethyltin(IV) dithiocarbamate compounds produced the least toxic effect for this series of organotin(IV) compounds toward Jurkat E6.1 cells. The cytotoxic graph of vincristine towards Jurkat E6.1 cells showed a plateau patent after the viability cell reach 40% at 0.30 µM concentration up to the highest concentration (5.00 µM). This clearly indicates that vincristine caused a cytostatic effect on the cell not cytotoxic like organotin(IV) dithiocarbamate compounds. Therefore, the organotin used is much better than vincristine in terms of cytotoxic effects. Cells will slowly proliferate or grow back once the cytostatic effects have faded away.The ICThe selectivity index (SI) showed that ODTC 3 has the highest value (SI = 1.11) followed by ODTC 1 and vincristine (SI = 0.96). ODTC 5 and 4 produced SI value of 0.86 and 0.71 respectively. The SI value obtained by ODTC 2 is 0.50. And finally, ODTC 7 has the lowest SI with a value of 0.15.The mode of cell death of Jurkat E6.1 cells treated with organotin(IV) dithiocarbamate is shown in 50 value at the tested range of concentration.From the results obtained, the triphenyltin(IV) series caused majority of cell death at apoptosis phase. ODTC 3 displayed a total of 87.5% apoptosis with 59.70 ± 1.96% of cell population in early apoptotic phase and 27.80 ± 2.17% in late apoptotic phase. ODTC 4 showed that 49.70 ± 2.84% of cells are in the early apoptosis stage while 38.63 ± 2.73% in late apoptosis. To sum up, the total apoptosis population caused by ODTC 4 on Jurkat E6.1 cells is 88.33%. As for ODTC 5, the total apoptosis population on Jurkat E6.1 cells is 82.97 ± 2.84%, with 56.50 ± 1.64% of it in early apoptosis and 26.47 ± 1.23% in late apoptosis. For the diphenyltin(IV) and dimethyltin(IV) series, the majority of the cell population detected is viable (52.23–73.97%). ODTC 1 only caused 25.4% of cell death at the apoptotic phase with 12.23 ± 1.93% of the cell population in early apoptosis and 13.17 ± 1.13% in late apoptosis. For ODTC 2, the cells were in the early apoptotic phase with 17.20 ± 2.69% and 30.07 ± 2.38% late apoptotic, with a total of the apoptotic population is 47.27%. ODTC 7 causes 28.06% apoptosis with 13.13 ± 2.62% in early apoptosis and 14.93 ± 1.35% in late apoptosis. ODTC 6 does not been tested for its mode of cell death since this compound only caused a cytostatic effect and does not produce ICTreatment of vincristine (positive control) towards Jurkat E6.1 cells revealed that the population of cells treated also died at the apoptotic phase (54.64%). Vincristine induced early apoptosis (36.07 ± 2.85%) of the cells while 18.57 ± 1.31% in the late apoptosis population. 50 value at the tested range of concentration.To investigate the cell cycle progression of Jurkat E6.1 cells treated with organotin(IV) dithiocarbamate compounds, evaluation by flow cytometry was conducted using the RNase/PI stain for 24 h of exposure. Different treatments cause different effects on Jurkat E6.1 cells as shown in The results showed that 59.37%  ± 3.07 Jurkat E6.1 cells that were exposed to ODTC 2 were arrested in the S phase while 19.03%  ±  0.20 and 21.63%  ±  3.17 of treated cells were distributed in G2/M and G0/G1 phase respectively. Treatment with ODTC 4 displayed that 64.10%  ± 4.46 of Jurkat E6.1 cells were accumulated in the S phase while cells in G0/G1 and G2/M phase were 13.22%  ±  2.30 and 22.67% ±  3.54 respectively. For treatment with ODTC 5, a number of Jurkat E6.1 cells accumulated in the S phase (58.70% ± 3.37). Only 9.84% ± 0.71 of the cell population accumulated in the G0/G1 phase and 31.46% ± 2.86 in the G2/M phase. As for ODTC 7, the treatment caused the arrestment of the cell cycle at the S phase with 57.19% ± 2.89. Cells distributed in G2/M and G0/G1 phase are 17.39% ± 4.52 and 23.41% ± 5.37 respectively. Other than that, 68.37% ± 4.78 of the Jurkat E6.1 cells treated with ODTC 3 were arrested at G0/G1 phase. In addition, a small percentage of the cells (27.29% ± 5.12 and 4.34% ± 0.99) were in S and G2/M phase. Jurkat E6.1 cells treated with vincristine (the positive control) also exhibited significant changes when compared to the negative control, specifically at the G0/G1 and S checkpoints at 24 h. The population of cells treated with vincristine detected in G0/G1 is 66.76% ± 1.39 with 25.24% ± 1.39 in S phase and 8.00% ± 0.00 in the G2/M phase.This study was performed to assess the cytotoxic effects of organotin(IV) dithiocarbamate compounds which are diphenyltin(IV) diisopropyl dithiocarbamate (ODTC 1), diphenyltin (IV) diallyl dithiocarbamate (ODTC 2), triphenyltin(IV) diisopropyl dithiocarbamate (ODTC 3), triphenyltin(IV) diallyl dithiocarbamate (ODTC 4), triphenyltin(IV) diethyl dithiocarbamate (ODTC 5), dimethyltin(IV) diisopropyl dithiocarbamate (ODTC 6) and dimethyltin(IV) diethyl dithiocarbamate (ODTC 7) and also vincristine (positive control) against Jurkat E6.1 human acute T lymphoblastic leukemia cell line using WST-1 assay . The mosThe cytotoxic effect and reaction of the organotin(IV) dithiocarbamate compounds are closely related to their structure and properties. The chemical structure of triphenyltin(IV) dithiocarbamate consists of three phenyl groups attached to the tin (Sn) atom. This makes the compound more lipophilic and exerts a more potent effect on the biological system compared to phenyltin or diphenyltin . The numThe longer the hydrocarbon chain, the more potent the compound . In thisGenerally, dithiocarbamate compounds have a lipophilic character that makes it easier for them to interact with cells and to cross the biological systems’ membranes . Also, t50 produced by the organotin(IV) compounds in their study ranged between 0.4–1.3 μM while doxorubicin caused a cytotoxic effect at 0.1 μM. Another study done by Kamaludin et al. [50 values of compounds that cause cytotoxic effects are in the range of 0.06–0.18 µM, while for the Jurkat E6.1, the range was 0.14–1.30 µM. Meanwhile, the compound produced an IC50 value in the range of 5.20–5.40 µM against K562 (myelomonocytic leukemia cells) because this cell is known as apoptosis-resistant cells. Awang et al. [50 values are ranged between 0.03–0.41 µM for exposure times of 24, 48, and 72 h. This showed that the synthesized organotin(IV) dithiocarbamate compounds could induce a cytotoxic effect on different leukemia cell lines even at low doses.A similar study by Kamaludin et al.  also shon et al.  showed tg et al.  carried The treatment of vincristine on Jurkat E6.1 cells caused cytostatic effects up to the highest concentration. Cytostatic effect means, a substance can slow down or arrest the growth of the cells without killing them . Once thSelectivity index (SI) is the reference value indicating the selectivity of the compound/substances towards cancerous cells over non-cancerous cells. According to Bartmańska et al.  the SI vCell death can be classified into a few different forms which are apoptosis, autophagy, necrosis, and entosis . HoweverFrom the mode of cell death assessment, it was found that the organotin(IV) dithiocarbamate compounds caused the Jurkat E6.1 cell death via apoptosis. This result is quite similar to many other studies where the organotin(IV) compound induced cell death via apoptosis  where itIn normal and healthy cells, there’s a structure called phosphatidylserine (PS) that is located in the inner membrane of the phospholipid bilayer. However, when apoptosis occurs, PS will be flipped and translocated to the outer leaflet of the plasma membrane, exposed it to the external cellular environment . It is sOriginally, apoptosis is defined by the morphology and characteristic of dying cells . HoweverThe cell cycle progression of Jurkat E6.1 cells treated with organotin(IV) dithiocarbamate compounds was evaluated using flow cytometry after reaction with the RNase/PI stain for 24 h. ODTC 3 and vincristine caused cell cycle arrest at G0/G1 checkpoint, a phase where the cell is growing and proteins and RNA are synthesized. Arrested cells at this phase cause the cell to not enter the S phase and this eventually stops the cells from duplicating. As for ODTC 2, ODTC 4, ODTC 5 and ODTC 7, the cell cycle arrest at the S-G2/M phase. S phase in the cell cycle is a phase where the genetic material is synthesized and replicated. G2/M checkpoint is a phase where it has the function of preventing cells with damaged DNA, lasting from the G1 and S phases or generated in G2, from entering mitosis phase. Arrested cells at these phases cause the cell unable to produce two new daughter cells. In this study, the treatment of ODTC 1 towards Jurkat E6.1 cells does not cause cell cycle arrest at any phase. This showed that ODTC 1 is not a