Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid “turn-on” response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity. They miormation ,4. HOCl At the same time, increased concentrations of MPO as well as of chlorination biomarkers , were reported in the development of oxidative/halogenative stress. Such a condition accompanies the inflammatory process in case of myocardial infarction, rheumatoid arthritis, renal insufficiency, cystic fibrosis, etc. ,7,8,9. TSince neutrophil activation leads to the production of various reactive oxygen (ROS), halogen (RHS), nitrogen (RNS) species and other molecules, it is important to distinguish the products of MPO catalysis among other oxidants. By this point, different methods of neutrophil MPO activity analysis exist. Currently, there are two main approaches. The first one is the analysis of MPO activity using mass spectrometry ,11,12,135 M−1 s−1 [Nowadays the problem of searching for a specific and sensitive probe for HOCl is being actively studied. Some of the commonly used probes, such as 2-nitro-5-sulfidobenzoate (TNB) ,20, hydr M−1 s−1 ). It stiSome currently available probes are aimed at «naked-eye» detection ,30 or ar2−) nor H2O2 oxidize CB. It was also shown that the oxidation of CB by HOCl and Tau-N-chloramine occurs with high-rate constants, such as 2.5 × 104 and 1.2 × 105 M−1 s−1, respectively.Previously we proposed a spectrophotometric method of measuring the kinetics of HOCl production by monitoring the oxidation of the dye Celestine blue B—C.I.51050 (CB)—in the presence of Tau . We provIn this study we made use of CB as a fluorescent probe of a “turn-on” type for HOCl detection in cellular systems by confocal microscopy, flow cytometry, and fluorescence spectroscopy assays.2, xanthine oxidase (XO), xanthine (X), superoxide dismutase (SOD), mannitol, poly-L-lysine in solution (0.1%), Tau, phorbol 12-myristate 13-acetate (PMA), EDTA, scopoletin, type II horseradish peroxidase (HRP), N-formyl-methionyl-leucyl-phenylalanine (fMLP), cytochalasin b (cytb), Hoechst 33342, diphenyleneiodonium chloride (DPI), histopaque-1077 and sodium azide (NaN3) . Other reagents were from Reakhim (Russia) and Belmedpreparaty (Belarus). Ceruloplasmin (CP) was isolated from the trisodium citrate-stabilized blood plasma of healthy donors as described at [Canavalia ensiformis (Con A), Caragana arborescens (CAA), Glycine hispida (SBA), Phaseolus vulgaris (PHA-L), Sambucus nigra (SNA), and Triticum vulgaris (WGA) were from Lektinotest . Solutions were prepared using apyrogenic deionized water with a specific resistance 18.2 MΩ × cm . The pH of buffer solutions was measured using a portative pHmeter  having the precision of measurements of 0.01 pH.The following chemicals were used: CB, APF, 4’,6-diamidine-2-phenyl indole (DAPI), 4-aminobenzoic acid hydrazide (ABAH), glycerol, sodium hypochlorite (NaOCl) in solution (1.78 M), KOribed at . MPO wasribed at . Plant lv/v) and stored in the dark at +4 °C.A concentrated solution of CB (1 mM) was prepared in 20% aqueous glycerol  containing 10 mM Naw/v) trisodium citrate was used as an anticoagulant at a ratio of 9:1. Neutrophils were isolated by centrifugation in the histopaque-1077 density gradient as described at [Venous blood samples were obtained from healthy donors at the Republican Scientific and Practical Centre for Transfusiology and Medical Biotechnologies . Then, 3.8%  or CM 2203 spectrofluorimeter . CB (20 μM) and HOCl (0.5–50 μM) in PBS were mixed. Excitation and emission maxima of CB and of its HOCl-oxidized form were scanned sequentially (220–520 nm for excitation and 550–610 nm for emission). The maxima chosen  were used to determine fluorescence intensity depending on the HOCl concentration.2− was generated by two ways: by introducing a weighed portion of KO2 into the test solution to reach the final concentration of 100 μM, and also in the XO/X system containing 140 mg/L XO and 1 mM X as described in [Oribed in .4 × 7H2O with 100 μM H2O2 as described in [2O2were prepared by diluting a commercial solution of NaOCl and H2O2 (30%) in deionized water. Solution of nitrite (NO2−) was obtained from NaNO2, dissolved in deionized water. Peroxynitrite (ONOO−) was synthesised from H2O2 and NaNO2, residual H2O2 was removed by MnO2. The concentration of ONOO− was determined spectrophotometrically at 302 nm, ε302 = 1670 M−1 cm−1 [Hydroxyl radical (•OH) was formed in the Fenton reaction by mixing 1 mM FeSOribed in . Stock sM−1 cm−1 .The fluorescence spectra of native probes (20 μM) were measured eleven times, and the standard deviation (σ) of a blank measurement was deduced. Next, different equivalents of HOCl were added and the fluorescence intensity at 590 nm (excitation at 430 nm) was recordedimmediately. Then, the data obtained were plotted as the increasing concentrations of HOCl, and the slope (k) was obtained. The LOD was calculated using the following equation: LOD = 3σ/k [2O2, 4 mM Tau, 10 µM KI, 500 mM NaCl, and 100 mM Na–acetate buffer, at pH 5.5, were added. To assess the generation of HOCl by MPO a 250 µM solution of HOCl in 100 mM Na–acetate buffer, pH 5.5, was diluted by half in series with 100 µL of H2O2-free 2× stock developer. The plate was incubated for 20 min at 37 °C with continuous stirring at 300 rpm. The fluorescence intensity of CB-oxidized product was measured at 573–583 nm (excitation at 482–492 nm) in the multimodal plate reader CLARIOstar  using the «Fluorescence intensity» mode.Chlorinating activity of MPO was assayed in 96-well plate: to 100 µL serial 2-fold dilutions of MPO from 125 ng/mL 100 mM Na–acetate buffer, with a pH 5.5, 100 µL of 2× stock developer containing 400 µM CB, 100 µM Hg to separate plasma for the MPO assay. For the SIEFED assay, MPO was adsorbed using monoclonal antibodies against MPO (clone 2F7) obtained by immunizing mice and producing a hybridoma as described earlier [2O2, 2 mM Tau, 5 µM KI, 250 mM NaCl, 100 mM Na–acetate buffer, pH 5.5, were added. The plate again was incubated for 60 min at 37 °C with continuous stirring at 300 rpm. The fluorescence intensity of CB-oxidized product was measured. Adding 250 mM NaCl and 100 mM Na–acetate buffer to the developer solution allowed us to neutralize the inhibitory effect of ceruloplasmin (CP) in plasma [To compare MPO concentration estimated by enzyme-linked immunosorbent assay (ELISA)  and by t earlier . Then, 1n plasma .2O2 generation by neutrophils was measured using the scopoletin/HRP fluorescent technique [6 cells/mL in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2) was supplemented with 1 μM scopoletin (a fluorescent substrate of peroxidase), 20 μg/mL HRP, and 1 mM NaN3  and fMLP at various concentrations was added to activate plasma membrane NADPH oxidase. The H2O2-mediated oxidation of scopoletin was recorded as a decrease of its fluorescence at 460 nm (excitation was at 350 nm) during 20 min at 37 °C using a CM2203 spectrofluorimeter .Hechnique . Briefly5 cells/mL) were incubated at 37 °C in a 96-well plate under continuous stirring  or 106 cells/mL in a quartz cuvette of CM 2203 spectrofluorimeter  in the presence of 20 μM CB and without it in the PBS, containing 1 mM CaCl2, 0.5 mM MgCl2, 5 mM D-glucose, and 20 mM Tau. After a short pre-incubation, cells were activated with PMA (0–100 nM), fMLP (0–2 μM), WGA (50 mg/L), CAA (75 mg/L), PHA-L (100 mg/L), Con A (100 mg/L), SNA (75 mg/L), or SBA (100 mg/L). Activation of neutrophils with fMLP and lectins was preceded by pre-incubation of cells with cyt b (2.5 mg/L) to provide the degranulation of the azurophilic granules. Fluorescent response via fMLP/lectins + cyt b system was compared with the appearance of fMLP/lectins in the absence of cyt b. To minimize the neutrophil proteins’ contribution in the fluorescence assay, the excitation was performed at 460 nm.Neutrophils  in the presence of 2 μM of APF with continuous recording of the fluorescence intensity changes  ,46.In both cases after 20 min of neutrophils stimulation at 37 °C, the fluorescence intensity was compared in the presence and absence of ROS/RHS production inhibitors or their scavengers.5 cells/mL) with various combinations of CB (20 μM) or APF (5 μM) and PMA (50 nM) was applied on the treated coverslips and incubated for 30 min at 37 °C. The glasses were carefully washed with PBS, after which cells were fixed in 4% paraformaldehyde (w/v) within 10 min at room temperature. In the following 5 min preparations were stained with DAPI (0.36 μM) at room temperature in the dark, after which the coverslips were washed two times with PBS and once with deionized water and mounted on the slides using FluoromountTM aqueous mounting medium. Images were obtained with an inverted confocal laser-scanning microscope, LSM 510 META or LSM 510 NLO , supplied with an objective-lens Plan-Apochromat 20×/0.8 M27 (LSM 510 META) or Zeiss Plan-Neofluar 40×/0.75 . Both CB and APF were excited at 488 nm, and emission was registered using a filter cutting off the waves below 505 nm. DAPI was excited at 405 nm (LSM 510 META) or DAPI was two-photon excited at 810 nm (effective excitation at 405 nm) by femtosecond laser radiation  and its fluorescence was registered at 420–480 nm. Images were elaborated by the software provided with the microscope.Neutrophils for confocal microscopy were prepared as follows. Coverslips (18 × 18 mm) were incubated for 1 h in 0.1% poly-L-lysine solution, rinsed with water and dried at room temperature. A suspension of neutrophils , after which they were rinsed with water and dried at room temperature. A suspension of isolated neutrophils was applied on the treated optical Petri dishes and incubated for 30 min at 37 °C. After that samples were treated with 20 μM CB and 20 mM Tau. To view the precise localization of RHS in neutrophils, we combined CB and Hoechst 33,342 for staining the RHS and nucleus, respectively. Hoechst 33,342 was added before the 30-min incubation mentioned above (for better staining). To obtain the maximum RHS output we added PMA at a concentration of 16.2 nM. Images were obtained with an inverted confocal laser-scanning microscope TCS SP5 SMD FLCS . CB was excited at 488 nm, and the emission was registered using a filter cutting off the waves below 515 nm. Hoechst 33,342 was excited at 405 nm and its fluorescence was registered in the range of 425–475 nm (band pass filter 450 ± 25 nm). The scanning started 15 min after PMA was added and lasted for 1 h. Images were elaborated by the software provided with the microscope.TM . Neutrophils (106 cells/mL) were incubated for 30 min with different combinations of CB (20 μM), PMA (50 nM), and ABAH (50 μM), washed by centrifugation , and the samples were analyzed by flow cytometry. Each sample contained at least 10,000 neutrophils, and the data obtained were elaborated using Kaluza Flow 2.3  software.The fluorescence intensity of CB in neutrophils  was measured using a flow cytometer, Naviost-test or one-way ANOVA followed by the posthoc Tukey’s HSD test to analyze differences between means. Values of p < 0.05 were considered to indicate statistically significant differences. To assess the compliance of actual and calculation data with linear dependence, the coefficient of determination R2  was computed.Statistical and graphical processing of data was performed using the software package OriginPro 2016 . The experiments were repeated thrice . The results are presented as mean ± SD, if not stated otherwise. Significant differences between means were identified