Selenoprotein glutathione peroxidases (GPX), like ubiquitously expressed GPX1 and the ferroptosis modulator GPX4, enact antioxidant activities by reducing hydroperoxides using glutathione. Overexpression of these enzymes is common in cancer and can be associated with the development of resistance to chemotherapy. GPX1 and GPX4 inhibitors have thus shown promise as anti-cancer agents, and targeting other GPX isoforms may prove equally beneficial. Existing inhibitors are often promiscuous, or modulate GPXs only indirectly, so novel direct inhibitors identified through screening against GPX1 and GPX4 could be valuable. Here, we developed optimized glutathione reductase (GR)-coupled GPX assays for the biochemical high-throughput screen (HTS) of almost 12,000 compounds with proposed mechanisms of action. Initial hits were triaged using a GR counter-screen, assessed for isoform specificity against an additional GPX isoform, GPX2, and were assessed for general selenocysteine-targeting activity using a thioredoxin reductase (TXNRD1) assay. Importantly, 70% of the GPX1 inhibitors identified in the primary screen, including several cephalosporin antibiotics, were found to also inhibit TXNRD1, while auranofin, previously known as a TXNRD1 inhibitor, also inhibited GPX1 (but not GPX4). Additionally, every GPX1 inhibitor identified  showed similar inhibitory activity against GPX2. Some compounds inhibiting GPX4 but not GPX1 or GPX2, also inhibited TXNRD1 (26%). Compounds only inhibiting GPX4 included pranlukast sodium hydrate, lusutrombopag, brilanestrant, simeprevir, grazoprevir (MK-5172), paritaprevir, navitoclax, venetoclax and VU0661013. Two compounds (metamizole sodium and isoniazid sodium methanesulfate) inhibited all three GPXs but not TXNRD1, while 2,3-dimercaptopropanesulfonate, PI4KIII beta inhibitor 3, SCE-2174 and cefotetan sodium inhibited all tested selenoproteins (but not GR). The detected overlaps in chemical space suggest that the counter screens introduced here should be imperative for identification of specific GPX inhibitors. With this approach, we could indeed identify novel GPX1/GPX2- or GPX4-specific inhibitors, thus presenting a validated pipeline for future identification of specific selenoprotein-targeting agents. Our study also identified GPX1/GPX2, GPX4 and/or TXNRD1 as targets for several previously developed pharmacologically active compounds. At moderate levels, ROS, like hydrogen peroxide and the hydroxyl radical, can cause substantial cellular damage by reacting with DNA, lipid membranes, and proteins, leading to tumorigenesis ,2. At hiBecause of the role of glutathione peroxidases in controlling hydroperoxide levels, counteracting oxidative stress, and regulating cell death pathways, inhibitors of GPXs have promising potential as therapeutics for several malignancies. Specific inhibitors will be essential to fully characterize the effects of GPX inhibition, and to validate the relevance of isoform specific targeting as anti-cancer treatments. However, due to the technical difficulties in producing active selenocysteine (Sec)-containing recombinant GPXs due to highly intricate selenocysteine translation machineries, large screening efforts have not been attempted and very few specific drug-like inhibitors have been identified .l-buthionine sulfoxamine (BSO), or selenoprotein translation machinery inhibitors, such as statins, are indirect inhibitors of GPX activity  and BSA [0.01%]) was added to columns 3–48 of 1536-well plates using a BioRAPTR Flying Reagent Dispenser (Let's Go Robotics (LGR), Carlsbad, CA). A no-enzyme control (0.01% BSA in TES assay buffer) was added to columns 1–2. Compounds  were added to each well with a pin tool , and the plates were incubated for 30 min at room temperature before 1 μL of master mix  was added. 1 μL of cumene hydroperoxide (CHP) ([0.5 mM] in 50% EtOH) was then added within 5 min to initiate the reaction. Fluorescence at 340/450 nm was measured using a ViewLux multimodal detector  at initial t = 0 and endpoint t = 15 min (GPX1) or t = 20 min (GPX4).Follow-up confirmation assays were also run in 96-well format using medium binding clear 96-well SpectraPlate microplates (PerkinElmer) and NADPH absorbance at 340 nm was measured every 30 s for 30 min using an Infinite® M Nano (Tecan).2.2.2To identify false positive compounds that interfere with the primary assay through inhibition of GR, ‘hits’ from the primary screening were run against a GR counter assay to identify this possible off-target inhibition C. Briefl2.2.3As an orthogonal assessment of GPX inhibition, a monobromobimane endpoint assay was used E. Briefl2.2.4TXNRD1 activity was assessed using a modified 1536-well TXNRD1 inhibition assay, based upon TXNRD1-catalyzed selenite reduction as previously described D [53]. B2.2.5Thermal stabilization of enzyme ± DMSO or ± compound was carried out in a Prometheus NT.48 instrument (NanoTemper Technologies) with an excitation wavelength of 280 nm. Intrinsic fluorescence at 330 nm and 350 nm was monitored while heating. Capillaries were filled with ∼10 μL of a solution of GPX1/4 (1 mg/mL), ± small molecule (100 μM) or DMSO, in TES buffer and placed into the instrument. The capillaries were subjected to a temperature range from 30 to 90 °C at a ramp rate of 2 °C/min. The ratio of the recorded emission intensities (350nm/330 nm) were plotted as a function of temperature. This ratio and first derivatives were calculated using the NanoTemper software and were plotted using GraphPad Prism.2.3Percent activity was determined by first calculating delta RFU , and normalizing enzyme activity using median delta RFU values for DMSO-treated enzyme control, and DMSO-treated no-enzyme controls.50 values, efficacy , and AUC (area under the curve), as previously described [50 value of ≤20 μM.Compound inhibitory activity in the qHTS assays was determined by plotting dose-response data for each sample and fitting with a four-parameter logistic fit