cell cycle specific.There are many research works conducted to study and investigate the effects of different organotin(IV) dithiocarbamate compounds on the cell cycle activities of different cell lines. For example, a cyclic trinuclear organotin(IV) complex with an aromatic oximehydroxamic acid group was able to cause cell cycle delay in the late S phase and G2/M checkpoint of HCT116 cells . MeanwhiRosewell Park Memorial Institute (RPMI) 1640 media , fetal bovine serum (FBS) , 1% penicillin-streptomycin , absolute ethanol , 2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt (WST-1) , dimethylsulfoxide solution (DMSO) , trypan blue 0.4% , phosphate buffer solution (PBS) , Vincristin , Annexin V-FITC/PI kit , RNase/PI stain , L-glutamate , organotin(IV) dithiocarbamate compounds.Seven organotin(IV) dithiocarbamate compounds, which are diphenyltin(IV) diisopropyl dithiocarbamate (ODTC 1), diphenyltin(IV) diallyl dithiocarbamate (ODTC 2), triphenyltin(IV) diisopropyl dithiocarbamate (ODTC 3), triphenyltin(IV) diallyl dithiocarbamate (ODTC 4), triphenyltin(IV) diethyl dithiocarbamate (ODTC 5), dimethyltin(IV) diisopropyl dithiocarbamate (ODTC 6) and dimethyltin(IV) diethyl dithiocarbamate (ODTC 7) were synthesized and characterized by our team  ,47,48 atAll stock solutions of the organotin compounds were prepared at 30 mM and dissolved in dimethyl sulphoxide (DMSO). The stock solutions were stored at 4 °C, fresh dilutions were made by adding the medium before the experiment.2 .The Jurkat E6.1 (TIB-152™) and WIL2-NS (CRL-8155™) cell lines were purchased from the American Type Culture Collection. These cells were cultured in Roswell Park Memorial Institute Medium 1640, RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin to obtain the complete growth medium. The cells were incubated at 37 °C with 5% CO2 flasks and seeded in 96-well culture plates with a final concentration of 1 × 106 cells/mL. The cells were exposed to organotin(IV) dithiocarbamate compounds with the highest concentration of 5.0 μM. Vincristine was used in this study as a positive control with its highest concentration, at 5.0 μM, while untreated cells (cells and complete medium only) were used as the negative control. Serial dilution methods are used to obtain the desired concentration gradient and the cells were then exposed to the compounds overnight (24 h). After the incubation period, the WST-1 solution  was added in a ratio of 1:10 to the suspension volume in the wells and incubated for another 4 h. Finally, the cells’ optical density (OD) was measured using an ELISA microplate reader  at 440 nm wavelength. The data obtained were analyzed using GraphPad Prism software (Dotmatics) to determine the IC50 values.Jurkat E6.1 and WIL2-NS cell lines were cultured in 75 cm50 value) of each compound on a non-cancerous cell line (WIL2-NS) to a leukemia cell line (Jurkat E6.1) using the following equation:The selectivity index is determined by dividing the cytotoxic activity (IC5 cell/mL. The cells were then treated with organotin(IV) dithiocarbamate compounds and vincristine at their respective IC50 concentrations and then incubated for 24 h. After the incubation period, 500 μL of cells were harvested and transferred into a microcentrifuge tube. The sample was centrifuged at 1500 rpm for 5 min at 4 °C  and the supernatant was discarded, and 500 μL of cold PBS was added to the sample followed by another centrifugation for 5 min, then the supernatant was discarded. Next, 150 μL of Annexin V binding buffer and 5.0 μL of Annexin V-FITC were added to the sample in the dark and then incubated for 15 min at room temperature. After that, 3.0 μL of propidium iodide (PI) was added to the samples. The samples then incubated for another 2 min at room temperature. Finally, 350 μL of Annexin V binding buffer was added to the samples and transferred into Falcon tubes. The samples were then analyzed using a BD FACS Canto II flow cytometer .Apoptosis assay was performed according to the manufacturer’s instructions using Annexin V-FITC/PI staining kit . The Jurkat E6.1 cells were seeded in a 6-well plate with a final concentration of 5.0 × 106 cells/mL and treated with organotin(IV) dithiocarbamate compounds for 24 h, with vincristine as the positive control. The concentration of each treatment used was based on the IC50 values obtained from the WST-1 assay. After the incubation period, 500 µL of cell suspension was washed twice with cold PBS and fixed overnight with 500 µL of 70% ethanol. The cells were then stained with 500 µL RNase/PI stain and measured using a BD FACS Canto II flow cytometer . The cell cycle phases were analyzed using the ModFit LT software (Verity Software House).Cell cycle analysis was performed according to the manufacturer’s instructions using PI/RNase Staining Buffer . For this assay, the Jurkat E6.1 cell line was cultured in 6-well culture plates with a final concentration of 1.0 × 10p-value of < 0.05 is considered statistically significant.All data are presented as the mean ± standard error of mean (SEM). Statistical analysis and evaluation of the percentage of viable cells and the concentration of compounds used to treat the cells were calculated using GraphPad Prism 9 software (Dotmatics) by employing a one-way analysis of variance (ANOVA). A 50 concentration. Further assessment revealed that the compounds demonstrated cell cycle arrest at different phases. Thus, this study indicates that the organotin(IV) dithiocarbamate compounds possess good potential to be developed into new anti-leukemic agents.This study has proven that the organotin(IV) dithiocarbamate compounds exhibited a cytotoxic characteristics because of their ability to inhibit Jurkat E6.1 cell growth at low doses with the most potent compound being ODTC 3. The compounds were also able to induce cell death via apoptosis after 24 h of treatment with the IC


However, the ferroptosis regulator acyl-CoA synthetase long-chain family member 4 (ACSL4) was found to be downregulated in RPE exposed to H2O2 and this effect was exacerbated when the RPE cells were simultaneously treated with DHA. Together, these results show a response of RPE when stressed which will likely be overwhelmed under disease conditions such as AMD resulting in cell death.Age-related macular degeneration (AMD) is a complex disease caused by different genetic and environmental risk factors leading to loss of cells in the central part of the retina. Oxidative stress appears to be an important environmental risk factor that contributes to both the initiation and progression of AMD. Retinal pigment epithelium (RPE) plays an important role in regulating oxidative stress in the retina and is one of the main retinal cell types affected in AMD. A main function of RPE is to phagocytose photoreceptor outer segments (POS) which are rich in the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), making this cell type potentially more susceptible to oxidative stress-induced lipid peroxidation which can lead to cell death. RPE is known to undergo necrotic cell death in response to oxidative stress. The aim of this study was to determine if DHA in POS can increase oxidative damage to RPE. It was found that RPE undergo increased lipid peroxidation and decreased cell viability when stressed with hydrogen peroxide in combination with DHA or POS. H Oxidative stress is commonly attributed to age-associated pathology in many diseases with damage from oxidative stress accumulating during ageing4. In addition, the antioxidant capacity diminishes and the efficiency of repair systems is compromised with age6. Oxidative stress also has an important role in the initiation and progression of AMD8. Moreover, treatments with antioxidants, such as vitamin C, vitamin E, β-carotene, and zinc, have been found to ameliorate AMD7. Various stimuli can induce different forms of oxidative stress in the eye9. Proportionally compared to the rest of the body, the retina consumes the most oxygen and therefore has several adaptations to deal with oxidative stress10. Dysregulation of pathways regulating oxidative stress are therefore likely to play a role in AMD pathology.Age-related macular degeneration (AMD) is the most common cause of visual loss in the elderly in developed countries11. The RPE has many important functions in the retina, including light absorption, transepithelial transport, buffers ions in the subretinal space, participates in the visual cycle, phagocytoses photoreceptor outer segments (POS), and secretes important factors to neighbouring cells12. In the retina, the RPE cells are continuously exposed to oxidative stress as RPE metabolises and recycles POS13. RPE cells also have large oxygen fluxes near the plasma membrane14 and are exposed to light12. Moreover, aged RPE have been found to have a reduced metabolic capacity, leading to increased susceptibility to oxidative stress15. Therefore, RPE need to be well adapted to handle oxidative stress.Retinal pigment epithelium (RPE) cell death is characteristic of late stage AMD17. POS are enriched with PUFAs, to the point that POS possess the highest relative concentration of PUFAs compared to all other body tissues18. The high amount of double bonds in PUFAs make them susceptible to lipid peroxidation by reactive oxygen species (ROS). Docosahexaenoic acid  is an omega-3 PUFA that is relatively rare in human tissue. However, DHA accounts for 60% of the PUFAs in the retina, with its highest concentration in POS18. DHA is an essential structural component of the cell membrane in POS19 and DHA is known to be able to enhance RPE survival20. However, DHA is also the most oxidizable fatty acid in humans18. RPE are constantly exposed to high concentrations of DHA through phagocytosis of POS and through transport of DHA from the choriocapillaris to the photoreceptors for photoreceptor membrane biogenesis21. Oxidative stress can therefore potentially cause lipid peroxidation of DHA in RPE, leading to RPE damage and possibly cell death.An important function of RPE is to phagocytose POS2O2) has been shown to lead to necroptosis, and not apoptosis, in RPE22. Necroptosis is a regulated form of necrosis23. Moreover, oxidative stress induced by tert-butyl hydroperoxide (tBHP) or by glutathione depletion were recently shown to cause ferroptosis in human cells, including RPE26. Ferroptosis is an iron- and PUFA-dependent form of regulated cell death, which is distinct from apoptosis and necrosis27. DHA is also known to be able to induce ferroptosis28. However, it is not yet known if DHA can contribute to RPE cell death through ferroptosis during oxidative stress.Oxidative stress caused by hydrogen peroxide  at 37 °C and 5% CO2. For experiments, cells were split and seeded in DMEM/F12 supplemented with 1% FCS and l-glutamine unless otherwise stated.ARPE-19  and hTERT RPE-1 (ATCC) cell lines were cultured and maintained in DMEM/F12  supplemented with 10% foetal calf serum  and 29, but with some modifications. Briefly, DHA and PA were dissolved in 50% ethanol to 150 mM at 70 °C and 90 °C respectively. The dissolved fatty acids were then immediately diluted, using prewarmed tips, in prewarmed 10% fatty acid-free BSA solution to 7.5 mM whilst stirring. This was incubated at 37 °C for 1 h to achieve BSA-fatty acid conjugation. The conjugated BSA-fatty acids were 0.22 µm filter-sterilised, aliquoted, and stored at − 20 °C. Conjugated BSA-fatty acids were used in cell culture medium at a final concentration of 500 µM fatty acid and 0.67% BSA unless otherwise stated.DHA  and palmitic acid  were conjugated to fatty acid-free bovine serum albumin  as previously described30, with some modifications. POS were washed three times with and resuspended in wash solution, consisting of 10% sucrose, 20 mM sodium phosphate buffer pH 7.2, and 5 mM taurine. To obtain a 2 mg/ml working solution of FITC, 10 mg of FITC was dissolved in 0.1 M sodium-carbonate buffer pH 9.5. To conjugate POS with FITC, 1.5 ml of FITC working solution was added to 5 ml of POS suspension and incubated at 21 °C for 1 h whilst rotating in the dark. The POS-FITC conjugate was then washed twice with and resuspended in DMEM with 2.5% sucrose.Bovine rod POS  were prepared and conjugated with fluorescein-5-Isothiocyanate  as previously described3 cells/well in a 96-well plate. The following day the cells were pre-treated for 24 h with 40 µM Z-VAD-FMK , 40 µM Necrostatin-1 , 1 µM GSK2791840B , or 5 µM Ferrostatin-1 . Pre-treatment with 500 µM deferoxamine  was 1 h before injury.RPE cell lines were seeded at 7.5 × 102. The cells were subsequently stained with 3 µM -4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; Invitrogen) in culture medium for 1 h at 37 °C and 5% CO2 to assess lipid peroxidation in RPE cell line membranes. C11-BODIPY581/591 incorporates into membranes and the excitation and emission fluorescence spectra of this lipid analogue both shift to shorter wavelengths when oxidised31. The cells were washed once with PBS and the medium with inhibitors was transferred back to the cells.Medium with inhibitors was transferred to a new plate and kept at 37 °C and 5% CO2O2 (Sigma-Aldrich) for 6 h or 500 µM DHA or 50 POS-FITC/cell for 18 h at 37 °C and 5% CO2.The cells were then treated with a final concentration of 500 µM HAfter treatment, the cells were washed with PBS and subsequently stained with 6 µg/ml Hoechst 33342 (Sigma-Aldrich). The cells were then fixed with prewarmed 4% PFA for 10 min at 21 °C and washed with PBS.2O2 treatment and subsequently stained with 3 µg/ml PI (Sigma-Aldrich) for 20 min at 21 °C. The cells were then fixed with prewarmed 4% PFA for 10 min and permeabilised with 0.1% Triton™ X-100 (Sigma-Aldrich) for 15 min at 21 °C. The cells were then incubated with 1:100 phospho S358 MLKL antibody  in 3% goat serum for 1 h at 4 °C. After two washes with PBS, the cells were incubated with 1:250 Alexa Fluor 647 antibody  for 30 min at 21 °C covered from light. After another two washes with PBS, the nuclei were counterstained with 300 nM DAPI  for 10 min at 21 °C covered from light, followed by another three washes with PBS.For pMLKL and PI staining, the cells were washed with PBS after HStained cells were imaged with a Leica DMi8 inverted microscope .2. The cells were then incubated for 1 h at 37 °C and 5% CO2 with a final concentration of 3 µM C11-BODIPY581/591 in DMEM/F12 without phenol red (Gibco) and no FCS. The cells were washed once with PBS and the medium with inhibitors was transferred back to the cells. The cells were then treated with a final concentration of 500 µM H2O2 for 3 h at 37 °C and 5% CO2. After H2O2 treatment, the cells were washed once with PBS, trypsinised, and transferred to Eppendorf’s containing medium with 10% FCS to inactivate the trypsin. The cells were then washed once with flow buffer, resuspended in 250 µl of flow buffer containing a final concentration of 2.4 µM DRAQ7 . The samples were kept on ice until analysis with a BD Accuri C6 flowcytometer .RPE cell lines were seeded and treated with inhibitors in 96-well plates as described above. Medium with inhibitors was transferred to a new plate and kept at 37 °C and 5% CO2O2 for 3 h at 37 °C and 5% CO2. After H2O2 treatment, the cells were washed once with PBS, trypsinised, and transferred to Eppendorf’s containing medium with 10% FCS to inactivate the trypsin.RPE cell lines were seeded and when stated treated with inhibitors in 96-well plates as described above. The cells were then treated with a final concentration of 500 µM HFor Annexin V and PI staining, the cells were stained using the Annexin V-FITC Apoptosis Detection Kit  according to the manufacturer’s instructions.3, the cells were permeabilised with 0.2% Triton X-100 for 5 min at room temperature. After washing with flow buffer the cells were incubated with 1:100 MLKL antibody  or 1:100 phospho S358 MLKL antibody 32 in 10% normal goat serum in flow buffer for 1 h on ice. After washing the cells were incubated with 1:250 Alexa Fluor 488 antibody  or 1:250 Alexa Fluor 647 antibody  for 30 min on ice. The cells were then washed once with flow buffer, resuspended in 250 µl of flow buffer. The samples were kept on ice until analysis with a BD Accuri C6 flowcytometer.For MLKL and pMLKL staining, the cells were fixed in prewarmed 4% paraformaldehyde for 10 min at room temperature. After washing with flow buffer, consisting of PBS with 1% BSA and 0.05% NaN4 cells per well in a 96-well plate. After 24 h, cells were then treated with 500 µM H2O2 or 500 µM BSA-PA or 500 µM BSA-DHA or H2O2 with BSA-PA or H2O2 with BSA-DHA for 6 or 18 h. Respective wells were pre-treated with 40 µM Nec-1 or 5 µM Fer-1 for 1 h before the treatment. Cells treated with 0.67% BSA and 0.17% ethanol were considered as the control (vector control). After treatment cellular viability was analysed using MTT. Briefly, cells were incubated with 0.5 mg/ml MTT (Sigma-Aldrich) for 4 h, and the formed formazan crystals were solubilised using 10% SDS-(0.01N)HCl solution. Absorbance at 570 nm and 690 nm was measured, and the 570/690 ratio was used for the comparison. The absorbance ratio of each group was compared to the control and expressed as a percentage.RPE cell lines were seeded at 105 cells/well in a 6-well plate. The following day the cells were treated with 300 µM H2O2 and 300 µM BSA-DHA for 24 h. RNA was extracted using TRIzol® Reagent  