using Student’s 2 = 0.999) was linear  caused such obvious changes in the fluorescence intensity of CB (Measurements of the fluorescence intensity provided by oxidized CB at varying HOCl concentrations in PBS (pH 7.4) and in its absence allowed assessing the ability of CB to recognize HOCl. Upon CB treatment with 50 μM HOCl, numerous excitation peaks were found at 260, 290, 312, 365 and 429 nm A. The fls linear C. The LOs linear . The tims linear . The selty of CB D. The abAssaying MPO chlorinating activity in the developer solution see  and compThe results of active MPO detection accomplished 30 min after separating plasma from heparin-stabilized blood incubated with PMA and without the activator at 37 °C for 4 h are demonstrated in Neutrophil activation can be triggered by a multitude of stimuli that activate various cellular signaling pathways, which allows one to analyze the mechanisms of neutrophil activation. Several agents, including fMLP and PMA, are commonly used to study neutrophil activation in vitro. PMA, a structural analogue of diacylglycerol, is a non-physiological stimulus. It is able to enter cells and directly activate protein kinase C, followed by a prolonged extra- and intracellular ROS generation ,48. In oOther inhibitors of ROS production were tested, such as SOD, DPI, mannitol and CP. All of them except mannitol suppressed CB oxidation by PMA-activated neutrophils C. The re2•− production by NADPH-oxidase, but not in azurophilic granules’ egress and degranulation [b, which destroys cortical filamentous actin (F-actin) [fMLP is another neutrophil activator that engages the surface formyl peptide receptor (a G-protein-coupled receptor), which results in a short-term Onulation ,54. To iF-actin) .b pre-incubation, the fluorescence of oxidized CB became more intense . Therefore, CB can be used to detect the production of HOCl by neutrophils that is caused by various stimuli.The most active stimulants of HOCl production by neutrophils among the used lectins were WGA and CAA, whereas SNA and SBA were unable to stimulate HOCl production. It should be noted that, as in the case with fMLP, the fluorescence intensity of CB in the suspensions of neutrophils, activated by lectins, increased only when cells had been pre-incubated with cyt Next, the confocal imaging was applied to assess the applicability of CB for endogenous HOCl detection in human polymorphonuclear neutrophils. The neutrophils incubated with CB revealed a very low basal level of fluorescence, most probably arising from their spontaneous activation upon adhesion to the glass a. Upon iIt is clearly known that the prolonged activation of neutrophils with PMA leads to NET formation. Such structures are currently viewed not only as part of anti-pathogen defenses , but alsp < 0.05) when cells were incubated with CB  and selectivity for HOCl and reactive halogenated species over other ROS/RNS . These rThe experiments accomplished showed that the SEIFED method allows using CB for the estimation of MPO concentration in the blood plasma samples activated by PMA. A significant correlation between SEIFED and ELISA has been shown .We also explored CB’s applicability for the detection of endogenous HOCl and reactive halogenated species in a suspension of living cells using receptor-dependent  ,55 and r2O2 production and exocytosis of MPO are prerequisites for an increase in the fluorescence intensity of the probe, which is indicative of HOCl and reactive halogenated species’ production in the cell suspension  and RHS production (as a result of both NADPH-oxidase activation and MPO exocytosis) by activated neutrophils with fMLP and lectins in the absence and the presence of cyt Furthermore, CB allowed viewing the formation of NETs using confocal microscopy. Pre-treatment of neutrophils with PMA led to the release of NETs, marked by an increase of the local CB fluorescence both in separately viewed activated cells and at the sites where intense CB fluorescence co-localized with NETs . It is kIn summation, a novel fluorescent probe CB for selective and specific HOCl and RHS detection is proposed. It is important to note that CB should applied only for in vitro studies, with MPO-containing cells, and control experiments with MPO inhibitors (such as ABAH) need to be used to confirm that any changes of fluorescence are due to MPO-derived oxidants (RHS). In our previous papers we showed the resistance of CB to HOSCN oxidation and a practically equal HOCl reactivity with HOBr , but the


In recent years, pomegranate oil has obtained more attention due to its content of conjugated linolenic acids and possible application in the prevention of many diseases. The purpose of this work was to evaluate the potential ability of pomegranate oil to modulate obesity-related metabolism and immune response using in vitro models. In this regard, pomegranate oil was characterized in terms of fatty acids profile, tocopherols and phytosterols, and antioxidant capacity. After evaluation of the safety profile, pomegranate oil’s capacity to modulate obesity-related metabolism was evaluated through adipolysis and adipokines secretion quantification in 3T3-L1 differentiated adipocytes and hepatic lipid accumulation assay in Hep G2 hepatocytes. The immunomodulatory activity was evaluated in Caco-2 cells by quantification of pro-inflammatory cytokines IL-6, IL-8, and TNF-α. This oil showed high antioxidant capacity and was mainly composed of conjugated fatty acid, namely punicic acid. Its chemical composition was responsible for its capacity to reduce the lipid accumulation in Hep G2 cells and 3T3-L1 differentiated adipocytes. In short, pomegranate oil shows great potential for the development of functional foods and nutraceuticals targeting obesity. Worldwide, obesity’s prevalence has been rising. From a simplistic standpoint it results from an imbalance between energy intake and expenditure that results in an excessive expansion of adipose tissue and a set of subsequent metabolic alterations. These include low-grade inflammation, characterized by higher levels of circulating pro-inflammatory cytokines and fatty acids, which can interfere with normal insulin function and thereby induce insulin resistance, which have been implicated in b-cell dysfunction ,2. IndeePomegranate by-products represent up to 50% of the total fruit weight and can be a sustainable source of phenolic compounds, carotenoids, and conjugated fatty acids . PomegraConsidering this, the purpose of this work was to evaluate the biological potential of organic pomegranate oil in terms of obesity-related metabolism and inflammatory response modulation supported by a detailed composition of key related bioactives.FL assay an activity of 583.04 ± 37.28 µmol Trolox equivalent/mg of oil was registered. Pomegranate oil was rich in PUFAS (1017.57 ± 2.09 mg/g), namely punicic acid (the main fatty acid corresponding to 70%), oleic (7%), and linoleic acid (6.8%) . The fatWhen regarding the genotoxic profile of the sample , no delep < 0.05) higher concentrations of glycerol released (range between 135 to 253 µg/mL) than the control (28 µg/mL), with the highest concentration of PO (20 mg/mL) exhibiting a release of glycerol 9 times higher than the control. The results obtained showed a dose-dependent response , as the p < 0.05) reduced leptin secretion in differentiated adipocytes (p > 0.05) between concentrations being found, as all concentrations promoted reductions of around 88% in leptin secretion. On the contrary, adiponectin secretion was stimulated by the presence of PO, with the lowest concentrations tested (10 and 15 mg/mL) presenting significantly (p < 0.05) higher values than the control and 10 mg/mL in particular exhibiting the highest values of all. Furthermore, in this case, an inverse dose-response was observed, which means that higher concentrations of PO promoted lower secretions of adiponectin. The presence of PO significantly  change the lipidic accumulation observed. The results obtained showed that PO’s presence led to a reduction in hepatic lipid accumulation of around 30% . The effp < 0.05) increased in the presence of PO samples, although the overall increase was relatively low when compared with basal levels, 9% for PO at 15 mg/mL and 5% at 10 mg/mL. On the other hand, a strong significant reduction (p < 0.05) of IL-8 secretion was verified in relation to the basal control, with both concentrations tested exhibiting reductions above 50%. Regarding TNF-α, no significant (p ˃ 0.05) differences were observed when compared with the basal control. The effect on pro-inflammatory cytokines IL-6, IL-8, and TNF-α was evaluated in Caco-2 . In basap < 0.05) reductions in the selected cytokines secretion. The presence of PO represented a significant (p < 0.05) reduction of IL-6 between 31% and 25%  in relation to the stimulated control. In the same way, a significant (p < 0.05) reduction (19%) in IL-8 secretion was observed for 10 mg/mL of PO-treated cells. When regarding TNF-α a significant (p <0.05) reduction of around 80% and 93% in the presence of 15 and 10 mg/mL of PO, respectively, was observed. In short, the presence of PO had a significant (p < 0.05) positive influence upon the inflammatory response in IL-1β stimulated Caco-2 cells. In cells stimulated with IL-1β, PO showed relevant anti-inflammatory effects demonstrated by significant . However, in the available literature, the results reported were related to the DPPH assay with emphasis on the phenolic fraction, and, for this reason, a direct comparison is not possible. In addition to the chemical composition, another important factor for the use of PO as a nutraceutical or functional food ingredient is its safety. In this way, no genotoxicity effects were verified in all tested concentrations. A similar result was obtained by Meerts et al. (2009) using concentrations of PO ranging between 3 and 5000 µg/plate [In the context of obesity, nutraceuticals and functional foods appeared as valuable strategies to complement the classic therapeutic and prevention solutions . In thisµg/plate . Furtherµg/plate ,26. On tµg/plate . These dµg/plate ,30,31.p < 0.05) inhibited the triglycerides accumulation in differentiated adipocytes, in a dose-dependent manner, through the increase in adipolysis. These results are in line with others reported by other authors whom demonstrated that punicic acid (and other conjugated linoleic acids) can increase adipolysis by the activation of peroxisome proliferator-activated receptors PPAR-γ and PPAR-α [p < 0.05) increase in adiponectin secretion, as adiponectin has been described as functioning as an insulin-sensitizing agent , and PO’s capacity to stimulate the production of this adipokine supports its potential use as a bioactive ingredient [p < 0.05) in the presence of PO for all tested concentrations. This is probably due to the up-regulation of PPAR-γ, as animal studies demonstrated that the activation of PPAR-γ-mediated mechanisms resulted in alteration in adiposity, reduced leptin levels and, as mentioned above, higher adiponectin levels [Regarding the bioactive potential of PO, it showed potential to modulate obesity-related metabolism. It significantly  reduced the secretion of IL-6 and IL-8 in non-stimulated cells as well as a significant reduction in all cytokines in in IL-1β stimulated caco-2 cells. This immunomodulatory effect can be related to the CLNAs presented in PO  as they have been considered as potential agonists of PPAR-γ and have a similar effect to those observed for conjugated linoleic acids (CLA) due to similarities in their chemical structural [Concerning the modulation of the immune response of Caco-2 cells, the presence of PO significantly  by Caco-2 cells through saturation reactions catalyzed by nicotinamide adenine dinucleotide phosphate (NADP) [The relationship between CLNAs and CLA is important because CLNAs can be metabolized into CLA by saturation reactions. In vitro studies demonstrated that punicic acid (and other stereo- or regioisomers) can be converted into CLA (namely e (NADP) ,54,55,56e (NADP) ,57,58. Te (NADP) ,60. In re (NADP) . The cape (NADP) . On the e (NADP) . Organic Pomegranate oil was supplied by All Organic Treasure . Tritridecanoin was obtained from Larodan ; HPLC grade methanol, hexane, dimethylformamide (DMF), ethanol, acetone, and dichloromethane were obtained from VWR , and sodium methoxide from Acros Organics . The standards Supelco 37 and CRM-164, trolox, phosphate buffered saline (PBS), 3--2,5-diphenyltetrazolium bromide (MTT), fluorescein, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) sulfuric acid (97%), and dimethyl sulfoxide (DMSO) were obtained from Sigma . Tocopherols and phytosterol standards  were obtained from Extrasynthese , IL-1β from Invitrogen . Random methyl-β-cyclodextrin (RMBC) kindly provided by CycloLab . Dulbecco’s Modified Eagle’s Medium (DMEM) and non-essential amino acids (NEAA) were obtained from Gibco , Fetal Bovine Serum from , Penicillin-Streptomycin-Fungizone from Lonza, , and Calf Bovine Serum (CBS), Iron Fortified from ATCC . Ames FT™ Mutagenicity Test Kit was obtained from Moltox , adipolysis Assay Kit from Abcam (Abcam ab133115), Leptin ELISA kit , Mouse Adiponectin ELISA kit  and ELISA using the Human IL-6 Elisa Kit High Sensitivity  were obtained from Abcam . Legend Max Human Elisa Kit IL-8 and the Legend Max Human Elisa Kit TNF-α were obtained from BioLegend  and BCA Pierce Assay Kit from Thermo Scientific.g; 18 °C; 5 min). The upper layer containing fatty acids methyl esters was collected and analyzed in a gas chromatograph HP6890A , equipped with a flame ionization detector and a BPX70 capillary column . Analysis conditions were as follows: injector , injector and detector temperatures were 250 °C and 275 °C, respectively; hydrogen was used as a carrier gas at a flow rate of 1 mL/min. The oven temperature was initially at 60 °C and then increased to a final temperature of 225 °C. Supelco 37 and CRM-164 were used for the identification of fatty acids. The determination of PO fatty acids profile was carried as previously described by Sousa et al. 2022  with sliFurthermore, atherogenic index (AI), thrombogenic index (TI), and hypocholesterolemic/hypercholesterolemic ratio (HH), concerning lipid quality were calculated according to Alba et al. 2019  using thv/v) in dichloromethane and filtered through a 0.22 µL syringe filter. A sample volume of 5.0 μL was used for the chromatographic analysis. Quantitative analysis of phytosterols was operated using an HPLC system consisting of an Agilent HPLC 1260 connected to a diode array detector . Tocopherols were analyzed using an HPLC system consisting of a Beckham 126 and a fluorescence detector (Varian detector). The separation column was ACE equivalence 5, C18, 250 × 46 mm , the mobile phase was methanol in an isocratic elution, the flow rate was 1.0 mL/min, and the column was kept at constant temperature (30 °C). Tocopherols were detected by fluorescence (excitation at 294 nm and emission at 326 nm) and phytosterols were detected by absorbance at 210 nm. Calibration curves (2.5–0.00025 mg/mL) of pure standards of α-tocopherol (RT: 14.93 min), γ-tocopherol (13.69 min) for tocopherols, and concentrations between (10–0.0625 mg/mL) of β-sitosterol (RT: 21.22 min), and cholesterol (RT: 18.19 min) were used for quantification.Tocopherols and phytosterols were quantified according to the method proposed by Pokkanta et al. 2019  with sliFL) through an adaption of the procedure first described by Poyato et al. 2013 [v/v) solution and added to 4.6 mL of a 7% (w/v) RMCD solution . The mixture was then shaken at 800 rpm in an orbital shaker for 1 h at room temperature. For standards preparation, a Trolox stock solution (1 mM) was prepared in PBS . From here, a Trolox work solution (100 µM) was prepared through dissolution with RMCD, and further diluted with PBS to obtain standards with concentrations ranging from 10 to 80 µM. RMCD solution was also used to dilute samples and as blank. Pomegranate oil’s antioxidant capacity was analyzed with lipophilic ORAC , with 20 µL of the sample (standards or blank) being added to 120 µL of fluorescein (116.66 nM), and equilibrated for 10 min at 37 °C. Afterwards, the reaction was initiated through the addition of 60 µL of AAPH (48 mM), and immediately placed in a microplate reader  with fluorescence being read (485 nm excitation and 580 nm emission), throughout 80 min in 1 min intervals. Results were expressed as µmol Trolox equivalent/mg.The L-ORACSalmonella typhimurium TA98 and TA100 strains were grown overnight, at 37 °C, after which they were placed in an orbital incubator until an OD650 between 1.0 and 1.4 was obtained. Samples were tested at 20, 15, 10, and 5 mg/mL. Two controls were assessed: one with chemical control capable of inducing bacterial mutation and another with water instead of test sample as solvent control. Assays were performed with and without metabolic activation. After 90 min of exposure at 37 °C, in exposure media and under constant stirring, reversion media was added and samples were transferred to a 384-well plate. Plates were then incubated for 48 h at 37 °C after which the number of wells that changed from purple to yellow were counted. According to the kit instructions a positive result (genotoxic effect) equaled an increase of ≥2-fold in a treatment group in relation to the corresponding solvent control.AMES assay was executed in accordance with the OECD guideline 471  using thv/v) of FBS and 1% (v/v) of Penicillin-Streptomycin-Fungizone. Caco-2 cells’ media were also supplemented with 1% (v/v) NEAA. Pre-adipocytes were cultured in DMEM with 10% (v/v) of CBS, Iron Fortified and 1% (v/v) of Penicillin-Streptomycin-Fungizone. All incubations were carried out 37 °C in a humidified atmosphere of 95% air and 5% CO2.Human Caucasian colon carcinoma epithelial cells, Caco-2 (ECACC 86010202), were obtained from the European Collection of Authenticated Cell Cultures. Human hepatocellular carcinoma cells, Hep G2 (ATCC HB-8065), and mouse pre-adipocytes 3T3-L1 (ATCC CL-173) were acquired from American Type Culture Collection. Caco-2 and Hep G2 cells were cultured using DMEM   and allowed to adhere for 24 h. Afterwards, the media was replaced by 100 µL of PO diluted in culture media at various concentrations (5–25 mg/mL). Media with DMSO (40%) was used as negative control and fresh medium as a positive control . After 24 h of exposure, 100 µL of MTT (solution (0.5 mg/mL) was added to each well and the plates were incubated at 37 °C, in the dark. After 2 h, MTT solution was removed and 100 µL of DMSO were added. The plates were shaken protected from light for 10 min and absorbance was read at 570 nm using a microplate reader .The cytotoxicity assay was performed according to ISO 10993-5 , with sl4 cells/mL in 96-well tissue culture plates  and allowed to grow until confluence. Two-day post confluence the media were replaced by differentiation induction media provided in the kit. Following induction, the media were replaced every three days by insulin media until more than 80% of the cells were differentiated. Afterwards, the medium was replaced by PO containing medium at various concentrations (10–20 mg/mL) and incubated for 24 h. Isoproterenol solution (10 µM) was used as a positive control and plain medium as a negative control. The glycerol concentration was measured by adding a glycerol-free reagent to 25 µL of cell supernatants, incubating the mixture for 15 min at room temperature, and measuring the absorbance (540 nm) in a microplate reader .The PO effect upon 3T3-L1 cells adipolysis was performed using the Adipolysis Assay Kit according to the manufacturer’s instructions. Briefly, cells were seeded at 3.0 × 104 cells/mL and differentiated until more than 80% of the cells were differentiated. Afterwards, the medium was replaced by a medium with PO (10–20 mg/mL) and incubated for 24 h. For basal activity plain media were used as control. At the end of the assay, supernatants were collected, centrifuged to remove debris, and stored at −80 °C until further analysis. Adiponectin and leptin detection was performed by enzyme-linked immunosorbent assay (ELISA) using the Mouse Leptin ELISA kit and Mouse Adiponectin ELISA kit according to the manufacturer’s instructions. The protein content of samples was determined using the BCA Pierce Assay Kit. Leptin values were obtained in pg/µg of protein in the sample and adiponectin in ng/ng of protein. Pre-adipocytes (3T3-L1 cells) were seeded at 2.0 × 104 cells/mL in a 96-well plate, were allowed to adhere. After 24 h the media were replaced by fresh media with PO (10–20 mg/mL) or chloroquine (25 μM steatosis induction control). After 72 h of exposure, cells were stained with Oil Red O and the absorbance at 490 nm was measured using a microplate reader . The results were expressed in relative percentage of control production. Hepatic lipid accumulation assay was performed using Hepatic Lipid Accumulation Kit according to the manufacturer’s instructions. Briefly, hepatocytes, seeded at 105 cells/well in 24-well microplates and incubated for 24 h. After 24 h, the culture media was carefully replaced with media supplemented with PO at 15 and 10 mg/mL, and the plate was re-incubated for another 24 h. As an inflammation control, IL-1β was used at 10 ng/mL while for basal activity control plain media were used. At the end of the assay, supernatants were collected, centrifuged to remove debris, and stored at −80 °C for further analysis. Caco-2 cells were seeded at 2.5 × 10Interleukins 6 (IL-6) and 8 (IL-8) and Tumor Necrosis Factor-alpha (TNF-α) detection were performed by ELISA using the Human IL-6 Elisa Kit High Sensitivity, the Legend Max Human Elisa Kit IL-8 and the Legend Max Human Elisa Kit TNF-αaccording to the manufacturer’s instructions. Protein content of samples was determined using the BCA Pierce Assay Kit. Interleukin values were obtained in pg/ng of protein in the sample. Minitab 17  was used to carry out statistical analysis. All data was reported as a mean ± standard deviation. Shapiro-Wilk’s test was used to confirm the normality of data distribution. The results obtained were tested at a 0.05 significance level using a one-way analysis of variance (ANOVA), followed by a multiple comparisons test (Tukey) to identify statistically significant differences.Pomegranate oil can be a valuable ingredient for functional foods or nutraceuticals due to its high content in bioactive molecules, particularly CLNAs. This bioactive oil plays an important role in modulating obesity-related metabolism through the reduction in lipid accumulation. The reduction in lipid accumulation combined with the increase in adiponectin can provide a benefit to the management of insulin resistance. Moreover, their capacity to modulate the immune response makes PO a great source of bioactive molecules to ameliorate a metabolic syndrome clinical condition.