to assess ICescribed . Hits weCompounds were further clustered and analyzed using TIBCO Spotfire 11.4.4  based on activity from both primary and follow-up screens, and figures and final curves were generated using log(inhibitor) vs. response for four-parameter variable slope GraphPad Prism version 9.3.1 .HTS data and assay information can be found at PubChem using ID numbers 1845191 for the GPX1 HTS and 1845192 for the GPX4 HTS.33.12O2  and MSA (IC50: 1.44 μM)  showed inhibitory activity in the GPX4 biochemical assay  were selected and tested against the optimized assay. Of the five GPX inhibitors noted in the literature ,42,54 on1.44 μM) E. MSA inal assay E, as repectively , Fig. 3EThe GR counter screen assay C was opt50 of 1.94 μM in the GR-coupled assay, and 5.3 μM in the mBBr assay). The lower GSH concentrations required similar incubations, whereby mBBr was added to react with remaining GSH at t = 15 min for both enzymes (Supp. Monobromobimane (mBBr) is a heterocyclic compound that reacts readily with low molecular weight thiols, like glutathione . In its es Supp. B. Perfores Supp. D.3.21280 library at five concentration points, and the MIPE 5.0, NPC 2.0, and NPACT libraries at seven concentration points, totaling 11,892 unique small molecules, using an automated screening platform. During the screens, plates were read at t = 0 min and t = 15 or 20 min for GPX1 and GPX4, respectively. A total of 64 positive control (enzyme + DMSO treatment), and 64 negative control (no enzyme + DMSO treatment) wells were included in each 1536-well plate to monitor assay performance. The activity of each compound was measured as the change in signal from an initial (t = 0) read and was normalized against control wells. Using Δ15 min signal for GPX1, the HTS assays resulted in an average Z′ of 0.67 ± 0.05, and a S/B of 6.7 ± 1.4 . Analysis of the CRCs resulted in 180 high-quality GPX1 actives  showed moderate inhibition of GPX1, reducing activity to 49% at the top dose (not shown).Auranofin was present in the screening libraries and inhibited GPX1 with an IC3.3The top compounds showing activity in the primary screen were reacquired, replated, and screened in an 11-point dose-response with freshly QC'ed stock to confirm activity. Of the 377 initial hits, 40 inhibited both GPX1 and GPX4 . Several secondary assays and counterscreens were selected to triage the initial screening hits, as summarized in the triage schematic in A GR activity assay was then run to identify ‘false positive’ compounds from the screen that interfered with the coupled primary assays. 94 compounds were found to inhibit the GR assay and were excluded from subsequent analysis. Of the 49 reconfirmed GPX1 hits, 24 (49%) of the compounds were GR actives, 54 of the 132 GPX4 actives showed activity in the GR assay (41%), and 59% of the pan-active hits were active in the GR assay B–C.The 121 compounds now remaining were subjected to further testing in a TXNRD1 activity assay to identify specific inhibitors of each GPX, pan-GPX inhibitors, and potentially also broad Sec-targeting compounds. Of the 121 GPX inhibiting compounds, 30 were found to inhibit TXNRD1 as well. This amounted to 14 of the 20 remaining GPX1 inhibitors and 11 of the remaining 42 GPX4 inhibitors , and 5 of the compounds inhibiting both GPX1 and GPX4. We found four compounds that showed pan-GPX inhibition and TXNRD1 inhibition, but not GR, suggesting they may be specific Sec-targeting agents.The 40 compounds remaining were then assessed for GPX activity in the orthogonal endpoint MBBr assay, and the inhibitory activity of all compounds tested was confirmed. Taken together, the assay panel suggested that the activities of these 40 compounds were indeed directly dependent on the GPXs, and not the coupling enzymes or assay formats B–C.A total of 5 GPX1-specific compounds, top 9 GPX4-specific compounds, and 2 pan-GPX active compounds  were then assessed in the final assays.3.4Previous work has shown similarities in the substrate specificities of GPX1 and GPX2, and a more unique substrate profile of GPX4 . The finFor complete activity profiles of the top novel inhibitors encompassing five GPX1 inhibitors (+ a reconfirmation of auranofin), nine GPX4 inhibitors, two pan-active GPX inhibitors, and pan-selenoprotein inhibitors see 3.5m) at which 50% of the enzyme is degraded [m for GPX1  in GR-coupled assays. All hits were additionally tested for inhibitory activity in a GR activity assay to remove any false positives from the primary screen, and subsequently also in TXNRD1 and GPX2 assays.Here, we report the development and implementation of several primary and secondary assays in 1536-well format, as well as several characterization assays, for the identification of novel small molecule inhibitors of GPXs from high-throughput chemical libraries. These assays used Sec-containing enzymatically active human recombinant GPX1 and GPX4, produced and purified using a novel method in After screening 11,892 compounds against the optimized qHTS GPX1 and GPX4 assays, hit compounds were defined as having >50% inhibitory activity at the top concentration. Auranofin was present in three of the libraries screened and was a high-quality active hit for GPX1 in all three, representing the high reproducibility of this assay. RSL3 and ML210 were present in the HTS libraries, but as expected were not active hits in either screen . Of the Of the novel GPX4 inhibitors we found in the HTS, only 8% showed cross-inhibition with TXNRD1, suggesting the promiscuity of the FINs (RSL3 and ML210) may not be representative of future development of specific GPX4 inhibitors. Surprisingly, almost a third of all GPX1-specific inhibitors also showed high-quality inhibition of TXNRD1, which suggests a much larger overlap in chemical space for inhibition of these two enzymes, or alternatively a common mechanism of action against the Sec residue. If the latter is the case, this suggests that the Sec residue of GPX1 