according to manufacturer’s instructions. cDNA was made using the High Capacity cDNA Reverse Transcription Kit  according to manufacturer’s instructions and primers were designed by Primerdesign Ltd. , see Table ® Green PCR Master Mix (Applied Biosystems) was used in combination with a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The relative gene expression data was analysed using the 2−∆∆Ct method as described by Livak and Schmittgen34.RPE cell lines were seeded at 3 × 105 cells/well in a 6-well plate. The following day the cells were treated with 500 µM H2O2, 500 µM BSA-PA, and 500 µM BSA-DHA for 6 h. The cells were then lysed in RIPA buffer supplemented with Complete Mini protease inhibitor cocktail (Sigma-Aldrich) and PhosSTOP phosphatase inhibitor  and passed through a 21-gauge needle  to shear the DNA. 20 µg of cell lysate in Laemmli sample buffer  supplemented with β-mercaptoethanol was heated to 98 °C for 5 min and subsequently loaded and ran on a 12% SDS-PAGE gel. The gel was then blotted onto a nitrocellulose membrane (Bio-Rad), blocked with TBS + 5% Marvel for 30 min at room temperature, and incubated with 1:500 ACSL4 antibody  overnight at 4 °C. The following day the membrane was incubated with 1:4,000 HRP-conjugated secondary antibody  for 30 min at room temperature. After incubation with Amersham ECL reagent , an Amersham Hyperfilm  was exposed to the membrane. The same membrane was reprobed with 1:5,000 β-actin antibody  for 1 h at room temperature followed by 1:4,000 HRP-conjugated secondary antibody  for 30 min at room temperature to check for loading control. Exposed films were developed in a Mi-5 X-ray developer machine .RPE cell lines were seeded at 2.5 × 10P-values of less than 0.05 were considered to be statistically significant.Experiments were repeated at least three times. Statistical analysis was performed using Microsoft Excel version 1905 and GraphPad Prism 6 software. 2O2 in order to investigate the role of apoptosis and necrosis in RPE cell death under oxidative stress conditions. H2O2-exposure did not induce apoptosis in RPE, although it did result in more necrotic cells . Nonetheless, 18-h exposure to PA with H2O2 lowered the cell viability to the same level as H2O2 alone and the inhibitors had no positive effect. Together, these results indicate an important role for lipids, and especially PUFAs, during oxidative stress in RPE.When exposed for 18 h, DHA alone significantly reduced RPE cell viability Fig. b. Moreov2O2 or DHA were studied. First, differential expression of common cellular stress markers was observed. SQSTM1, also known as p62, is a multifunctional protein involved in cellular processes including autophagy and stress. SQSTM1 expression in RPE was significantly downregulated upon H2O2 exposure  and lysophosphatidylcholine acyltransferase 3 (LPCAT3) were significantly downregulated in RPE treated with H2O2  forms after oxidative stress and promotes RPE survival20. However, increasing dietary DHA uptake has shown to improve intermediate AMD, but not advanced AMD39. Moreover, with age, phagocytosis of POS can lead to lipid accumulation in RPE, leading to ROS production40. This is likely caused by carboxyethylpyrrole (CEP) protein adducts, which are formed by an oxidation product unique to DHA41. CEP adducts are found to be more abundant in AMD retinal tissue, specifically in the RPE, Bruch’s membrane, and choroid42. In addition, CEP adducts are also found in AMD drusen42 and in lipofuscin43. This indicates that DHA peroxidation is increased and that ferroptosis could be a common form of cell death in AMD. CEP adducts have been shown to stimulate angiogenesis45, thus possibly contributing to neovascularization in geographic atrophy AMD. Moreover, mice immunised with CEP adducts develop a geographic atrophy AMD-like phenotype, indicating a role for CEP adducts in the aetiology of AMD46. CEP adducts were also found to be elevated in the plasma of patients with AMD41. CEP adducts could therefore play a role in AMD pathology and possibly function as a biomarker for AMD. It is therefore possible that in a disease setting like AMD, with prolonged oxidative stress in combination with high concentrations of DHA, the negative consequences of the formation of CEP adducts outweigh the positive benefits of the formation of NPD1, leading to an overall negative impact on cell viability by promoting necroptotic and ferroptotic cell death.Several studies have reported numerous health benefits of dietary DHA, such as supporting fetal development, preventing preterm birth, cardiovascular disease prevention, and improving cognitive function28. The amount of lipid ROS increased in RPE injured with H2O2, DHA, and POS  in ARPE-19 cells51. Additionally, RPE cells are reported to have the ability to oxidize PA, generating acetyl CoA and β-hydroxybutyrate, a process that increases the mitochondrial oxygen consumption rate52. This increased mitochondrial metabolic activity or proliferation, possibly due to EMT, could be responsible for the elevated cellular viability observed with PA at 18 h. However, 18-h treatment with the PUFA DHA showed a significantly more decreased cell viability, indicating that peroxidation of DHA could exacerbate oxidative stress in RPE. Importantly, subsequent treatment with necroptosis or ferroptosis inhibitors significantly alleviated this effect. This indicates that oxidative stress in RPE can lead to both necroptosis and ferroptosis, and that this is influenced by DHA.RPE treated with fatty acids and subsequently injured with Hone Fig. . When exDHA Fig. a. Howeve PA Fig. b. Prolon2O2 or DHA (Fig. SQSTM1 expression and thereby increases autophagy53. Important genes involved in apoptosis did not change expression necessary for apoptosis in RPE exposed to H2O2 or DHA (Fig. 6. The important ferroptosis inhibitor GPX4 is GSH-dependent. However, oxidative stress caused by H2O2 or DHA injury did not change the expression of GPX4 or GCLM in RPE (Fig. 2O2 compared to DHA in terms of the expression of stress-related genes, but a similar response in terms of apoptosis-related genes and ferroptosis-related genes.A differential transcriptomic response was found in RPE injured with HDHA Fig. , with a DHA Fig. a. SQSTM1DHA Fig. b, suggesACSL4 was found to be downregulated in expression in RPE injured with H2O2 (Fig. 2O2 and this was exacerbated when DHA was added under oxidative stress (Fig. LPCAT3 was found to be downregulated in RPE injured with H2O2 or DHA (Fig. 54. ACSL4 catalyses the activation of PUFAs by esterification, producing acyl-CoA. Acyl-CoA is an intermediate in various metabolic pathways, including phospholipid production for membrane biogenesis. ACSL4 is found to change lipid composition which affects ferroptosis sensitivity55. ACSL4 promotes the incorporation of omega-6 PUFAs in cellular membranes, which makes cells more sensitive to ferroptosis. ACSL4 has been proposed as a biomarker for ferroptosis and inhibition of ACSL4 also promotes necroptosis56. Downregulation of ACSL4 in response to H2O2 and downregulation of LPCAT3 in response to H2O2 or DHA could indicate that RPE have a defensive response towards oxidative stress and PUFAs to prevent ferroptosis and induce necroptosis. However, the concentrations that were used in this study could have been too high or too prolonged to prevent ferroptosis from occurring. Similarly, this defence mechanism could also be overwhelmed in a disease setting like AMD.Interestingly, ess Fig. . The mos56. H2O2-induced oxidative stress in RPE in this study caused both increased phosphorylation of MLKL and increased lipid ROS formation. However, the important regulators ACSL4 and LPCAT3 were found to be downregulated after H2O2 injury and the additional presence of excess DHA decreased the expression of ACSL4 even more. Inhibition of ACSL4 is known to promote necroptosis56. This shows that there is a complex regulation of the different cell death pathways in response to oxidative stress that may be interconnected. The proclivity of RPE cells to undergo cell death through necroptosis and ferroptosis after oxidative injury have both been found before24 but a role for DHA in this process has not been fully established in RPE. Ferroptosis in particular seems a logical form of cell death in RPE during AMD because of the accumulation of DHA through phagocytosis of POS and the increased oxidative stress. Here, we showed that the detrimental effects of H2O2-induced oxidative stress in RPE was exacerbated with DHA which led to both necroptosis and ferroptosis.It is thought that necroptosis and ferroptosis can compensate each other when either one is inhibitedSupplementary Information.