Leishmania braziliensis infection have an exacerbated inflammatory response associated with tissue damage and ulcer development. An increase in the rate of patients who fail therapy with pentavalent antimony has been documented. An adjuvant therapy with an anti-inflammatory drug with the potential of Leishmania killing would benefit CL patients. The aim of the present study was to investigate the contribution of peroxisome proliferator-activated receptor-γ (PPAR-γ) activation by pioglitazone in the regulation of the inflammatory response and L. braziliensis killing by monocytes. Pioglitazone is an oral drug used in the treatment of diabetes, and its main mechanism of action is through the activation of PPAR-γ, which is expressed in many cell types of the immune response. We found that activation of PPAR-γ by pioglitazone decreases the inflammatory response in CL patients without affecting L. braziliensis killing by monocytes. Our data suggest that pioglitazone may serve as an adjunctive treatment for CL caused by L. braziliensis.Patients with cutaneous leishmaniasis (CL) due to  Leishmania braziliensis causes a clinical spectrum of diseases in which cutaneous leishmaniasis (CL) is the most prevalent clinical form. CL lesions present an intense inflammatory infiltrate with the predominance of lymphocytes, mononuclear phagocytes, and a few parasites  is the first drug of choice. However, an elevated rate of therapeutic failure rates has been reported, reaching 70% depending on the clinical form. Moreover, patients in the pre-ulcerative phase of the disease, as well as elderly subjects, are more likely to fail SbV therapy  in association with standard doses (20 mg/kg/20 days) of SbV decreased healing time in refractory CL patients   . To dateLeishmania killing. These findings suggest that pioglitazone is a strong candidate for adjuvant therapy in CL patients.Classically activated monocytes/macrophages produce pro-inflammatory soluble factors such as TNF, IL-1β, IL-6, and MMP9, while the alternative activation induces the expression of CD163, playing a pivotal role in wound healing . CD163 iL. braziliensis DNA by PCR  and the Brazilian Commission of Ethics in Research (2.512.434). All subjects provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki and subsequent revisions. Fifteen CL patients were recruited from an endemic area of leishmaniasis—Corte de Pedra, Bahia-Brazil. Diagnostic criteria consisted of the presence of an ulcerated skin lesion, with no evidence of mucosal involvement, and the detection of A by PCR . A contrL. braziliensis (MHOM/BR/LTCP11245) was obtained from a skin lesion of a CL patient and identified as L. braziliensis by multilocus enzyme electrophoresis , and streptomycin (100 µg/ml) . Leishmania parasites in the stationary phase were used.An isolate of phoresis . FollowiLeishmania antigen (SLA) was prepared from an isolate of L. braziliensis as previously described ] were determined. The acceptable endotoxin concentration was 0 EU/ml. SLA was employed at 5 μg/ml in all assays.Soluble escribed . Briefly6cells in 1 ml of RPMI-1640 (low glucose)  supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) . PBMCs were dispensed into 24-well plates and incubated at 37°C under 5% CO2 for 24 h in the presence or absence of SLA (5 µg/ml), lipopolysaccharide (LPS) (10 ng/ml), Pam3Cys (100 ng/ml), pioglitazone (1 μM) (Sigma-Aldrich), or GW9662 (10 μM) (Sigma-Aldrich).Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by Ficoll-Paque  gradient centrifugation. After being washed in saline, the cells’ concentration was adjusted to 3 × 102 for 72 h in the presence or absence of pioglitazone (1 μM) (Sigma-Aldrich). Cell culture and biopsy culture supernatants were collected and stored at −70°C until use. Cytokine levels were quantified by ELISA, and DNA levels (double stranded) were measured using a spectrophotometer. DNA extracted from the biopsy of patient CL was used as a positive control.CL patients and HSs were submitted to tissue biopsy using a 4-mm punch. Biopsied material was cultured in complete RPMI medium (low glucose) at 37°C and 5% CO6/ml) were maintained in RPMI-1640 (low glucose)  supplemented with 10% FBS , penicillin (100 U/ml), and streptomycin (100 µg/ml). After being washed, cells were infected with L. braziliensis (5:1) in the presence of pioglitazone (1 μM) at 37°C, 5% CO2. After 4 h, non-internalized parasites were removed by washing, and cells were incubated for 24 h. Parasite load was evaluated by optical microscopy, following H&E staining, by 3 different observers at 4 and 24 h post-infection.Monocytes were purified from PBMCs by negative selection using MACS columns . Monocytes were prepared following a method previously described by our laboratory to yield a purity of 99% and identified by flow cytometry as CD14+CD3−CD19− . BrieflyTNF, IL-6, IL-10 , IL-1β, MMP9, and sCD163  levels were determined by ELISA using commercial kits, following the manufacturers’ instructions. Results are expressed in pg/ml.https://go.drugbank.com/) was used to obtain the pharmacokinetics and pharmacodynamics information of pioglitazone . Protein–protein interaction (PPI) network analysis and Gene Ontology (GO) enrichment were performed using STRING v11.0 (https://string-db.org/). All interaction networks were limited to Homo sapiens species, and a minimum confidence score (high confidence: 0.700) was used for the interactions. The interaction networks that presented PPI enrichment p-value <0.05 were considered significant.DrugBank v5 (6 monocytes (HSs) were treated with dihydrorhodamine-123 (DHR) at 10 ng/ml  for 10 min. After that, cells were infected with L. braziliensis (5:1 ratio) for 24 h and then stained with anti-CD14 and anti-MHC-II fluorochrome-conjugated antibodies. The mean fluorescence intensity (MFI) of DHR in the cells was assessed by flow cytometry, and data were analyzed through FlowJo®.To evaluate the reactive oxygen species (ROS), 1 × 106 cells/ml of RPMI-1640 (low glucose)  supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) . PBMCs were dispensed into 24-well plates and incubated at 37°C under 5% CO2 for 24 h in the presence or absence of pioglitazone (1 μM) (Sigma-Aldrich). Monocytes were obtained as previously described. Briefly, 10 × 106 PBMCs was dispensed into 6-well plates and maintained in RPMI-1640  supplemented with 10% FBS , penicillin (100 U/ml), and streptomycin (100 µg/ml). Next, cells were washed two times with saline and maintained in RPMI-1640  supplemented with 10% FBS , penicillin (100 U/ml), and streptomycin (100 µg/ml) for 24 h in the presence or absence of pioglitazone (1 μM).PBMCs were isolated from heparinized venous blood by Ficoll-Paque  gradient centrifugation. After being washed in saline, the cells’ concentration was adjusted to 3 × 106 cells, and cells were incubated for 4 h in the dark. The reaction was stopped, and the formazan salt crystals were solubilized by adding 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Next, the samples and blanks were transferred to 96-well flat-bottomed plates and read in a spectrophotometer at 570 nm. As a positive control, 3% paraformaldehyde (PFA) (Sigma-Aldrich) was used in all experiments.Cell viability was determined by MTT. Briefly, 0.5 mg of Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) was added to 3 × 10in vitro assays were statistically analyzed using GraphPad Prism v8. All the results are shown as mean and SD. The paired t-test was used to compare two dependent continuous variables, whereas three or more dependent continuous variables were compared by the ANOVA test. The results were considered statistically significant when p < 0.05.All data obtained in To investigate potentially relevant interactions of PPAR-γ, we performed a confidence analysis (high confidence 0.700) based on a PPI network, which allowed us to choose inputs (proteins) in addition to providing enrichment pathways (GO) of the generated interaction network. Briefly, we chose 12 proteins  that are known to induce or downregulate the inflammatory response. We then identified five proteins  that directly interact with the nuclear receptor PPAR-γ. Furthermore, GO enrichment analysis showed that PPAR-γ is related to the following biological processes: upregulation of DNA-binding transcription factor activity, cellular response to LPS, inflammatory response, immune response, response to cytokine, and positive regulation of inflammatory response  . To testL. braziliensis induces the expression of TLR4 and TLR2 and the production of inflammatory cytokines by monocytes from CL patients , whereas cytokines such as IL-4, IL-13, and IL-10 and the dexamethasone corticoid induce an “alternative” activation profile (M2) . In CL, L. braziliensis-infected patients. The lack of effect of pioglitazone regarding IL-10 levels in SLA-stimulated PBMCs and lesion cells is somehow different from the results observed in LPS- and Pam3Cys-stimulated cells, where pioglitazone reduced IL-10 levels. The most likely explanation for this disparity is that Leishmania infection may induce cell types other than monocytes/macrophages to produce IL-10. Interestingly, although no decrease in IL-10 was detected, we found a lower parasite load in monocytes between 4 and 24 h of infection in the presence of pioglitazone. We thus evaluated whether pioglitazone treatment would interfere with ROS quantification. Pioglitazone also did not interfere in ROS production within infected monocytes. It has been shown that alternatively activated macrophages can produce ROS  can be found in the article/The studies involving human participants were reviewed and approved by Institutional Review Board of the College of Medicine of Bahia (protocol no. 2.471.185) and the Brazilian Commission of Ethics in Research (2.512.434). The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Institutes of Health AI136862 and CAPES.