is more accessible to compounds than that in GPX4. It is well known that the Sec residue of TXNRD1 is solvent exposed and easily reacts with electrophilic compounds . It is iFollowing triage assays, auranofin  and five novel compounds were identified as GPX1/GPX2-specific inhibitors. Of these six compounds, auranofin, omapatrilat, and tenatoprazole showed significant shifts in the melting temperature of GPX1 and can thus be considered direct binders of the enzyme. The two cephalosporins, cefoxitin sodium salt, and ceftibuten did not show any thermal stabilization. The final three compounds do not share structural similarity, nor overlap in mechanism of action. Omapatrilat was developed as a dual neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibitor that has been experimentally assessed as a hypertension treatment, while tenatoprazole was a clinical candidate for peptic ulcer and reflux functioning as a proton pump inhibitor. Based on our findings, targeting of GPX1 and/or GPX2 could also be considered as a potential off-target mechanism of action for these compounds.The GPX2 isoform of human selenoprotein GPXs is found in epithelial cells and has similar substrate specificity with GPX1, but not as much with GPX4 . Additioin vivo remains to be investigated.We identified 13 novel biochemical inhibitors of GPX4. These inhibitors proved less promiscuous in our suite of assays, showing little to no cross-inhibition with GPX1, GPX2, or TXNRD1. Although the final high-quality inhibitors were varied in mechanism of action and structure, several categories of structurally similar hits were identified, helping to validate the assay and pipeline. Unsurprisingly considering the affinity of GPX4 for phospholipid hydroperoxides, the largest category of inhibiting compounds contained fatty acids and surfactants. These tended to inhibit primarily at top doses and were not considered high-quality hits, however several of the final hits, namely lusutrombopag and pranlukast, contain longer carbon chains. Of the more drug-like classes, these included three NS3-4A serine protease inhibitors produced for treatment of hepatitis C: simeprevir, grazoprevir, and paritaprevir. Furthermore, the Bcl-2 family protein inhibitors, navitoclax and venetoclax, both showed potent GPX4 inhibition. The final set of GPX4 inhibitors included GSK-3965 and CDIBA, liver X receptor (LXR) agonist and a cytosolic phospholipase A2 (cPLA2) inhibitor, respectively. The remaining top high-quality hits, namely brilanestrant and VU0661013 were considered singleton hits and remain structurally different from the other compounds. Important to consider when assessing these hits is our choice to use the substrate CHP in our assay. This helped to facilitate the difficulties of HTS, so these hits have not been assessed using other lipid hydroperoxides as GPX4 substrates . If GPX4 inhibition contributes to the pharmacological effects of any of these compounds when used in vivo for these compounds should be assessed next, with a focus on GPX inhibition. This is especially important considering that the chemical libraries utilized here encompassed compounds having previously confirmed pharmacological activities and presumed mechanisms of action. GPX inhibition has, however, typically not been considered as a possible activity for most of these compounds.The purpose of this study was to establish an HTS discovery pipeline for GPXs and to evaluate the workflow for identifying specific probes inhibiting GPX1 and GPX4. Though the compounds screened have other established targets and are known to be promiscuous, and were selected for that purpose, the identification of auranofin, a known GPX1 inhibitor, using these assays helps to validate the screening process. During this pilot, many compounds were identified that interfere with GR and TXNRD1 in secondary assays, demonstrating the relevance of these follow-ups to exclude false-positive hits and more general Sec-targeting activity. In total, 20 novel GPX inhibitors, two of which were pan-GPX inhibitors, five inhibited GPX1 (and GPX2) and 13 specifically inhibited GPX4. Furthermore, the overlap in compounds that inhibit GPX1 and GPX2 suggests the importance of assaying GPX2 inhibition, and possibly other GPX isoforms, to fully characterize GPX1 inhibition. With our studies confirming several compounds as inhibitors of GPXs, the effects and possible mechanisms of action Our study provides a basis for future identification, development, and characterization of novel GPX inhibitors for the potential treatment of malignancies by establishing a series of robust assays designed for detecting and triaging hits from high-throughput small molecule library screens.DMC, ESJA, and MDH conceived the study and designed the experiments. DMC performed the experiments. DMC and QC produced recombinant proteins, including GPXs and TXNRDs. JT and CKT assisted with automation and library screens. HG and MS analyzed the data. DMC wrote the final manuscript, which was revised by QC, HG, JT, CKT, MS, ESJA, MDH.10.13039/100006108National Center for Advancing Translational Sciences, National Institutes of Health. DMC was supported through a joint NIH-Karolinska Insitutet graduate training program. ESJA acknowledges funding from 10.13039/501100004047Karolinska Institutet, The Knut and Alice Wallenberg Foundations (KAW 2019.0059), The 10.13039/501100002794Swedish Cancer Society (21 1463 Pj), The 10.13039/501100004359Swedish Research Council (2021–02214), National Laboratories Excellence program under the National Tumor Biology Laboratory project (2022–2.1.1-NL-2022-00010) and the Hungarian Thematic Excellence Programme (TKP2021-EGA-44) and The 10.13039/501100011019National Research, Development, and Innovation Office (NKFIH) grant ED_18-1-2019-0025.This work was partially supported by the intramural research programs of the ESJA and QC are shareholders in Selenozyme AB, a company selling recombinant selenoproteins.