Cr1 and Cr2) containing the natural flavonoid chrysin were synthesized. The metal complexes were characterized using UV-Vis, Fourier-transform infrared, MS, SEM-EDX, XRD, and molar conductance measurements. Based on experimental and DFT/TD-DFT calculations, octahedral geometries for the synthesized complexes were suggested. The powder XRD analysis confirms that the synthesized complexes were polycrystalline, with orthorhombic and monoclinic crystal systems having average crystallite sizes of 21.453 and 19.600 nm, percent crystallinities of 51% and 31.37%, and dislocation densities of 2.324 × 10−3 and 2.603 × 10−3 nm-2 for Cr1 and Cr2, respectively. The complexes were subjected to cytotoxicity, antibacterial, and antioxidant studies. The in vitro biological studies were supported with quantum chemical and molecular docking computational studies. Cr1 showed significant cytotoxicity to the MCF-7 cell line, with an IC50 value of 8.08 μM compared to 30.85 μM for Cr2 and 18.62 μM for cisplatin. Cr2 showed better antibacterial activity than Cr1. The higher EHOMO (−5.959 eV) and dipole moment (10.838 Debye) values of Cr2 obtained from the quantum chemical calculations support the observed in vitro antibacterial activities. The overall results indicated that Cr1 is a promising cytotoxic drug candidate.Organic–inorganic hybrid salt and mixed ligand Cr(III) complexes ( Coordination compounds (CCs) have a more important place in chemistry as functional materials owing to their exceptional structural arrangements . CCs havin vitro and in vivo findings have shown that trivalent chromium, Cr(III), has biological benefits, such as enhancing the antioxidant enzyme defense system and facilitating insulin signaling or sensitivity (Following the first suggestion that Cr(III) compounds could act as a nutritional enhancement to glucose metabolism in 1959 by Schwarz and Mertz , there hsitivity , suppressitivity , decreassitivity , lowerinsitivity , reducinsitivity , lowerinsitivity , and inhsitivity . Howeversitivity  as the ssitivity . Howeversitivity .3.6H2O salt was used as a metal precursor. Moreover, metformin-coordinated Cr(III) complexes reportedly exhibit antibacterial activity and cytotoxicity. We previously reported the synthesis, antibacterial, and cytotoxicity profiles of Cu(II) complexes using 1,10-phenanthroline and the drugs metformin and ciprofloxacin chromate (III) as Lewis base and acid, respectively, where CrClfloxacin , which s6.6H2O), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were purchased from Loba Chemie Pvt. Ltd. . Triethylamine, NaHCO3, Mueller–Hinton agar, methanol (MeOH), and diethyl ether were purchased from . Ascorbic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich.All chemicals and reagents were of analytical grade and used without any treatment. 1,10-phenanthroline , chrysin (Sigma Aldrich), chromium chloride hexahydrate (CrClCr1) of 1,10-phenanthroline and an oxo ligand, chrysin  was prepared as reported previously by 3.6H2O (0.266 g) was added slowly and refluxed at 90°C in an oil bath. The resulting light green solution was refluxed for 1 h 20 min; then, 1 mmol of methanolic solution of 1,10-phenanthroline was added slowly. A drop of triethylamine was added to the reaction mixture to maintain the alkaline pH of the solution and the reflux was continued for an additional 6 h 40 min. The resulting brown solution was filtered and cooled. Next, 10 mL of diethyl ether was added to the filtrate and allowed to stand overnight. The resulting jelly-like brown precipitate was left to dry slowly at room temperature.A heteroleptic Cr(III)complex  was added slowly and stirred, resulting in a faded green solution. To the reaction mixture, 1 mmol of chrysin (0.254 g) dissolved in a mixture of methanol:ethanol (1:3) was added in the presence of a deprotonating agent, 1 mmol NaOH. Triethylamine  was used to maintain the alkaline pH of the solution and the reaction was refluxed for an additional 8 h. The resulting greenish-brown solution was filtered, treated with diethyl ether, and allowed to stand for 2 weeks. A light green powder was obtained after the slow evaporation at room temperature. The overall synthesis scheme is presented in For n et al.  with min−1 in KBr pellets on a PerkinElmer BX FTIR spectrometer. The UV-Vis spectra were recorded from 200–800 nm on an SM-1600 spectrophotometer using dilute solutions (1.0 × 10−4 M) of the test compounds. The thermogravimetric/differential thermal analyses (TGA/DTA) data were recorded from 25 to 800°C at a heating rate of 10°C/min under nitrogen-atmosphere (20 mL/min) on a Shimadzu DTG-60H thermal analyzer. X-ray diffraction  was used to determine the crystalline nature of the synthesized metal complexes. The diffraction was recorded with 2θ = 10 to 80 using CuKα (λ = 1.5406 Å) radiation at 40 kV and 30 mA (d), Miller indices (hkl), and lattice parameters  were estimated using QUALX2.0 (Fourier-transform infrared (FTIR) spectroscopy was recorded from 4,000 to 400 cmnd 30 mA . The intQUALX2.0 . SEM-EDXCr1 and Cr2) were evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. The detailed cell culture conditions and procedure for the cytotoxicity assay were reported previously  and Streptococcus pyogenes (ATCC19615)) and Gram-negative (Escherichia coli (ATCC25922)) bacterial strains and Pseudomonas aeruginosa (ATCC27853) using the disk diffusion method in Mueller–Hinton agar (MHA) (The ar (MHA) . All proar (MHA) . Known cCr1 and Cr2) were evaluated as we described previously, with minor modifications  employing the B3LYP  hybrid fS. aureus (PDB: 2w9h) (E. coli (PDB: 6F86)  , S. aureB: 2w9h) , and E. B: 6F86)  proteinsB: 6F86) . DockingB: 6F86)  were useCr1 and Cr2, respectively  nature 2Cl2](C15H9O4) and [C19H20Cl2CuN5O4].5H2O, for Cr1 and Cr2, respectively.Molar conductance analysis was performed to ascertain the electrolytic and non-electrolytic natures of the synthesized metal complexes. Their molar conductances for ectively . The res) nature . The higolecules   center, the ν-OH vibration appeared at 3,399 and 3,404 cm-1 for Cr1 and Cr2, respectively (ν-OH of chrysin in Cr1 (87% transmittance) and Cr2 (85% transmittance) were relatively large compared to that for free chrysin (52%). This confirmed that only one of the hydroxyl groups participated in the bonding. Furthermore, the free chrysin vibrations  observed at 1,640/1,608 and 2,930/3,123 cm-1 occurred due to ν-C=O and ν-C-H stretching, respectively, similar to previous reports  between the experimental and B3LYP calculated ν-OH could be due to the presence of an intermolecular hydrogen bond in the experimental measurement. In addition, the experimental/computational stretching vibrational bands of the 1,10-phenanthroline monohydrate ligand for ν-C=C  and ν-C=N  functional groups shifted to 1,630/1,608 and 1,521/1,429 cm-1, respectively, in Cr1, with a decrease in intensity. The stretching vibrational bands of the ν-OH  associated with the water of crystallization of 1,10-phenanthroline were diminished following the metal-ligand coordination. The decreased intensity indicated the formation of a rigid and symmetric structure with the metal center  was evidenced by the changes in the intensity and vibrational frequency of the free metformin  and 330 nm (less intense), which are associated with the benzoyl system and cinnamoyl system in DMSO . In thisCr1 and 330 nm for Cr2), possibly due to the extension of the conjugated system due to complexation. New stronger absorption peaks appearing at 413 nm for Cr1 (Cr2 (via a Cr(III) metal center (Following the reaction of Cr(III) with the ligands 1,10-phenanthroline and chrysin, bathochromic shifts to the benzoyl and cinnamoyl bands of chrysin were observed, whereas the bands of 1,10-phenanthroline were observed at a longer wavelength (278 nm). The broad peak of chrysin that appeared at 320/325 nm diminished with a slight bathochromic shift (330/335 nm for  for Cr1  and at 4Cr1 (Cr2  were assl center .Cr1 exhibited a peak at m/z = 738.109 to 741.114 (found = 735.070), which was attributed to the complex cation [C39H25Cl2CrN4O4]+, and a base peak at m/z = 516.077 (found = 514.040) associated with the coordination sphere and methanol  corresponded to the chemical entities in the coordination sphere, the Cr-metal center, two 1,10-phenanthroline and two chlorides [M = C24H16Cl2CrN4]+, and an m/z value of 255.0653 (found = 254.060) corresponding to the ligand in the ionization sphere of the complex, which is a chrysin fragment [C15H10O4]. The presence of m+4 peaks (peaks observed around m/z = 482.015 and 516.077) in the mass spectrum confirmed the coordination of two chlorides to the Cr-metal center. In Cr2, a molecular ion peak at m/z = 595.000 (found = 594.080) attributed to the complex [C19H30Cl2CrN5O9], a peak at m/z = 576.049 (found = 576.070), and a base peak at m/z = 558.039 (found = 558.060) were associated with [C19H28Cl2CrN5O8] and [C19H26Cl2CrN5O7] fragments, respectively. Two base peaks at m/z = 255.065 (found = 253.050) and m/z = 130.109 (found = 129.100) were associated with the chrysin (C15H9O4) and metformin (C4H911N5) ligands, respectively, confirming the coordination of the utilized ligands according to the designed complex (m+4 type peaks to the m/z peaks confirmed the coordination of two chlorides to the Cr(III) center  of the complexes were calculated from their relation to the average crystalline size (D) of the complexes  mixed ligand complexes showed 66.51% and 31.37% crystallinity for omplexes  using Eq−3 and 2.603 × 10−3 nm-2 for Cr1 and Cr2 complexes, respectively, representing a crystalline material with relatively less dislocation density and less irregularity within the structure. Previous research work by −4–2.10 × 10−3 nm-2 for Cu(II), Ni(II), Mn(II), and UO2II) mixed ligand polycrystalline complexes.The calculated dislocation density values were 2.324 × 10Cr1 and Cr2 complexes were identified based on the thermogravimetric curves in the temperature range between 25°C and 800°C  from 51.2°C to 213.2°C (DTGmax = 126°C), the weight loss was 7.21% , corresponding to the release of water molecules, ii) from 295.2°C to 590.4°C (DTGmax = 465°C) due to C15H8O3 + 2Cl groups with a total weight loss of 41.63%   .Cr2 complex thermally decomposed into three main degradation steps: i) from 55.56°C to 267.41°C (DTGmax = 243°C), the weight loss was 9.11% , corresponding to the release of water molecules; ii) from 275.57°C to 411.07°C (DTGmax = 400°C), associated with the decomposition of the two chlorine moiety, with a total weight loss of 11.76% ; iii) from 420°C to 524°C (DTGmax = 441°C) and a weight loss of 24.42% , corresponding to the decomposition of C4H11N5 + OH organic moiety with the final residue of chromium oxide (The um oxide .Cr1 and Cr2) were obtained from energy-dispersive X-ray (EDX) analysis. The SEM image of the synthesized complexes showed agglomerated particles and the presence of non-uniform-sized small grains. The EDX spectra of Cr1 and Cr2 showed characteristic signals for carbon, nitrogen, oxygen, chlorine, and chromium, which were associated with the atoms present in the ligands and the metal center, confirming the presence of the complexes waving the CHCrNOCl chemical composition of the complexes .Quantum chemical descriptors vital for correlating the energy, structure, and reactivity characteristics of the synthesized Cr(III) complexes were calculated from the frontier molecular orbitals (FMOs) HOMO, and LUMO . The bansoftness , inferriAccording to the hard–soft–acid–base (HSAB) theory, hard acids prefer to coordinate with hard bases and soft acids with soft bases . Hence, μ) is the tendency of an electron to leave a stable system. A negative chemical potential indicates that a stable complex that does not spontaneously decompose into its constituent element is stable, whereas hardness (η) measures the resistance to changes in the distribution of electrons within a molecule. It is characterized by compounds with large HOMO–LUMO gaps possessing less reactivity (μ) of −3.684 and −4.234 eV, respectively, for Cr1 and Cr2, inferring that Cr2 followed a stable system.Chemical potential (activity . In thisCr1 and Cr2) are presented in Cr1, the uniform distribution of the HOMO–LUMO confirmed the presence of ligand–ligand charge transfer via the Cr(III) metal center. This dominant charge transfer masked the expected d→d electronic transition. In the Cr2 complex, the HOMO–LUMO isodensity showed the concentration of the HOMO on chrysin and LUMO on the chrysin and metal center, inferring the possible intra-ligand and ligand-to-metal charge transfer.The B3LYP-calculated HOMO–LUMO distributions of the ligands and the corresponding metal complexes (in vivo and in vitro via the reactive oxygen species (ROS) generation mechanism (Cr(III) complexes have been used as efficient cancer therapeutic agents both echanism . In contechanism  and 1,10echanism . A low iechanism , suggestechanism . The viaechanism .Cr1 complex showed better cytotoxic activity, with an IC50 value of 8.08 μM compared to Cr2 (IC50 = 30.85 μM) and cisplatin (IC50 = 18.62 μM). A lower cell viability indicates a higher cytotoxic potential of the test compound  complexes were synthesized and subjected to cytotoxicity evaluation against the human breast adenocarcinoma cancer cell line (MCF-7) at a range of concentrations (3.125–100 µM). Although both complexes showed promising cytotoxicity results, the compound ; i.e., acompound . Thereforeported . Compariomplexes . The celCr1, Cr2, and cisplatin for 24 h, the morphology changed, with the cells appearing round to oval in shape, while the cell density was also reduced. The effect was more pronounced for the Cr1 complex even at lower concentrations (12.5 μM). Generally, the microscopic and cell viability results obtained from this work demonstrated that the reported metal complexes are cytotoxic, as reported for other metal complexes  with estrogen receptor alpha  residual amino acids together with a binding affinity  and the inhibition constant (Ki) parameters are given in Cr1 demonstrated a binding energy and an inhibition constant of −6.83 kcal/mol and 9.93 µM, respectively, with a total of 14 van der Waals  and π–alkyl/π–ion  interactions (Cr2 showed two hydrogen bonding (Glu 323 and His 356), 16 van der Waals , and π–alkyl/π–ion  interactions. Arg 394, Glu 353, Leu 387, and Leu 391 are the most important active site amino acids of ERα that participate in hydrogen bonding and π–alkyl interactions , minimum inhibition constant (2.78 µM), two hydrogen bonds (Gly 77 and Asn 46), eight van der Waals interactions , and seven π–alkyl/π–ion interactions   . The obtCr1 and Cr2) were evaluated using a DPPH assay  and cisplatin (IC50 = 18.62 μM). The overall results showed that the Cr(III) complexes reported in this study are promising cytotoxic drug candidates. However, further in vivo cytotoxicity studies against the MCF-7 cell line and toxicity studies are recommended.Organic–inorganic hybrid salts of Cr(III) complexes containing the natural flavonoid chrysin were synthesized. Spectroscopic (FTIR and UV/Vis), microscopic (SEM-EDX), thermogravimetric (TGA/DTA), mass spectrometric, pXRD, and molar conductance measurements were used to characterize the synthesized complexes. The coordinations of the ligands to the metal ions were confirmed using FTIR and UV/Vis spectral data. A molar conductance study was used to confirm the ionic/non-ionic nature of the complexes. The molecular weights of the metal complexes were confirmed by mass spectrometry. SEM-EDX was used to confirm the composition of the elements in the synthesized metal complexes. The pXRD patterns of the complexes in their powder showed polycrystalline natures with crystalline average sizes of 21.453 and 19.600 nm, for


PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood.Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night.We find that We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.The online version contains supplementary material available at 10.1186/s13059-023-03082-w. Arabidopsis thaliana, long days (LD) result in the stabilization of the floral activator CONSTANS (CO), which activates the expression of the florigen encoding gene FLOWERING LOCUS T (FT) , w, wFT andelf3-1 in SD, we observe a phenocopying of the LD gene expression response, for example, clusters 3 and 5 that are up-regulated by LD are also up-regulated in elf3-1 in SD. Consistent with the non-flowering phenotype of phyC-4, this background shows constitutive activation of SD responsive clusters even in LD. Finally, overexpression of ELF3 in LD causes repression of the LD activated expression clusters. The importance of ELF3 and PHYC for the expression of photoperiodism can clearly be seen when individual genes from representative clusters are observed directly. Consistent with the early flowering response of LD and elf3-1 plants, we see that FT1 and FT2, two florigen encoding genes, are strongly upregulated in these conditions, as is the key floral regulator AP1 and VRN1. The circadian regulators GI, LUX, PRR7, and TOC1 show oscillating behavior in WT with repression at the end of the day, with the evening repression being stronger in SD  class of zinc-finger transcription factors. These genes all share a common transcriptional pattern, being more highly expressed in LD and being repressed at dusk, particularly under short day conditions  