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Under electrochemical conditions, a series of alcohols (21 examples) produces high selectivity in good yields (up to 100%). Usage the current method, 10 mmol (1060 mg) of benzaldehyde is also successfully reduced to benzyl alcohol  without any by‑products. This route to alcohols matched several green chemistry principles: (a) atom economy owing to the use of H2O as the solvent and the source of hydrogen, (b) elimination of the homogeneous metal catalyst, (c) use of smooth reaction conditions, (d) waste inhibition due to low volumetric of by-products, and (e) application of safe EtOH co-solvent. Moreover, the ability of the system to operate with alkyne and alkene compounds enhanced the practical efficiency of this process.Artificial photosynthesis, an umbrella term, is a chemical process that biomimetics natural photosynthesis. In natural photosynthesis, electrons from the water-oxidation reaction are used for carbon dioxide reduction. Herein, we report the reducion of aldehydes and ketones to corresponding alcohols in a simple undivided cell. This reaction utilized inexpensive nickel foam electrodes (1 cm The existing methods for carbonyl reduction frequently require metals  or stoichiometric reductant reagents  that commonly show limited substrate scope  furan, 2,5- Bis(hydroxymethyl) tetrahydrofuran, and 2,5-dimethylfuran21. Li et al. pointed to mechanisms of electrochemical reduction of furfural on metallic Cu electrodes in acidic media. This mechanism may happen through either electrocatalytic hydrogenation or electro-reduction paths22. In a subsequent study, Attard’s group examined the reduction of ketopantolactone (KPL) and ethyl pyruvate (EP) on platinum electrodes24. Koper and co-workers reported the hydrogenation of acetophenone, 4-acetylpyridine, and acetone at platinum single-crystal electrodes  by water-oxidation reaction is a fundamental electrode reaction owing to its essential applications in electrochemical energy apparatus such as regenerative fuel cells and rechargeable metal-air batteries31. Ru and Ir oxides have been employed in the OER reactions as catalysts, though low abundance and high cost have limited their widespread use32. Recently, increasing attempts have been made to design Ni, Co, Mn and Fe-based transition metal oxides or hydroxides that replaced Ru- and Ir-based OER catalysts39. Ni and Cu foams have been widely noticed as electrodes in OER reactions due to their 3D conductive frameworks and porous structures43. The high porosity and surface area of 3D nanoporous foams show increased conductivity compared to 2D planar electrodes44. For example, Li et al. thermally synthesized a 3D NiO/Ni foam as OER catalysis. The oxidation of nickel compounds under thermal conditions led to the NiO film45. Also, a simple one-step process for synthesizing NiO on the surface of Ni foam during electrochemical alcohol oxidation, without the need for an external thermal or nickel source, is reported in our previous article46.Artificial photosynthesis is a chemical process that biomimetic natural photosynthesis. In the mentioned process, water and carbon dioxide are transformed into carbohydrates and oxygen using sunlightWhile carbonyl compounds are reduced on solid-metal electrodes using expensive homogeneous and heterogeneous metals, we propose an alternative electrochemical methodology without requiring specialized reductants via low-cost OER electrons Fig. c.Changing several electrodes as anode and cathode seriously affected the reaction efficiency Table . Running4 with other electrolytes such as NaCl, LiBr, and KIO3 led to a notable drop in the yield (entries 9–11). Unsurprisingly, no reaction occurred without electrolytes (entry 12). Moreover, by changing the electrolyte to 0.2 mmol, the amount of product decreased to 59% (entry 13). While CH3CN and ethanol were almost equally efficient, the latter choice was for its superior safety profile (entry 14). However, the reaction did not proceed when ethanol was used instead of a mixture of ethanol and H2O (entry 15). Changing the voltage to 2.3 V or 1.3 V decreased the yield of the reaction (entries 16 and 17).Replacing LiClO4 as the supporting electrolyte (0.5 mmol) in H2O: EtOH (6 mL) under Constant- potential conditions in an undivided cell.So, the final optimized conditions for reducing benzaldehyde to benzyl alcohol are as follows: LiClOWith the optimized reaction conditions in hand, we probed a wide range of aldehydes and ketones to explore the ability and compatibility of this electrochemical hydroxylation reaction. As shown in Table o-bromo, o-chloro, and m- chloro benzaldehyde worked well in this system and provided the products  in 75–100% yields. Our results indicated that electron-withdrawing groups such as cyano, chloro, and bromo at the para position afforded 21%, 59%, and 90% isolated yields. Electron-donating groups like isopropyl, methyl, and methoxy at the para position were all converted in 30–100% yields (2 h–2j). The reduction of 2, 6-dichlorobenzaldehyde was more challenging than the 2, 4-dichlorobenzaldehyde due to its satirically hindered. 2, 4-Dimethylbenzaldehyde and 2, 4-dichlorobenzaldehyde provided the corresponding alcohols in 100% yield (2 l and 2 m). Notably, 2, 4-dimethoxybenzaldehyde produced the desired alcohol in a trace yield (2n). The 1-naphthaldehyde and 2-naphthaldehyde substrates provided the desired 2o and 2p in 45% and 57% yield, respectively. As examples of ketones, Acetophenone successfully transformed into 1-phenylethanol in 88% yield (2q), while the reduction of benzophenone failed (2r). No product was detected when cyclohexanone and hexanal were utilized as reaction substrates (2 s and 2t). In all cases, the yield obtained from this method has been very successful compared to the results of previous reports.Simple benzaldehyde could furnish the desired benzyl alcohol in a good yield (2a). Fortunately, 49. However, these reactions often require the use of noble transition-metal catalysts (e.g. Pd and Pt) or Renay-Ni, which is less expensive albeit hard to handle52. The summarized -electrochemical hydrogenation of unsaturated organic compounds reports were collected in a review article by Ye in 202153. Under the present system, styrene and phenylacetylene afforded ethylbenzene in a 99% yield. Further investigation indicates a low level of selectivity found under our method for the C=O bond reduction over C=C hydrogenation. To verify this claim, we investigated the reaction of cinnamaldehyde and 3-phenylpropiolaldehyde. The GC results showed that hydrogenation of C=C was faster than C=O reduction 18.We observed a featureless cyclic voltammogram without any cathodic peak for a mixture of EtOH/ benzaldehyde and EtOH/ H2 V Fig. a. Likewi2O or EtOH), alcohol is the favorite product.Although the mechanism of carbonyl reduction was not discussed herein, the experimental and theoretical study of benzaldehyde reduction by Rousseau and Lilga’s group strongly implied that in protic solvents , benzaldehyde, 4-methoxybenzaldehyde, 4-isopropyl benzaldehyde, 4-chlorobenzaldehyde, 3-chlorobenzaldehyde, 2,4-dichlorobenzaldehyde, 2,6-dichlorobenzaldehyde, 2, 4-dimethylbenzaldehyde, 2, 4-dimethoxybenzaldehyde, cyclohexanone, hexanal, 2-chlorobenzaldehyde, 3-chlorobenzaldehyde, 4-cyanobenzaldehyde, 4-methylbenzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, acetophenone, benzophenone, cinnamaldehyde, and solvents were purchased from Sigma-Aldrich or Merck Company. 3-phenylpropiolaldehyde was synthesized according to the procedure followed by Kim47. The products were characterized by gas chromatography (GC) and thin-layer chromatography (TLC). Preparative thin layer chromatography (PTLC) separations were carried out on 0.25 or 0.5 mm E. Merck silica gel plates (60F-254).All chemical materials were used without further purification. Lithium perchlorate . 13C NMR spectra were measured in CDCl3 at 101 MHz. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiple.The nickel foam (Nanobazar), iron foam , iron-nickel foam , aluminum foil , and titanium foil  purchased from commercial sources. The electrochemical reactions were conducted through a Model JPS-302D tracking dual DC power supply. Cyclic voltammetry analysis was carried out on a Palmsens (Emstat3 +) electrochemical workstation, using a nickel foam as the working electrode, a Pt electrode as a counter electrode, and an Ag/AgCl as a reference electrode. The cyclic voltammogram was recorded at a scan rate of 20 mV/s. GC yield was performed on a Model TOF LC/MS gas chromatograph spectrometer. 4 (0.5 mmol) in 6.0 mL H2O, and EtOH (5:1) were added successively. The electrolytic cell was equipped with nickel foam (1 cm × 1 cm) as an anode and a cathode. The solution was electrolyzed at ambient temperature under a constant voltage (3.4 V). After completing the reaction, the mixture was extracted with ethyl acetate. The obtained organic layer dried over Na2SO4 and was used for GC and NMR spectroscopy.In a 10 ml beaker with a stirring bar, the starting material (0.5 mmol), LiClO4 (5.0 mmol) in 60 mL H2O, and EtOH (5:1) were added successively. The electrolytic cell was equipped with nickel foam (5 cm × 5 cm) as an anode and a cathode. The solution was electrolyzed at ambient temperature under a constant voltage (6.8 V). After completing the reaction, the mixture was extracted with ethyl acetate. The obtained organic layer dried over Na2SO4 and was used for GC and NMR spectroscopy.In a 100 ml beaker with a stirring bar, the starting material (10.0 mmol), LiClOSupplementary Information.