As a central epigenetic modification, histone acetylation affects the expression of genes with a wide range of functions across all life forms . HistoneThe Arabidopsis genome harbours 18 HDAs, which are organized in three superfamilies comprised of the HDA classes 1-4 . HISTONE35S::HDA19-GFP plants.In this Data Report, we provide an immunoprecipitation (IP)-based analysis of the HDA19 interactome in etiolated Arabidopsis seedlings. HDA19 is directly involved in hypocotyl elongation during photomorphogenesis and, based on our findings, also in hypocotyl elongation during skotomorphogenesis . The exaArabidopsis thaliana Col-0 and the transgenic line 35S::HDA19-GFP were used in this study. 35S::HDA19-GFP lines have been described previously 2, 2.5 mM KH2PO4, 70 μM H3BO3, 14 μM MnCl2, 1 μM ZnSO4, 0.5 μM CuSO4, 0.01 μM CoCl2, 0.2 μM Na2MoO4, and 40 μM Fe-EDTA, solidified with 0.4% Gelrite Pure. MES (1 g/L) and 1.5% (w/v) sucrose were added, and the pH was adjusted to 5.5 with KOH. Seeds were stratified on plates for 2 days at 4°C in the dark and grown at 22°C in vertical position in the dark with 70% relative humidity.eviously . Seeds wExperiments were carried out with the μMACS Epitope GFP tag protein isolation kit (MACSmolecular) following the manufacturer’s instructions with minor modifications. Hypocotyls were collected by dissection in dark conditions within ca. 5 minutes per plate (~25 seedlings). Samples from each plate were frozen immediately in liquid nitrogen. A total of 25 plates were used to obtain 0.5 gram of tissue. Hypocotyls were ground with liquid nitrogen and resuspended in 500 μL of extraction buffer , 1 mM PMSF, and 50 μM MG132). The samples were incubated on ice for 30 minutes with occasional mixing and centrifuged for 20 minutes at 10,000 x g at 4°C. The supernatants were individually collected in fresh tubes, and 400 μL of each input was added to 50 μl of anti-GFP microbeads and incubated for 1 hour and 30 minutes in a mixer set to 60 rpm at 4°C, while 100 μL of each input was kept to be used for Western blots using anti-GFP as a control. The samples were eluted in 50 μL denaturing elution buffer supplied with the kit.2O) and elution buffer 3 (50% acetonitrile (ACN) in ultrapure water). The column was centrifuged at 4,000 g for 2 min and the elution solution was collected in a new tube, dried by speed vacuum, resuspended in 100 µL of 0.1% formic acid, desalted, and loaded into a C18 Ziptip pipette tip. The elution was dried down under vacuum, the pellet was re-dissolved in 10 µL of 0.1% (v/v) formic acid (FA) with 3% (v/v) acetonitrile (ACN), and the liquid chromatography was performed by injecting 4 µL of sample in the LC-nESI-Q Exactive mass spectrometer model (Thermo Fisher Scientific) coupled with an on-line nanoUHPLC (Dionex UltiMate 3000 Binary RSLCnano). The Acclaim PepMap 100 C18 trap column  and the Acclaim PepMap RSLC C18 nano LC column  were used to deliver solvent and separate tryptic peptides with a linear gradient from 5% to 35% of acetonitrile in 0.1% (v/v) formic acid for 60 min at a flow rate of 300 nl/min. The acquisition cycle for MS data was performed in the data-dependent mode with a full survey MS scan followed by 10 MS/MS scans of the top 10 precursor ions from the scan. The mass spectrometer was operated in full scan mode  in the Orbitrap analyser at a resolution of 70,000. Data-dependent MS/MS acquisitions were performed with a 2 m/z isolation window, 27% NCE , and 17,500 resolving power.The protocol was conducted as described previously . BrieflyRaw data were analysed with the Proteome Discoverer™ Software 2.2 (Thermo Fisher) using the Sequest search algorism. The Arabidopsis protein database (Araport11) was used to conduct the searches; only high confidence proteins were selected for the analysis. All peptide spectrum matches were filtered with a q-value threshold of 0.05 (5% FDR), and the proteins were filtered with high confidence threshold . Nuclear proteins identified in more of two biological replicates were considered as putative interacting partners of HDA19. Localization of the proteins was gathered from published experimental evidence or–in cases where such information was unavailable–prediction inferred from the Subcellular Location of Proteins in Arabidopsis Database (SUBA).Gene Ontology (GO) analysis was conducted using the AgriGO v2.0 toolkit web-server . Signifihttps://string-db.org). Only nucleus-located partners of HDA19  expression through histone deacetylation (A protein-protein interaction (PPI) network constructed from this subset of proteins shows a suite of well-known partners of HDA19, including five members of the SIN-LIKE (SNL) family. SNL proteins were shown to be involved in the repression of AP2 family transcription factors that repress tylation . We alsotylation . A bimoltylation , which wtylation , and PROtylation  were ideThe current dataset identified a large suite of putative novel interacting partners of a key regulator of plant development and stress responses, HDA19. The identification of SNL members, HDC1, and histone H1 can be considered as validation of the current IP assay. A surprisingly large subset of (predicted) non-nuclear proteins was identified with high confidence, suggesting that some of these proteins may transiently associate with chromatin. Besides expected binding partners such as HCD1, H1, and SNLs, we found that HDA19 interacts with proteins involved in chromatin remodelling, nuclear protein export/import, protein ubiquitination associated with DNA damage repair, and chloroplast-nucleus signalling, suggesting a wide range of largely unexplored functions of HDA19 in etiolated Arabidopsis seedlings.http://www.ebi.ac.uk/pride/archive/projects/PXD045454.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner WS: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. IV: Conceptualization, Data curation, Formal Analysis, Methodology, Visualization, Writing – review & editing.