is repressed by ELF3, and it loses photoperiodism becoming constitutively expressed in elf3-1. Since PIF4 overexpressors in Arabidopsis are very early flowering Mean TPM values were transformed into log2(TPM + 1). Genes with the maximum log2(TPM + 1) > 2 were kept. To investigate transcriptomic response towards a particular treatment, timecourse perturbation matrices were constructed as the difference of log abundances between paired conditions. For example, clusterR (https://github.com/mlampros/ClusterR), was used for performing the clustering. The expectation-maximization algorithm was used for fitting GMM to the given matrices. The Bayesian information criterion was used for estimating the number of clusters.Gaussian Mixture Models (GMM), a distribution-based clustering method and implemented by an R package Seeds were sterilized and sown on ½ X Murashige and Skoog-agar (MS-agar) plates at pH 5.7 and grown in Magenta™ GA-7 Plant Culture Box (Thomas scientific) and harvested at the indicated time.43−, 137 mM NaCl, and 2.7 mM KCl) containing 1% formaldehyde (F8775 SIGMA). The reaction was quenched by adding glycine to a final concentration of 62 mM. Chromatin immunoprecipitation (ChIP) was performed as described [Three-gram seedlings for each set were fixed under vacuum for 20 min in 1xPBS (10 mM PObwa (v0.7.17-r1188) was used to map raw reads to Brachypodium genome Bdistachyon_314_v3.1. Unmapped reads, mate unmapped reads, non-primary alignment, and duplicate reads were removed. Peaks were identified using MACS2 (v2.2.7.1) and filtered by q-value < 0.01. Bigwig files for IGV tracks were generated using deeptools function bamCoverage and normalized using RPKM.For processing ChIP-seq fastq files, ELF3 bound genes were determined if ELF3 peaks overlap with the genomic regions of gene body extended by 2 kb towards upstream and downstream.E-value < 0.05 : GSE147373 , GSE1282phyAB mutant background (plant 5).Line A: pUBI-BdPHYC-OX in an Arabidopsis B. distachyon WT background.Line B: pUBI-BdPHYC-OX 19-7 (homozygous) in B. distachyon seeds were incubated between 2 sheets of wet filter paper for 2–3 days in darkness at 4 °C. After removal of the lemma, the seeds were plated on ½ MS agar supplemented with 5 μM Norflurazon to inhibit greening during the red light irradiation. The seedlings were grown for 6 days at 22 °C in darkness. In order to induce the degradation of PHYA and PHYB, the seedlings were irradiated with constant red light  for 16 h. Subsequently, the seedlings were transferred to darkness at 22 °C to monitor dark reversion of PHYC. At time points 0, 4, 8, and 12 h after dark transfer, relative levels of active PHYC (Pfr/Ptot) were measured using a dual wavelength ratio spectrophotometer (Ratiospect) as described previously [B. distachyon seedlings were used per measurement. To inhibit oxidation, the seedlings were incubated for 20 min in ice-cold 50 mM DTT solution prior to the measurement.eviously . The shoA. thaliana seeds were sterilized before plating them on 4 layers of Whatman® filter paper saturated with 4.5 ml ddH2O. For sterilization, the seeds were washed first shortly with 70% ethanol and then twice with 100% ethanol. The seeds were stratified for at least 2 days at 4 °C in darkness. To induce germination, the seeds were incubated during 4 to 8 h in white light at 22 °C. Subsequently, the seedlings were grown in darkness at 22 °C for 4 days. Prior to Ratiospect measurements, the seedlings were irradiated for 20 min with constant red light  to convert PYHC into the active Pfr form. Afterwards, the seedlings were transferred into darkness at 22 °C to monitor dark reversion of PHYC. At time points 0, 4, 8, and 12 h after red light irradiation, relative levels of active PHYC (Pfr/Ptot) were measured using a dual wavelength ratio spectrophotometer (Ratiospect) as described previously [A. thaliana seedlings (freshweight) were used per measurement.eviously . One hun6-3xFLAG (negative control) transgene. After stratification in dark at 4 °C for 4 days, transgenic Brachypodium plants were grown in a growth chamber with a photoperiod of 14 h of light (200 umol·m−2·s−1) and 10 h of darkness, at 24 °C during daytime and 18 °C at night. Leaves from 45-day-old (old) or 21-day-old (young) plants were harvested at ZT0 in darkness and flash frozen in liquid N2. The protein extraction was performed in darkness with dim green safety light. About 30 mg (for old plants sample and YFP negative control) or 10 mg (for young plants sample) of total protein were used for purification via FLAG immune-precipitation , using the method as previously described [https://phytozome.jgi.doe.gov/pz/portal.html). All proteins identified in YFP control were subtracted from the identifications and a curated list containing ELF3 specific interactors was presented, showing names of their Arabidopsis homolog proteins.Plant materials for affinity purification coupled with mass spectrometry (AP-MS) were made from Brachypodium plants expressing either pUBI-ELF3-GFP-FLAG or pMDC32-YFP-Hisescribed , 66. Aftescribed . MS dataAdditional file 1: Supplementary Dataset S1. Proteins Co-Purified by BdELF3-GFP-FLAG AP-MS.Additional file 2: Supplementary Dataset S2. Gene expression analyzed over 24 h in both LD and SD growth conditions.Additional file 3: Supplementary Dataset S3. Genes identified, which fall into major clusters.Additional file 4: Supplementary Dataset S4. Significantly bound ELF3 peaks at ZT20.Additional file 5: Supplementary Dataset S5.ppd1-1 transcriptome.Additional file 6: Supplementary Dataset S6. Coincidence between ELF3 and PHYC ChIP-seq peaks.Additional file 7: Supplementary Dataset S7. Gene names used in this study.Additional file 8: Supplementary Dataset S8. BLAT on proteins.Additional file 9: Supplementary Dataset S9. Primers used in this study.Additional file 10: Fig. S1. Phytochromes are necessary for LD activation of flowering. A. phyC-4 does not flower in inductive conditions. B. phyA-1 is late flowering in long days. Fig. S2. Night length determines flowering phenotype in Brachypodium and a night break promotes early flowering A. Plants grown under 12L:4D condition flower nearly at the same time as plants grown und 20L:4D (LD) conditions at after about 3 weeks, whereas plants grown under 12L:12D, 20L:12D or 12L:20D did not flower during the course of the experiment. Experiment was terminated after 75 days, as plants started to senesce. A night break triggers flowering under non inductive short day conditions in Brachypodium. B. Introducing a night break of 1h or 2 nigh breaks of 30min each leads to a flowering phenotype similar to plant being grown under inductive long day conditions . Fig. S3. Transcriptional and ELF3 bound pattern of representative genes. RPKM was used to show ChIP signal in IGV screenshots. Fig. S4. Transcriptional and ELF3 bound pattern of representative genes. RPKM was used to show ChIP signal in IGV screenshots. Fig. S5. Transcriptional and ELF3 bound pattern of representative genes. RPKM was used to show ChIP signal in IGV screenshots. Fig. S6. Transcriptional and ELF3 bound pattern of representative genes. RPKM was used to show ChIP signal in IGV screenshots. Fig. S7.ppd1-1 transcriptome shows a similar behavior to phyC-4. A. Transcripts were clustered according photoperiod response, same with Fig. ppd1 and PPD1 OX under SD and LD. Fig. S8. ELF3 protein is degraded in response to light. Independently of photoperiod Plants were grown under 12L:12D (SD) or 20L:12D condition as indicated and samples taken 12 DAG at the indicated time . We used wild type plants  or plants overexpressing ELF3 (pUBI:ELF3_GFP_FLAG) (lane 3 to lane 7) and probed with an antibody raised in rabbit against ELF3 peptide . Fig. S9. ELF3 protein is stabilized in phyC-4 A. Plants were grown under 20L:12D condition samples taken 12 DAG at the indicated time . We used Bd21-3 , phyC-4  or plants overexpressing ELF3 (pUBI:ELF3_GFP_FLAG) (lane 5) and probed with an antibody raised in rabbit against ELF3 peptide (Agrisera). ELF3 accumulates in phyC-4 background at the end of the long day, but can not be detected in Bd21-3 background. ELF3 was detected using custom anti-ELF3 . B. Transcript level of ELF3 in phyC background are unchanged, indicating that phyC controls ELF3 in the protein level. Fig. S10. A. Western blot for 2 independent lines overexpressing pUBI:phyC-GFP-Flag in Brachypodium wild type background. B. Western blot for 6 independent lines overexpressing 35S-NFlag-BdPHYC in Arabidopsis background. Western blot was probed with an antibody against Flag epitope . Plants were grown under 20L:4D (LD) condition and samples taken 12 DAG at the indicated time .Additional file 11. Review history.