Although the 13 RM Y STRs commonly found in commercial assays provide higher levels of paternal lineage differentiation than conventional Y STRs, there are many male paternal relatives that still cannot be differentiated. This can be improved by increasing the number of Y STRs or choosing those with high mutation rates. We present a RM Y STR multiplex comprising 19 loci with high mutation rates and its developmental validation . The multiplex was found to be robust, reproducible, specific and sensitive enough to generate DNA profiles from samples with inhibitors. It was also able to detect all contributor alleles of mixtures in ratios up to 9:1. We provide preliminary evidence for the ability of the multiplex to discriminate between male paternal relatives by analyzing large numbers of male relative pairs (536) separated by one to seven meioses. A total of 96 mutations were observed in 162 meioses of father–son pairs, and other closely related male pairs were able to be differentiated after 1, 2, 3, 4, 5, 6 and 7 meiosis in 44%, 69%, 68%, 85%, 0%, 100% and 100% of cases, respectively. The multiplex offers a noticeable enhancement in the ability to differentiate paternally related males compared with the 13 RM Y STR set. We envision the future application of our 19 RM Yplex in criminal cases for the exclusion of male relatives possessing matching standard Y STR profiles and in familial searching with unknown suspects. It represents a step towards the complete individualization of closely related males.Rapidly mutating Y-chromosomal short tandem repeats (RM Y STRs) with mutation rates ≥ 10 Y STRs are important genetic markers commonly employed for the detection of male DNA in a female DNA background, paternity testing and lineage searching . Paterna® Y23 System , have mutation rates ≥ 10−2/locus/generation, in comparison to the standard Y STRs with mutation rates of 10−3/locus/generation or lower [Rapidly mutating Y-chromosomal short tandem repeats (RM Y STRs), as found in assays like Yfiler™ Plus  and PowePlexor lower . RM Y STor lower . Furtheror lower . These cor lower . Severalor lower ,9,10, Itor lower , Serbia or lower , China [or lower  and Turkor lower . Using 1or lower  were ablor lower  identifihttp://www.pbscensus.gov.pk (accessed on 6 September 2021)”. Such trends elicit a challenge for the differentiation of related males from Pakistan. The discrimination capacity of Y STR markers can be improved either by increasing the number of markers or by selecting markers with high mutation rates. We have employed both strategies and instigated a multiplex for male differentiation in forensic cases involving males with shared ancestry. Here, we present the developmental validation of a RM Y STR multiplex comprising 12 known core loci [The Pakistani population is endogamous, as 40% of marriages are among first cousins “available online: ore loci  and 7 adhttps://www.ncbi.nlm.nih.gov/ (accessed on 4 January 2019)” and all primer sequences were designed using web-based software Primer3 v4.0, “available online: http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 4 January 2019)”. Forward primer sequences were labeled with a florescent dye compatible with the matrix standards, and all primers were synthesized by ThermoFisher Scientific.A total of 19 loci comprising 12 core RM-YSTRs: DYF399S1, DYF387S1, DYF404S1, DYS526 (b), DYS449, DYS518, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627, along with 7 additional markers: DYS442, DYS635, DYS464, DYS389 (II), DYF393S1, DYS385 and DYS549, were chosen based on their high mutation rates, as published in other populations ,16,17. ABuccal swabs of 302 healthy male individuals  were collected, with informed consent from the Bahawalpur District of Pakistan . The pedTM thermocycler  with the following thermocycler conditions: initial denaturation for 3 min at 95 °C; 30 cycles of 95 °C for 45 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min; a final extension at 60 °C for 10 min and a hold at 4 °C indefinitely. PCR products were scanned with GeneScan™ 500 LIZ® size standard (Applied Biosystems) and Hi-Di formamide and genotyped on a ABI 3130xl genetic analyzer  using the manufacturer’s recommended protocols. Genotyped data were analyzed using GeneMapper ID software, and allele numbers were designated following the guidelines of the International Society of Forensic Genetics (ISFG) [Co-amplification of the 19 RM Y STR markers was carried out in a 5-dye florescence-based multiplex assay, ensuring marker balance and no overlapping allelic size ranges . PolymerValidation studies were performed using male control DNA, including 9948 for precision and allelic designation and 2800 M for the sensitivity, stability and mixture analysis . For sensitivity testing, serial dilutions of 2800 M  were amplified in triplicate where DNA was diluted using autoclaved nuclease-free water. For stability testing, humic acid  was added to the PCRs at concentrations of 25 ng/µL, 75 ng/µL, 85 ng/µL, 100 ng/µL, 125 ng/µL, 175 ng/µL and 225 ng/µL in triplicate and hematin  at concentrations of 50 µM, 65 µM, 85 µM, 100 µM, 125 µM, 150 µM, 250 µM, 500 µM and 1000 µM in triplicate. Precision studies were performed in 10 genotyping runs of 9948 standard DNA by calculating the standard deviations for the fragment size variations at each STR locus. The mixture studies were evaluated by preparing (a) male/female mixtures; keeping the female DNA (9947 A) consistent (300 ng) and male DNA serially diluted ; (b) a mixture of three males at equal concentrations (2800 M and population samples M100 and M103) and (c) various ratios of male/male DNA (2800 M and population sample M103), including 1:1, 3:1, 6:1, 9:1 and 18:1.ix is the (relative) haplotype frequency of the ith haplotype, and n is the total number of haplotypes [PowerMarker software v3.25  was usedplotypes . The mutplotypes . DiffereOptimization of the multiplex was achieved by varying the primer concentrations (0.31 µM–0.65 µM) in a final reaction volume of 8 µL . SensitiTo test the performance of the multiplex in the presence of inhibitors, stability studies were carried out with two inhibition models: hematin and humic acid. The DNA template input amount was 0.5 ng, and the allele peak height threshold was set at 100 RFU. A total of 27 alleles (complete genotype) was obtained at 50 µM, 65 µM, 85 µM, 100 µM and 125 µM concentrations of hematin and at 25 ng, 75 ng, 85 ng, 100 ng and 125 ng concentrations of humic acid. At a concentration of 150 µM of hematin, 19 alleles were ≥100 RFU, while DYS626 dropped to 60 RFU. At a concentration of 250 µM, eight alleles were ≥100 RFU, while three alleles of DYF399S1 were ≤100 RFU, and 13 loci  were completely dropped out. For humic acid at a concentration of 175 ng, 21 alleles were ≥100 RFU, while 6 alleles were entirely dropped out. At a concentration of 225 ng, 18 alleles were ≥100 RFU, and 9 alleles were dropped out completely.Mixture studies were employed to test the reliability of the multiplex for mixed stains. Firstly, a high but constant female DNA concentration (300 ng) was combined with lower male DNA concentrations in a dilution series (mixture a). Full Y STR profiles were obtained down to 0.05 ng regardless of the female DNA. For the mixture of three males at equal concentrations (mixture b), all expected alleles from the three males were present. For mixtures of two males in various ratios (mixture c), all expected alleles from both males were present at all ratios except 18:1, for which 93% of the expected alleles were present.n = 97) was defined by at least one unique haplotype and therefore achieved 100% discrimination capacity (DC) with HD = 0.991512. The global HD of RM Y STRs has been previously reported by Ballantyne et al. in 2014 as 0.9999985, while, from Pakistan, it has been reported to be 0.9921 in the Sindhi, Brahui and Punjabi regions [Each male-rooted pedigree n = 9 was defi regions ,24,25,26−1), and the lowest mutation rate was at DYF393S1 and DYS442 (6.17 × 10−3). However, no mutations were seen at DYS385 (ab) and DYS549, while the average mutation rate over all the loci was 2.92 × 10−2, as shown in Mutation rates were calculated for the 19 RM Y STRs based upon 162 father–son pairs selected from 97 pedigrees. The highest mutation rate was observed at DYF399S1 , two meiosis (brothers and grandfather/grandson), three meiosis (great-grandfather/grandson or uncle/nephew), four meiosis (first cousins and great-uncle/nephew), five meiosis , six meiosis (second cousin) and seven meiosis (second cousin once removed). At least one mutation of the 19 RM Y STR markers was observed in 44%, 69%, 68%, 85%, 0%, 100% and 100% of these cases, respectively, as shown in −2/locus/generation. According to The proportions of pairs that could be differentiated therefore increased with the number of meiosis , with fiIn 2022, Franz and collaborators  were ablThe differentiation potential of 19 RM Y STRs was calculated in 97 pedigrees at all numbers of meiosis. The highest differentiation potential was observed at DYF399S1, DYS626 and DYS547 by the total number of mutations , respectively , whereasThe current data comprises 196 copy number variations at DYF387S1, DYS464 and DYF404S1 . HexapliGenotypes from two paternally related males for DYF404 that typically have two copies on the Y chromosome (CNV = 2) are shown in The newly designed RM Y STR multiplex presented here is a useful complement to conventional Y STR kits for the lineage identification and individualization of paternally related males. Validation studies have revealed that it is a robust, efficient and sensitive system for RM Y STR genotyping. A total of 97 endogamous pedigrees were analyzed with a newly designed multiplex for differentiating related males. This 19 RM Y STR panel shows a high level of differentiation between one and seven meiosis. An overall differentiation rate of 66% was obtained for paternally related male pairs separated by one to seven meiosis in 97 endogamous pedigrees. This is far in excess of the differentiation rates obtainable from commercially available multiplexes (PowerPlex Y23 and Yfiler Plus) and at least as effective as other multiplexes with more STRs. Triplicate, quadruplicate, pentaplicate and hexaplicate alleles were observed at two loci (DYS464 and DYF387S1). Further studies should be conducted for more data in major global populations to gain insights into the haplogroup effects on the mutation rate estimates at these loci. Moreover, carefully ascertained additional Y STR markers and population-specific mutation rates can improve the differentiation power among male relatives as a step towards the complete individualization of paternally related males. This study contributed data to expanding the global databases of RM Y STRs.