The forthcoming generation of materials, including artificial muscles, recyclable and healable systems, photochromic heterogeneous catalysts, or tailorable supercapacitors, relies on the fundamental concept of rapid switching between two or more discrete forms in the solid state. Herein, we report a breakthrough in the “speed limit” of photochromic molecules on the example of sterically-demanding spiropyran derivatives through their integration within solvent-free confined space, allowing for engineering of the photoresponsive moiety environment and tailoring their photoisomerization rates. The presented conceptual approach realized through construction of the spiropyran environment results in ~1000 times switching enhancement even in the solid state compared to its behavior in solution, setting a record in the field of photochromic compounds. Moreover, integration of two distinct photochromic moieties in the same framework provided access to a dynamic range of rates as well as complementary switching in the material’s optical profile, uncovering a previously inaccessible pathway for interstate rapid photoisomerization. Rapid switching between discrete states in the solid state is a cornerstone for the technological development of devices based on stimuli-responsive materials. Here authors break the speed limit of common classes of photochromic molecules through tailoring framework environments. However, even in solution, where the molecules exhibit more degrees of freedom in comparison with the solid state, the rate constants of such processes are typically varied from 10−5 to 100 s–1 using light as an external stimulus.14 By utilizing solvent polarity and viscosity as variables, the speed of the photoisomerization process can be enhanced, but typically within one order of magnitude.15 In the solid state, switching between distinct states is usually further constrained compared to solutions due to close packing, π-π stacking, or hydrogen-bonding interactions, especially for molecules for which isomerization is accompanied with large structural transformations or formation of zwitterionic species .17Rapid switching between two (or more) discrete states in the solid state is a cornerstone for the technological development of devices based on stimuli-responsive materials. For example, the development of on-demand-activated drug delivery platforms, photochromic heterogeneous catalysts, molecular motors, recyclable and healable materials, artificial muscles, multilevel anticounterfeiting and information encryption systems, and tailorable supercapacitors fully relies on fast changes between distinct states occurring in the solid state.18 as a variable or by grafting certain types of photochromic molecules to a specific substrate14, the presented conceptually different approach realized through construction of the spiropyran environment results in ~1000 times switching enhancement even in the solid state compared to its behavior in solution, setting a record in the field of photochromic compounds , but also environment tunability due to matrix modularity. Furthermore, this approach led to a drastic enhancement of photoswitch isomerization ability in the solid state, addressing challenges associated with limited photoisomerization due to strong intermolecular interactions typically pronounced in the solid state and impeding development of stimuli-responsive materials. To the best of our knowledge, such a rapid response observed in the solid state, through integration of spiropyran derivatives into the confined solvent-free space of rigid frameworks, has no analogs for these classes of photoswitches either in solution or in the solid state. While slight enhancements in photoisomerization rate could be anticipated and achieved using organic solvent23 Selection of the previously studied compounds with reported photophysics was an avenue to deconvolute the influence of sample preparation and instrumentation  on the targeted photophysical performance, especially considering the importance of wavelength selection for light-responsive molecules.23Comparative analysis of the photochromic responses was performed for compounds belonging to three distinct classes possessing different photoisomerization mechanisms: spiropyran, hydrazone, and diarylethene derivatives Fig. . Photois24 They undergo isomerization between neutral  and charge-separated  forms through excited-state C–O bond cleavage. Formation of the cis-merocyanine isomer through C–O bond cleavage is followed by thermal relaxation and rotation to the trans-merocyanine isomer )50 and post-synthetic modification57 that includes integration of targeted molecules after the scaffold synthesis. In the de novo approach, photoresponsive moieties were incorporated as pillars between two-dimensional layers constructed from zinc paddlewheel nodes connected by tetratopic H4DBTD linkers -−4,4″-dicarboxylic acid, Fig. 6O4(OH)4(BPDC)6 , which possesses “defects”,  spectroscopy performed on the digested (destroyed in the presence of acid) MOF samples (DBTD) was carried out using the de novo approach (see Supplementary Note 4− layers connected through Zn2(O2C–)4 nodes, enables photoisomerization of sterically-demanding 3 by installation of 3 as pillars with photochromic moieties extending into the MOF pores (DBTD) matched the pattern simulated based on single-crystal X-ray diffraction data  confirmed integration of 2 within UiO-67, resulting in a framework containing 0.1 photoswitches per 10 metal nodes . Preparation of Zn19 For instance, the MC-to-SP conversion can be classically treated with a monoexponential decay function as shown before.61 We compared the behavior for photoswitches integrated within the framework with the results obtained for the same photoswitches in solution. Notably, all solid-state samples  were prepared such that the thickness of the samples did not exceed the penetration depth of the excitation wavelength. We analyzed the photoisomerization rates in the same solvents which were used for the framework synthesis, i.e., a photoswitch in solution and inside the MOF pores was surrounded by the same solvent molecules. Table –2 s−1) and Zn2(3)(DBTD) (5.4 × 10–2 s−1) are similar to the rates of 2 (3.6 × 10–2 s−1) and 3 (8.0 × 10–2 s−1) observed in N,N-dimethylformamide (DMF). Both spiropyran-based photoswitches integrated within the porous scaffolds exhibit reversible photoisomerization which also coincides with their behavior detected in solution, demonstrating that solution-like photoisomerization can be maintained in bulk solid materials. As a next step, we evaluated the possibility to modulate the