Saccharomyces cerevisiae does not efficiently grow and ferment at high-temperature conditions. The sterol composition alteration from ergosterol to fecosterol in the cell membrane of S. cerevisiae affects the thermotolerant capability.The thermotolerant yeast is beneficial in terms of efficiency improvement of processes and reduction of costs, while S. cerevisiae ERG5, ERG4, and ERG3 were knocked out using the CRISPR–Cas9 approach to impact the gene expression involved in ergosterol synthesis. The highest thermotolerant strain was S. cerevisiae ERG5ΔERG4ΔERG3Δ, which produced 22.1 g/L ethanol at 37 °C using the initial glucose concentration of 50 g/L with an increase by 9.4% compared with the wild type (20.2 g/L). The ethanol concentration of 9.4 g/L was produced at 42 ℃, which was 2.85-fold of the wild-type strain (3.3 g/L). The molecular mechanism of engineered S. cerevisiae at the RNA level was analyzed using the transcriptomics method. The simultaneous deletion of S. cerevisiae ERG5, ERG4, and ERG3 caused 278 up-regulated genes and 1892 down-regulated genes in comparison with the wild-type strain. KEGG pathway analysis indicated that the up-regulated genes relevant to ergosterol metabolism were ERG1, ERG11, and ERG5, while the down-regulated genes were ERG9 and ERG26. S. cerevisiae ERG5ΔERG4ΔERG3Δ produced 41.6 g/L of ethanol at 37 °C with 107.7 g/L of corn liquefied glucose as carbon source.In this study, ERG5, ERG4, and ERG3 resulted in the thermotolerance improvement of S. cerevisiae ERG5ΔERG4ΔERG3Δ with cell viability improvement by 1.19-fold at 42 °C via modification of steroid metabolic pathway. S. cerevisiae ERG5ΔERG4ΔERG3Δ could effectively produce ethanol at 37 °C using corn liquefied glucose as carbon source. Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ had potential in ethanol production at a large scale under supra-optimal temperature.Simultaneous deletion of The online version contains supplementary material available at 10.1186/s13068-023-02312-4. Saccharomyces cerevisiae (S. cerevisiae) is of great importance in various biotechnological applications with unique biological characteristics of fermentation capacity, ethanol production, and CO2 release [S. cerevisiae at comparatively low temperatures (30–32 °C) owing to its limited thermotolerance [Saccharomyces cerevisiae growing at a normal temperature, there are also yeasts in nature that can tolerate higher temperatures, such as Ethanol Red [Fungus  release . In the  release . Bioethaolerance . In addianol Red , 6. The anol Red , 6. The Conocarpus erectus [S. cerevisiae. In addition, high temperature enables more efficient feedstock hydrolysis, thus resulting in ethanol productivity increase in simultaneous saccharification and fermentation [The comparatively high temperature meets the requirement in the release of fermentable sugars from lignocellulosic biomass  like sta erectus , and ric erectus . Additioentation , 11. Thuentation , 13.S. cerevisiae [ERG enzymes [S. cerevisiae was formed from zymosterol catalyzed by C-24 sterol methyltransferase (Erg6p) [ERG3), C-24 (28) sterol reduce (ERG4), and C-22 sterol desaturase (ERG5) [Thermotolerant capability of yeast could be improved by various factors of sterols , heat shrevisiae . The prerevisiae . De novo enzymes  Fig. 1)S. cerevi enzymes . In the  enzymes , which m (Erg6p) . Fecostee (ERG5) .Fig. 1DeERG3, ERG4, and ERG5 genes in S. cerevisiae contributed to the thermotolerance improvement of yeast [ERG3, ERG4, and ERG5 in S. cerevisiae were compared using the Clustered Regularly Interspaced Short Palindromic Repeats–Cas9 (CRISPR–Cas9) approach . OD600 nm values of S. cerevisiae ERG5Δ(8.26), ERG4Δ(8.54), ERG5ΔERG4Δ(8.41), and ERG5ΔERG4ΔERG3Δ(8.55) were 1-, 1.03-, 1.02-, and 1.03-fold in comparison with the wild-type strain (8.27) , ERG4Δ(8), ERG5ΔERG4Δ(14), and ERG5ΔERG4ΔERG3Δ(23) were 1.7-, 2.7-, 4.7-, 7.7-fold in comparison with the wild-type strain (3), respectively. In addition, after culture for 24 h at 42 °C, the growth of all the strains was severely inhibited. However, after culture for 48 h, most of the strains grew on the solid medium. The density and quantity of colonies from engineered yeasts were higher than the wild-type strain. Under a dilution of 10–4, many colonies from engineered yeasts were detected on the plate; meanwhile, no colony from the wild-type strain was detected. Thus, S. cerevisiae ERG5ΔERG4ΔERG3Δ represented the strongest thermotolerance capacity among engineered S. cerevisiae strains.The thermotolerance of four engineered S. cerevisiae ERG5ΔERG4ΔERG3Δ at 30 and 42 °C based on the tinting color degree after dyeing of methylene-blue solution  was considerably lower than the wild-type strain (23.5%). Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ with the simultaneous deletion of ERG5, ERG4, and ERG3 possessed improved thermotolerance.The cell viability was investigated by the morphology observation of S. cerevisiae ERG5ΔERG4ΔERG3Δ were investigated using the wild-type strain as the control at 30, 37, and 42 °C . The deletion of ERG5, ERG4, and ERG3 did not affect glucose consumption and ethanol production under regular fermentation conditions.The glucose consumption and ethanol production of  °C Fig. . At 30 °ion Fig. A. The etS. cerevisiae ERG5ΔERG4ΔERG3Δ at 37 ℃ was higher (22.1 g/L) than that of the wild-type strain (20.2 g/L)  Fig. B. At 42 /L) Fig. C. TherefS. cerevisiae ERG5ΔERG4ΔERG3Δ  was far higher than up-regulated genes, which indicated that the simultaneous deletion of three genes of ERG5, ERG4, and ERG3 could cause the reduction of overall cell metabolism in engineered yeast.Scatter plot was used to reflect the difference of 2170 genes in total between the wild-type strain and S. cerevisiae ERG5ΔERG4ΔERG3Δ and the wild-type strain. The categories of significant differences in gene expression were investigated using the number and percent of up-regulated and down-regulated genes in each item (p < 0.05) . Most of the gene categories exhibited down-regulated characteristics. Overall, S. cerevisiae ERG5ΔERG4ΔERG3Δ after gene deletion resulted in down-regulated expression of genes.In this study, GO was classified into three categories of biological process (15 items), cellular component (12 items), and molecular function (14 items) between 05) Fig. . The numUp-regulated and down-regulated genes were analyzed based on the significantly different functions using the KEGG enrichment pathway Fig. . KEGG enS. cerevisiae ERG5ΔERG4ΔERG3Δ is drawn in Additional file ERG1, ERG11, and ERG5 and down-regulated genes of ERG9 and ERG26 in S. cerevisiae ERG5ΔERG4ΔERG3Δ were tested based on the KEGG pathway analysis. The results showed that ERG5, ERG4, and ERG3 gene deletion resulted in significant expression differences of genes involving the steroid metabolic pathway based on transcriptomics.Steroid biosynthesis from terpenoid backbone biosynthesis in S. cerevisiae ERG5ΔERG4ΔERG3Δ was used to produce ethanol at 37 °C with corn as material using the industrialized production processes. The concentrations of ethanol and sugar were measured during the fermentation of corn saccharified liquid  used in this experiment was a haploid strain. The plasmid gRNA-trp-HyB carrying hygromycin B resistance gene for guide RNA synthesis and Cas9-NTC carrying nourseothricin resistance gene for S. cerevisiae genomic DNA digestion were from Addgene . Escherichia coli DH5α for plasmid amplification and enzymes were from Sangon Biotech (China). The primer synthesis and gene sequencing were performed by Sangon Biotech (China). All biochemical reagents are analytically pure.S. cerevisiae ERG3, ERG4, and ERG5 target genes, 20-bp nucleotide sequences were obtained based on the efficiencies of target sequences from website (http://chopchop.cbu.uib.no/system) for CRISPR/Cas9 knocking-out. Three pairs of primers containing target sequences and gRNA-trp-HyB sequences were synthesized by Sangon Biotech (China). The prepared primers were used to amplify the plasmid gRNA-trp-HyB to obtain the corresponding linear vectors for the gRNA target  at the wavelength of 600 nm. The thermotolerance of S. cerevisiae was tested on the solid and liquid YPD media. The wild-type and engineered S. cerevisiae solution were diluted by gradient and transferred to the solid YPD plate for culture at 37 °C and 42 °C. The viability assessment of S. cerevisiae was performed under an optical microscope using the methylene-blue dyeing method [S. cerevisiae ERG5ΔERG4ΔERG3Δ were compared to analyze the effect of gene deletion on the thermotolerance of S. cerevisiae.The growth profile of engineered g method . The lifS. cerevisiae ERG5ΔERG4ΔERG3Δ were investigated using the wild-type strain as the control. The high-performance liquid chromatography (HPLC) method was used to measure the glucose and ethanol contents as the following parameters of Waters 2410 Refractive Index detector, Shodex SH1011 chromatographic column, a mobile phase of 0.01 mol/L H2SO4, a flow rate of 0.6 mL/min, injection volume of 5 μL, and column temperature of 50 °C.The effect of different temperatures of 30, 37, and 42 °C on the glucose consumption and ethanol production of engineered S. cerevisiae and the wild-type strains were carried out under the liquid culture conditions at 30 ℃. S. cerevisiae cDNA library was constructed via the processes of total RNA extraction, mRNA purification, and cDNA synthesis. cDNA was subjected to end A tail addition. Linker ligation reactions were finally performed [q-value < 20) were removed by a Trimmomatic tool, and the clean data were obtained for use [The cell proliferations of engineering erformed ; the proerformed . Transcr for use , 35. Aft for use . After t for use , Transcr for use , 39.S. cerevisiae ERG5ΔERG4ΔERG3Δ strain in a 20-L fermentation tank using fed-batch fermentation technology. The ethanol production of engineered S. cerevisiae ERG5ΔERG4ΔERG3Δ was used to evaluate the application feasibility under a high-temperature condition of 37 °C. The detailed processes were as follows: (1) raw material crushing. Dry corn as raw material after being crushed into fine powder was mixed with amylase and water; (2) liquefaction treatment. The uniform solution was heated and transported into a liquefaction tank to enter the liquefaction process; (3) fermentation. The liquefied solution was transferred into the fermentation tank after cooling. Then, the fermented mature solution containing ethanol was obtained after fermentation; (4) distillation. The fermented mature solution was sent into a distillation tower for the purification and dehydration of ethanol  and DEG analysis (B) between S. cerevisiae ERG5ΔERG4ΔERG3Δ and wild-type strain.Additional file 2. Steroid biosynthesis modification of S. cerevisiae ERG5ΔERG4ΔERG3Δ from terpenoid backbone biosynthesis after gene deletion.Additional file 3. Technical route of ethanol production from corn by liquefaction, high-temperature fermentation, and distillation.Additional file 4. S. cerevisiae DEGs.