Yield loss due to abiotic stress is an increasing problem in agriculture. Soybean is a major crop for the upper Midwestern United States and calcareous soils exacerbate iron deficiency for growers, resulting in substantial yield losses. Fiskeby III is a soybean variety uniquely resistant to a variety of abiotic stresses, including iron deficiency. Previous studies identified a MATE transporter (Glyma.05G001700) associated with iron stress tolerance in Fiskeby III. To understand the function of this gene in the Fiskeby III response to iron deficiency, we coupled its silencing using virus-induced gene silencing with RNAseq analyses at two timepoints. Analyses of these data confirm a role for the MATE transporter in Fiskeby III iron stress responses. Further, they reveal that Fiskeby III induces transcriptional reprogramming within 24 h of iron deficiency stress, confirming that like other soybean varieties, Fiskeby III is able to quickly respond to stress. However, Fiskeby III utilizes novel genes and pathways in its iron deficiency response. Identifying and characterizing these genes and pathways in Fiskeby III provides novel targets for improving abiotic stress tolerance in elite soybean lines. Strategy I plants also release other compounds, including coumarins, which either chelate Fe3+ or reduce Fe3+ to Fe2+ [Soybean is the second most valuable crop grown in the US, with 4.44 billion bushels produced in 2021 from over 87 million acres . Plants zosphere . This re to Fe2+ ,6.Glyma.05G001700, conferred an altered phenotype in iron sufficient conditions when silenced. Glyma.05G001700 encodes a multidrug and toxic compound extrusion (MATE) transporter protein. Glyma.05G001700 is homologous to Arabidopsis DTX14 (AT1G71140), which is known to export the antibiotic norfloxacin [FRD3, which mediates xylem loading of citrate for transport from roots to shoot, ELS1 which is iron responsive and regulates leaf senescence, and a golgi-localized MATE, BCD1, which reestablishes iron homeostasis in cells when it’s been released by stress or physical damage [Fiskeby III is a soybean variety identified from Sweden which exhibits high tolerance to a multitude of abiotic stresses, including −Fe ,9,10,11.floxacin . MATE prfloxacin ,17,18. Il damage ,20,21,22Previous work by our group has shown soybean gene expression can change within 30 min of stress exposure . AdditioGlyma.05G001700. In analyses examining the silencing effect, all comparisons are VIGS_Glyma.05G001700 vs. VIGS_EV grown under the same conditions. Second, the iron treatment effect; the same VIGS treatment (VIGS_Glyma.05G001700 or VIGS_EV at 1D or 7D) in +Fe vs. −Fe. Third, the effect of time; the same VIGS treatment (VIGS_Glyma.05G001700 or VIGS_EV) under either +Fe or −Fe conditions, but identifying differences in expression between 1D vs. 7D of −Fe stress. Combining the findings from these three factors provides more detailed insights into the Fiskeby III iron stress response than each factor alone.The experiment was designed to measure iron stress responses in either VIGS_Glyma.05G1700 or VIGS_EV (empty vector control) infected Fiskeby III leaves and roots exposed to either one day or seven days of −Fe stress . Iron stGlyma.19G001500, was differentially expressed in all four VIGS_Glyma.05G001700 vs. VIGS_EV comparisons in leaves and three of the four comparisons in roots. In all comparisons, silencing Glyma.05G001700 causes significant up-regulation of Glyma.19G001500. Glyma.19G001500 is homologous to Arabidopsis gene AT3G23590 (REF4/Medd33a/Med5a/b), a member of the mediator transcriptional coactivator complex [p < 0.05) of the genes associated with specific expression clusters. Cluster TL1 corresponded to a single significantly overrepresented GO term (p < 0.05) overrepresented with the GO terms very-long chain fatty acid metabolism (GO:0000038) and wax biosynthesis (GO:0010025)  . In leav complex ,31,32,33 complex . The rem GO term , alterna0010025) . No signGlyma.19G001500, which was DE in all but the 1D+Fe comparison, no other DEGs were shared by multiple comparisons  at 1D−Fe, 21 genes were DE at 7D+Fe and a single gene was DE at 7D−Fe . As in lparisons A. Like lparisons A, with TGlyma.17g140400, which was differentially expressed in both VIGS_EV_1D, VIGS_EV_7D, and in VIGS_ Glyma.05G001700_1D. Glyma.17G140400 encodes a lipid transfer protein, the Arabidopsis homolog  is involved in basal defense responses, responding to changing reactive oxygen species levels [Glyma.17G140400 expression was suppressed after 1D−Fe stress but increased after 7D−Fe stress. The increased expression after 7D−Fe stress in Fiskeby III might explain the high level of stress tolerance observed in Fiskeby III. In VIGS_Glyma.05G001700 silenced plants at 1D−Fe genes known to be associated with iron stress responses  were differentially expressed, with NAC73 down-regulated under −Fe stress and the remaining genes up-regulated under −Fe stress. In VIGS_Glyma.05G001700 silenced plants exposed to −Fe stress for 7D, over 50% of the DEGs were associated with phosphate deficiency responses, all of which were down-regulated by −Fe stress.Comparing plants treated with the same VIGS vector (either VIGS_EV or VIGS_Glyma.05G001700) but grown in either +Fe vs. −Fe  revealeds levels . Multipls levels . In our Glyma.05G001700 plays a role in the Fiskeby III iron homeostasis and stress response. Analyzing all 1,097 DEGs from all four comparisons together generated a heatmap with eight expression clusters  deficiency responses, reaffirming the overlapping responses of −Pi and −Fe responses in soybean. One gene, Glyma.04G218900  was up-regulated in −Fe at both 1D and 7D of −Fe stress. In Arabidopsis, GCN5 directly regulates key genes controlling Fe and Pi homeostasis including FRD3 and PHO2 to maintain a steady state of Pi and Fe in cells [In roots, comparing +Fe and −Fe treated plants identified far more DEGs than in leaves at both timepoints  is involved in chloroplast movement . SPX1 reGlyma.05G001700 in Fiskeby III has a dramatic and varied response to −Fe stress at 1D.We were also interested in the 486 root genes unique to 1D and the 130 genes unique to 7D of −Fe stress in VIGS_Glyma.05G001700 silenced plants . We spliAmong the 130 DEGs unique to 7D, there are no over-represented GO categories associated with the 52 up-regulated genes, but there are four over-represented GO categories associated with the 78 up-regulated genes . These GO categories are important for cell wall modification, lignin catabolism, regulating transcription, and cellular phosphate ion homeostasis.Glyma.08G153600, Glyma.08G101500, Glyma.09G115100) and expression profiles are conserved in all four analyses. Glyma.08G153600 has no Arabidopsis homolog but is thought to encode a transmembrane protein. Glyma.08G101500 expression increases over time and is homologous to Arabidopsis gene MRP3, an ABC-transporter induced by cadmium, nickel, arsenic, cobalt, and lead, but not iron [Glyma.09G115100, is homologous to the Arabidopsis BRUTUS (BTS) gene, a major iron regulatory gene [TIFY10A homologs (Glyma.05G027600 and Glyma.09G071600), which are down-regulated in VIGS_Glyma.05G001700 exposed to either +Fe or −Fe while TIFY10B homologs  are only down-regulated in VIGS_Glyma.05G001700 exposed to −Fe. The total 2951 DEGs identified in leaves organize into a heatmap with five clusters  in +Fe or −Fe and compared gene expression differences between 1D and 7D after treatment C. For exnot iron . It is eory gene . Other Dclusters C. Clusteclusters .Comparing 1D−Fe stress to 7D−Fe stress in roots revealed the most DEGs of any analysis, 7608 DEGs in total . HoweverGlyma.05G001700 plays a role in Fe homeostasis under +Fe conditions [Glyma.05G001700 resulted in 136 DEGs in leaves and 21 DEGs in roots when comparing VIGS_EV and VIGS_Glyma.05G001700 at both timepoints and both iron conditions. In both leaves and roots, the majority of DEGs (132 and 21) were identified at 7D in +Fe conditions. A single gene, Glyma.19G001500, was differentially expressed in all but one treatment comparison (1D +Fe in roots). While silencing impacts Glyma.19G001500, it has no effect on its homolog Glyma.05G001600, which is located adjacent to the VIGS target gene Glyma.05G001700. However, in both leaves and roots in +Fe and −Fe conditions and at both 1D and 7D of −Fe stress, silencing Glyma.05G001700 results in significant up-regulation of Glyma.19G001500. Glyma.19G001500 is homologous to the Arabidopsis gene AT3G23590, also known as REF4/Med33a/Med5a/b, hereafter referred to as Med5a/b. Med5a/b is a subunit of the Mediator complex which functions as a central coordinator of plant responses to abiotic stresses, with different subunits playing unique roles for different stresses [Med5a/b results in increased phenylpropanoid biosynthesis [3+ reduction to improve iron availability, highly up-regulated in Fiskeby III roots after 14 days of −Fe stress [PDF1.2 in Clark (iron tolerant soybean) after 6 hrs of −Fe stress [PDF1.1 in Arabidopsis activates ethylene signaling pathways to induce an iron deficiency response in roots [A previous study by our group revealed that nditions . This fistresses . The Medstresses . In Arabynthesis . Flavonoynthesis ,45. Up-rynthesis . Phenylpynthesis . Our pree stress . Med5a/be stress ), which e stress . It is ae stress , suggestin roots . This prGlyma.05G001700 is silenced, it is logical that Glyma.19G001500 plays an important role in the Fiskeby III iron stress response. However, silencing of Glyma.05G001600, which would also silence Glyma.19G001500, failed to induce a phenotypic response in Fiskeby III grown under either +Fe or −Fe conditions [AtbHLH038, AtbHLH039, have failed to result in phenotypic changes. Recently, Assefa, J. Zhang, R.V. Chowda-Reddy, A.N. Moran Lauter, A. Singh, J.A. O’Rourke, M.A. Graham and A.K. Singh [Glyma.05G001700 and Glyma.05G001600  and that silencing both genes simultaneously would significantly alter the Fiskeby III response. Alternatively, Glyma.05G001600 and Glyma.19G001500 may have adopted distinct functions