photoisomerization kinetics of spiropyran derivatives through changes in solvent viscosity and polarity. Based on literature precedent, we anticipated that the presence of a polar solvent would stabilize the zwitterionic merocyanine isomer and impede the MC-to-SP isomerization process.63 For instance, aqueous solutions of spiropyran derivatives are known to exist primarily in the merocyanine form because of the stabilizing hydrogen-bonding interactions.64 Likewise, the presence of viscous solvent can inhibit the structural rearrangement upon spiropyran isomerization.65 Indeed, we observed slight changes in the rate constant of MC-to-SP conversion depending on solvent choice  to 31.2 ± 0.5 s–1 (UiO-67 + 2)), i.e., led to ~1,000-fold enhancement in comparison with the values measured in solution as demonstrated in Fig. 67 or our studies performed for the exact same photoresponsive molecules, 2 and 3 (vide supra). A comparison of the found rates of related compounds reported in the literature and the rates obtained herein is reported in Supplementary Table Since we observed that the solvent  can impede the photoisomerization process by ~4-fold . Similar to 2 and 3, the isomerization of 5 and 6 requires significant structural transformations, however, it does not occur through formation of zwitterionic species  was used to verify and quantify photoswitch installation (DBTD) resulted in framework degradation; therefore, this material was not suitable for studies of photoisomerization kinetics in a solvent-free environment.To elaborate further, we also probed the kinetics of the coordinatively-integrated diarylethene compounds, 11 and 12, for which isomerization does not involve formation of charged species (similarly to 5 and 6 hydrazone derivatives) and occurs within the plane, i.e., without even significant structural rearrangements associated with isomerization of hydrazone or spiropyran derivatives. For instance, X-ray crystal structures of two photoisomers, demonstrating the minimal structural rearrangement associated with diarylethene isomerization, are presented in Supplementary Fig. 2(3)(DBTD) containing spiropyran derivatives. Upon evacuation of the hydrazone-integrated MOFs, UiO-67 + 5 and UiO-67 + 6, we observed only a slight enhancement in the photoisomerization rate (~1.5-fold) in comparison with the rates found for the same photochromic compounds in DMF  are most likely associated with the transition from the zwitterionic merocyanine to neutral spiropyran form, allowing for the suppression of the solvent stabilization effect upon evacuation.As described above for the spiropyran derivatives, photophysical studies of hydrazone and diarylethene compounds integrated within the porous matrices revealed that each photoswitch maintained reversible photoisomerization with a rate constant comparable to the value obtained in solution. The rate constants determined in these experiments are summarized in Table on rate ~.5-fold i1–O and adjacent C2–O bond lengths  were fixed as shown in Supplementary Fig. Similar to the discussed hydrazone compounds, diarylethene derivatives undergo photoisomerization between two neutral forms Fig. . In addi68 For that, 2 and 5 were successfully integrated within the UiO-67 scaffold through post-synthetic modification as described above. PXRD analysis showed that the framework integrity was maintained throughout the post-synthetic modification procedure, and 1H NMR spectroscopic analysis in combination with TGA confirmed integration of both photoresponsive units, resulting in preparation of UiO-67 containing 0.1 and 0.6 of 2 and 5, respectively, per metal node 2 in DMF under UV irradiation. It is known from our previous studies and literature reports70 that the merocyanine photoisomer exhibits the ability to coordinate metal cations upon its exposure to solutions containing metal salts.70 We used this approach for coordination of copper cations to the merocyanine form of 2. After a very thorough washing procedure to remove the residual copper cations, EDX spectroscopic analysis revealed a relatively homogeneous distribution of copper throughout the material  was estimated to be 0.084 ± 0.002 s−1 while the rate constant for 5 (λmax = 363 nm) was estimated to be 0.0004 ± 0.0001 s−1. Both of these values are in line with the photoisomerization rate constants obtained for the same photoresponsive moieties integrated within UiO-67 individually. Furthermore, incorporation of two classes of photochromic molecules in one material allowed for complementary modulation of distinct regions of the material’s absorption profile. As anticipated, exposure to UV irradiation resulted in a simultaneous increase in absorbance in the visible region due to photoisomerization of 2 and decrease in absorbance in the UV region due to photoisomerization of 5. Thus, this material demonstrates a pathway for tuning the material’s optical properties across a wide range of the UV-visible spectrum  but also provides a possible avenue to control, for instance, release rates of multicomponent drugs in photopharmacology10 or multilevel anticounterfeiting and information encryption.11 Therefore, the presented studies foreshadow a conceptually distinct avenue to achieve required breakthroughs in device performance, including development of probes for real-time monitoring of self-assembly processes71 or biochemical pathways,72 as well as near-instantaneous detection of, for instance, toxins.73 However, these exciting applications only scratch the surface of the forthcoming generation of materials with tunable and tailorable properties that can be designed by leveraging the reported design principles and which can operate within a broad dynamic range.To summarize, we demonstrated a conceptually distinct approach to engineer photoswitch environment on the example of sterically-demanding spiropyran derivatives, resulting in ~1000-fold photoisomerization rate enhancement in the solid state, providing a pathway for the development of devices based on stimuli-responsive materials. Historically, achieving solution-like photoisomerization in a solid-state material has been a benchmark in the field of stimuli-responsive materials; however, the strategic engineering of the photoswitch environment through its integration in a rigid solvent-free matrix shown in this work is a pathway for surpassing solution-like behavior, leading to diminishment of stabilizing electrostatic interactions which are dominant and cannot be avoided in conventional solution-based studies. The presented approach also allows for integration of more than one type of photochromic unit within the same scaffold, preserving the achieved breakthrough in the photoisomerization rate of corresponding photochromic units. Moreover, incorporation of two distinct classes of photoresponsive units into the same host framework not only allows for selective tunability of one part of the absorption spectrum but also offers the opportunity to create a single material with a wide dynamic range of photoisomerization rates from 10H-indole-5-carboxylic acid was prepared based on a modified literature procedure.74 To a 250-mL round bottom flask, 3-methyl-2-butanone  and 4-hydrazineyl benzoic acid , and acetic acid (25 mL) were added and heated at reflux for one day. After cooling to room temperature, the solvent was evaporated under reduced pressure. Then, 100 mL of acetone was added to the flask, and the reaction mixture was sonicated for 30 min. The filtrate was collected, and the solvent was removed under reduced pressure. Next, 100 mL of dichloromethane was added to the flask, and the resulting reaction mixture was sonicated for 30 min. The filtrate was collected, and the solvent was removed under reduced pressure, affording a red solid . The collected 1H NMR spectrum matched the reported one74 : δ = 12.78 , 7.99 , 7.93–7.89 , 7.52–7.49 , 2.25 , and 1.27  ppm.The compound 2,3,3-trimethyl-31H NMR spectrum matched the reported spectrum74 : δ = 8.38 , 8.21–8.18 , 8.03–8.00 , 3.98 , 2.79 , and 1.56  ppm.To a 250-mL round bottom flask, containing 100 mL of a mixture of diethyl ether and hexane (3:2 v/v), I : δ = 12.35 , 8.24 , 8.03–8.00 , 7.83–7.80 , 7.68 , 7.27–7.24 , 6.92–6.90 , 6.70–6.68 , 6.03–6.00 , 2.76 , 1.24 , and 1.13  ppm. 13C NMR (DMSO-d6): δ = 167.37, 158.96, 151.20, 140.72, 135.88, 130.83, 128.50, 125.80, 122.94, 122.88, 121.60, 120.88, 118.78, 115.47, 106.27, 105.90, 51.56, 28.39, 25.48, and 19.53 ppm. Melting point: 285–287 °C. HR-ESI-MS: m/z found [M + H] for C20H19N2O5+ 367.1290 .To a 100-mL round bottom flask, II  δ = 11.77 , 7.97 , 7.91 , 7.79 , 7.44 , 4.40 , 1.32 . 13C NMR (DMSO-d6) δ = 167.53, 162.92, 147.45, 139.74, 131.65, 131.52, 130.59, 129.67, 127.95, 124.25, 114.07, 62.13, 14.31. Melting point: 272–273 °C. HR-ESI-MS: m/z found [M + H] for C18H17N2O6+ 357.1079 .To a 25-mL round bottom flask, 4-(2-ethoxy-2-oxoacetyl)benzoic acid  and 4-hydrazinobenzoic acid  were added and dissolved in 5.00 mL of ethanol. The resulting mixture was then stirred at room temperature for eight hours. After eight hours, the reaction mixture was diluted with 10.0 mL of water. The resulting precipitate was collected by filtration, and the solid was purified by column chromatography (0–50% ethyl acetate to hexanes), affording 6 as a yellow solid . 59 The UiO-67 powder was collected by filtration, thoroughly washed with DMF (3 × 10 mL), and then dried in air for 10 min. After drying, UiO-67  was placed in a 20-mL vial. Then, 1.00 mL of a 30.0 mM DMF solution of 2 was added. The vial containing the resulting mixture was placed in a preheated aluminum block at 75 °C for 24 h. After cooling to room temperature, the resulting powder was collected by filtration and washed with DMF (3 × 10 mL). The PXRD patterns of parent UiO-67 and UiO-67 + 2 are shown in Supplementary Fig. 1H NMR spectroscopic analysis of the digested sample, which corresponds to 0.01 of 2 integrated per metal node. The 1H NMR spectrum of the digested MOF sample confirming integration of 2 is shown in Supplementary Fig. To postsynthetically integrate 2 into UiO-67, parent UiO-67 was first synthesized based on a literature procedure.59 The UiO-67 powder was collected by filtration, thoroughly washed with DMF (3 × 10 mL), and then dried in air for 10 min. After drying, UiO-67  was placed in a 20-mL vial. Then, 0.500 mL of a 30.0 mM DMF solution of 5 was added, and the mixture was heated at 75 °C for two hours. After two hours, 0.500 mL of a 30.0 mM DMF solution of 2 was added. The vial containing the resulting mixture was heated at 75 °C for 24 h. After cooling to room temperature, the resulting powder was collected by filtration and washed with DMF (3 × 10 mL). The PXRD patterns of parent UiO-67 and UiO-67 + 2 + 5 are shown in Supplementary Fig. 1H NMR spectroscopic analysis of the digested sample, which corresponds to 0.1 of 2 and 0.6 of 5 integrated per metal node. The 1H NMR spectrum of the digested MOF sample confirming integration of both 2 and 5 is shown in Supplementary Fig. To postsynthetically integrate both 2 and 5 in UiO-67, parent UiO-67 was first synthesized based on a literature procedure.λex = 365 nm, distance = 2.5 cm, and LEDD1B power supply set at 700 mA).UV-vis absorbance spectra of hydrazone-containing samples in solution were collected using a ThermoFisher Evolution 350 UV-vis spectrometer with quartz cuvettes. Samples were irradiated with a mounted high-powered LED  was used for in situ irradiation of the samples. Diffuse reflectance spectra of hydrazone-containing samples were collected using a ThermoFisher Evolution 350 UV-vis spectrometer paired with Harrick Scientific Praying Mantis Diffuse Reflection accessory. For studies conducted under ambient atmosphere, the samples were prepared by filling the sample holder with BaSO4 and placing 1–2 mg of the hydrazone-containing sample on top of the BaSO4 layer. For studies conducted under vacuum, the samples were loaded into a high-temperature reaction chamber (Part HVC-VUV-5) and evacuated for 24 hours on a Schlenk line prior to collection of the spectra. A mounted high-powered LED  was used for in situ irradiation of the samples.Diffuse reflectance spectra for spiropyran- and diarylethene-containing samples were collected using an Ocean Optics JAZ spectrometer. An Ocean Optics ISP-REF integrating sphere was connected to the spectrometer using a 450-μm SMA fiber optic cable. For studies conducted under ambient atmosphere, the sample was loaded inside a 4.0-mm quartz sample holder. For studies conducted under dynamic vacuum, the sample was loaded into a quartz flow cell and connected to a Schlenk line with Tygon tubing. In all cases, a 400-nm longpass glass filter  was placed between the quartz sample holder and the integrating sphere to filter any UV light from the internal tungsten-halogen lamp. The quartz sample holder and longpass filter were attached to the top of the integrating sphere with electrical tape to prevent sample displacement. A mounted high-powered LED . Images of the samples before and after UV irradiation 2 (0.60 mM in DMF) under UV light (λex = 365 nm) for three days to promote coordination of copper cations by merocyanine.22 After three days, the sample was washed thoroughly with DMF and ethanol. Following the washing procedure, the sample was dried on vacuum overnight. The sample was mounted on a carbon substrate and analyzed with a Tescan Vega 3 SBU variable pressure SEM equipped with a backscattered electron detector. EDX analysis was performed with an accelerating voltage of 20.0 kV, a beam intensity of 13, a working distance of 10 mm, and 1962× magnification. EDX analysis revealed an equal distribution of copper in the sample , using the residual solvent peak as the reference standard. FTIR spectra were collected on a Perkin-Elmer Spectrum 100. PXRD patterns were recorded on a Rigaku Miniflex II diffractometer at a scan rate of 5°/min with accelerating voltage and current of 40 kV and 15 mA, respectively.NMR spectra were obtained on Bruker Avance III-HD 300, Bruker Avance III 400 MHz, Oxford 500 MHz, and Bruker Ascend 600 MHz NMR spectrometers. Supplementary InformationPeer Review File