following genome duplication, perhaps for different stress responses. This is the subject of ongoing research in the lab.Given the vast documentation for the role of Med5a/b in Arabidopsis and the up-regulation of the soybean homolog when nditions . TherefoK. Singh  used a gi (GO:0016036). The statistical significance of these GO categories becomes obvious when the individual annotations of the genes are examined. Three purple acid phosphatases, an SPX1 gene, and a phospholipase D gene are all down-regulated under −Fe conditions. Conversely, a TIFY10B transcription factor is up-regulated in −Fe conditions in VIGS_Glyma.05G001700_1D. These changes in gene expression reinforce that Fiskeby III is utilizing phosphate deficiency responses to compensate for the iron-deficient conditions. In addition, these analyses confirm that Fiskeby III was experiencing and responding to −Fe stress within 24 h. Future studies in Fiskeby III will focus on even earlier transcriptional responses to −Fe stress.Analyses comparing iron stress treatment (+Fe and −Fe) revealed fewer total DEGs in leaves (91) but more DEGs in roots (1097) than comparing silencing treatment . Genes in cluster IR6, are associated with eight GO categories involved with photosynthesis  and one associated with detecting biotic stimulus (GO:0009595). These photosynthesis GO categories represent the importance of photosynthesis in the roots and also photosynthetic products being transported to the roots. In cluster IR6 Glyma.04g218900, a homolog of AtGCN5/AtABCF5 is up-regulated by −Fe conditions in VIGS_Glyma.05G001700 silenced plants at 1D and 7D. In Arabidopsis, GCN5 is involved in response to multiple abiotic stresses including maintaining a steady state of Pi and Fe [The 1097 DEGs identified in roots in response to +Fe vs. −Fe comparisons cluster into eight unique clusters B. Clustei and Fe . AtGCN5 i and Fe . Using ai and Fe , AtGCN5 i and Fe ,54,55,56i and Fe . ABA sigBRUTUS (BTS), which exhibits increasing expression as exposure to −Fe stress increases. In Arabidopsis leaves, BTS functions as an iron sensor to regulate energy metabolism in the shoot, while in roots BTS serves as a negative regulator of Fe homeostasis [BTS is up-regulated as the duration of −Fe stress increases (1D to 7D) in both VIGS_EV and VIGS_Glyma.05G001700 plants exposed to both +Fe and −Fe, PYE is up-regulated only in VIGS_Glyma.05G001700 silenced plants exposed to both +Fe or −Fe, but not in VIGS_EV plants. In contrast, TIFY10, a transcription factor that responds to multiple abiotic stresses including bicarbonate stress, −Pi, ozone, drought, high salinity, UV radiation, and cold [TIFY10 is up-regulated early to induce the Fiskeby III Fe- stress response, then returns to normal levels as Fiskeby III achieves homeostasis under −Fe conditions.The majority of the DEGs from this experiment were identified when we compared gene expression after 1D of −Fe stress to expression after 7D of −Fe stress. These comparisons illustrate how Fiskeby III responds to stress over time, the response to a relatively short-term stress exposure (1D) and the response to an extended (7D) stress exposure. In total, 3308 DEGs were identified in leaves and 7662 DEGs were identified in roots. Regardless of the VIGS construct, +Fe plants have more DEGs than −Fe plants. As the plants after 1D and 7D of −Fe stress are the same age, the increase in DEGs in plants moved to new +Fe solutions may reflect the surge of nutrients the 1D +Fe plants access with their new nutrient solutions. In leaves, the largest number of DEGs (2797) are differentially expressed in VIGS_EV+Fe. This is immediately evident from the heatmap , where teostasis . POPEYE eostasis . While Band cold  was downi stress. In the DAS root heatmap  and VIGS_Glyma.05G001700+Fe (4783) . Interes heatmap C, statisbilayers ,63. Whilbilayers , it may bilayers ,24,26,64There was a significant overlap of the statistically significant GO categories in roots and leaves, which is unique for soybean iron deficiency responses. Of the 36 statistically significant GO categories in DL1, 20 were also statistically significant in either DR4 or DR5. Similarly, of the 45 GO categories associated with DL5, 23 were also statistically significant in DR4 or DR5. These overlapping responses could be because these comparisons have not been made before in soybean. Alternatively, the conservation of responses could reflect the differing speed of the response in Fiskeby III compared to Clark (the major soybean line used for −Fe research). The altered timing in Fiskeby III allows us to capture the responses in both leaves and roots simultaneously.To provide additional validation for the genes identified by RNA-seq, we compared the genes identified in this study to genes identified as differentially expressed in the tolerant soybean line Clark exposed to −Fe stress for 30 min, 60 min, 120 min, 1 h, 6 h, 1 day, 2 days, and 10 days ,24,26,50Glyma.05G001700, and how the response of Fiskeby III changes from an initial stress response (24 h) to extended stress (7 days). Through our analyses, we discovered that Fiskeby III invokes all aspects of the canonical soybean iron deficiency response, though the timing of the responses may differ from that of Clark. It was surprising to identify only classic iron uptake/homeostasis genes when comparing 1D vs. 7D of −Fe stress in both leaves and roots. This suggests that upon initial stress exposure Fiskeby III induces a wide array of responses that are subsequently shut off as the period of stress continues.Our previous work showed soybean has a suite of conserved responses identified in −Fe studies, including iron uptake/homeostasis, defense, DNA replication/methylation, and photosynthesis. The majority of earlier soybean −Fe studies have relied on comparing +Fe-grown plants to −Fe-grown plants and measuring gene expression at different timepoints. These previous studies were also limited to the Clark genotype. This study reaffirms the need to conduct these types of studies on a variety of lines to identify new pathways for crop improvement. This study allowed us to not only compare +Fe to −Fe, but also the silencing of our candidate gene, 4*7H2O, 3 mM Mg(NO3)2*6H2O, 2.5 mM KNO3, 1 mM CaCl2*2H2O, 4 mL Ca(NO3)2*4H2O, 0.02 mM KH2PO4, 542.5 μM KOH, 217 μM DTPA, 20 μM MnCL2*4H2O, 50 μM ZnSO4*7H2O, 20 μM CuSO4*5H2O, 0.2 μM Na2MoO4*2H2O, 1 μM CoSO4*7H2O, 1 μM NiSO4*6H2O, 10 μM H3BO3. Full nutrient solutions (+Fe) are provided with 100 μM Fe(NO3)3 while iron deficient (−Fe) solutions are provided with 50 μM Fe(NO3)3. To reflect the calcareous soil conditions 16.8 g of NaHCO3 is added to each hydroponic unit. Finally, all plants are provided a daily solution composed of 10 μmol K2HPO4, 222 μmol NH4NO3, and 0.179 μmol H3BO3. All plants were moved to full nutrient solutions 3). On day 8, plants were rub-inoculated with either the VIGS_EV vector or the VIGS_Glyma.05G001700 vector. The development of these vectors is described in our previous publication [®, Germantown, MD, USA) to release viral particles into the buffer. Unifoliate leaves of Fiskeby III plants were dusted with carborundum, 20 μL of the inoculum was applied, and leaves were rubbed. Gloves were changed between constructs to ensure no cross-contamination. At the time of inoculation, cotyledons were removed from all plants to force them to utilize nutrients provided by the hydroponics, which results in more consistent gene expression results. Post-VIGS infection, all plants remained in a full nutrient solution. Seven days post inoculation 25% of the plants were moved to iron deficient (Fe− (50 μM Fe(NO3)3)) conditions and 25% of the plants were moved to new +Fe conditions, the remaining 50% of plants remained in the original solution. Thirteen days post-inoculation, the remaining 50% of plants were moved with half the plants to −Fe conditions and half the plants to +Fe conditions. All plants were harvested 24 h later. At harvest, all leaf tissue and total root systems were collected in separate 50 mL tubes and immediately flash-frozen in liquid nitrogen. Tissue was stored at −80 °C until RNA extraction.Fiskeby III seeds were germinated in vermiculite and allowed to grow for 6 days. On day 7, plants were transferred to hydroponics, set up as previously described ,66,67,68lication . For rub® RNeasy® Plant Mini Kit , following the manufacturer’s instructions. Any contaminating DNA was removed using the Ambion® TURBO DNA-free kit . Qiagen® RNeasy MinElute CleanUp Kits  were used to further purify and concentrate samples. Sample quality and purity were measured on an Implen NanoPhotometer N60 . RNA was extracted from four biological replicates and the three with the highest quality were submitted for sequencing. RNA was shipped to the University of Minnesota Genomics Center where quality was again checked using an Agilent BioAnalyzer . Libraries were constructed from 500 ng of RNA using the Illumina TruSeq Stranded mRNA kit. Samples were sequenced on a NovaSeq 6000  generating 20 million reads per sample of paired-end reads, each 150 base pairs long. All reads were submitted to the NCBI SRA database under BioProject accession PRJNA878827.RNA was extracted from flash-frozen tissue using the Qiagenhttps://phytozome-next.jgi.doe.gov/, accessed on 25 December 2022) using hisat2 [Reads were loaded into the USDA SciNet high-performance computational system for analysis. Read quality was checked using FastQC . VIGS ing hisat2  with defg hisat2 . Data weg hisat2  for furtg hisat2  and Genog hisat2  were useg hisat2 ,77,78. UArabidopsis thaliana proteins  using BLASTP (E>10–6). The best hit and the most informative hit are reported. Additionally, reported are the gene ontology (GO) annotations associated with each of the Arabidopsis best hits. Over-represented GO terms were identified using custom perl scripts as described in [DEGs identified by these analyses were assigned annotations using custom perl scripts as described previously . The priribed in  using thribed in  with a Bribed in . Transcrribed in .