Lycopersicon esculentum Mill.) CGFS-type GRX, is not yet fully understood. We discovered that LeGRXS14 is relatively conserved at the N-terminus and exhibits an increase in expression level under salt and osmotic stress conditions in tomatoes. The expression levels of LeGRXS14 in response to osmotic stress peaked relatively rapidly at 30 min, while the response to salt stress only peaked at 6 h. We constructed LeGRXS14 overexpression Arabidopsis thaliana (OE) lines and confirmed that LeGRXS14 is located on the plasma membrane, nucleus, and chloroplasts. In comparison to the wild-type Col-0 (WT), the OE lines displayed greater sensitivity to salt stress, resulting in a profound inhibition of root growth under the same conditions. Analysis of the mRNA levels of the WT and OE lines revealed that salt stress-related factors, such as ZAT12, SOS3, and NHX6, were downregulated. Based on our research, it can be concluded that LeGRXS14 plays a significant role in plant tolerance to salt. However, our findings also suggest that LeGRXS14 may act as a negative regulator in this process by exacerbating Na+ toxicity and the resulting oxidative stress.Salt stress represents a significant abiotic stressor for plants and poses a severe threat to agricultural productivity. Glutaredoxins (GRXs) are small disulfide reductases that can scavenge cellular reactive oxygen species and are crucial for plant growth and development, particularly under stressful circumstances. Although CGFS-type GRXs were found to be involved in various abiotic stresses, the intrinsic mechanism mediated by LeGRXS14, a tomato ( Salt stress also triggers an excess of reactive oxygen species (ROS) in plants, such as singlet oxygen (1O2), superoxide anions (O2·−), hydrogen peroxide (H2O2), and hydroxyl radicals (HO·), which act on DNA, lipids, and proteins, leading to irreversible oxidation events and oxidative damage and ultimately leading to cell death , salt stress (100 mmol·L–1 NaCl), heat stress (40 °C), and chilling stress (14 °C). The sampling time points were 0, 10, 20, and 30 min, and 1, 2, 3, 6, 12, and 24 h after the start of the stress treatment. Following treatment, plants were snap-frozen in liquid nitrogen and refrigerated at –80 °C.Tomato seeds (‘Micro-Tom’) were sterilized using a combination of 70% ethyl alcohol and 4.5% sodium hypochlorite before being cultivated in Petri dishes. After seven days of growth in an MS medium, the seeds were then transferred to soil, where they continued to be cultivated under controlled conditions. Maintenance of the environmental rooms saw a consistent temperature of 25 ± 2 °C, with a photo fluency rate of white light of approximately ∼110 µmol mLeGRXS14 accession numbers for the Solyc02g082200.3 full-length complementary region were obtained from tomato genomes . The method of Gateway cloning was employed for the construction of p35S::GRXS14 and p35S::GRXS14-GFP. PCR amplification of DNA fragments was carried out from cDNA, followed by cloning into the pENTR vector (Invitrogen). Subsequently, coding sequences from the entry clones were transferred to gateway-compatible destination vectors. The generation of transgenic A. thaliana plants was established through Agrobacterium-mediated transformation using homozygous transgenic T3 lines that contained a single insertion.The GRXS14-cDNA_Fw, 5′-ATGTCGCTCGGAATTTCGC;Cloning primers: GRXS14-cDNA_Rev, 5′-TCAGGAGCACAATGTCCT, GRXS14-cDNA (no stop code) _Rev, 5′-GGAGCACAATGTCCTTTCT; vectors: p35S::GRXS14:pGWB402Ω, p35S::GRXS14-GFP:pGWB405.t-test was used.RNA extraction was completed with a Total RNA Kit , with 100 mg of each section handled separately. Subsequently, 1 microgram of RNA was used for reverse transcription, followed by the use of the FastKing RT Kit (Tiangen) for cDNA synthesis. The quantitative real-time (qRT) PCR reaction was performed with the Real Universal Color PreMix (Tiangen) and involved an eight-well optical PCR plate for each reaction. Each reaction comprised 7.5 µL premix, 0.75 µL forward primer, 0.75 µL reverse primer, and 6 µL cDNA template. A set of PCR cycle parameters was initiated, which included one cycle of 30 s at 95 °C and 40 cycles of 10 s at 95 °C and 30 s at 60 °C. To ensure that gene expression levels were normalized, actin was utilized as an internal control. Arabidopsis seedlings, we generated transgenic plants with 35S promoter-driven GRXS14 . To carry out confocal imaging, seven-day-old seedlings grown on ½ MS medium in Petri dishes were studied using a Zeiss LSM 710 microscope. Blue excitation  was used for the excitation of GFP. For confocal imaging, a ×20 water immersion objective was used. Our examination included more than 10 independent lines, and we induced plasmolysis using 0.8 M sorbitol. Protoplasts were generated through the enzymatic digestion of plant material using a solution consisting of 20 mM MES (pH 5.7), 1.5% cellulase R-10, 0.4% macerozyme R-10, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 0.1% BSA, and during the incubation process.To investigate GRXS14-GFP in Agrobacterium tumefaciens-mediated transient expression in N. benthamiana was used to detect the co-localization of LeGRXS14 with a plasma membrane marker (mCherry-plasma membrane) and a nucleus marker (mCherry-Nucleus) [Agrobacterium tumefaciens GV3101 transformed with the target gene and silencing suppressor strain 19K at 28 °C, collected the cultures, and resuspended them with transformation solution (10 mM MES/KOH (pH 5.6), 10 Mm MgCl2, and 150 μM acetosyringone), so that the OD600 was adjusted by 0.5. We mixed equal volumes of 19K, LeGRXS14-GFP, and markers separately and left this for 3 h before injecting it into one-month-old tobacco leaves. A Zeiss LSM 710 microscope was used to observe the co-localization. For confocal imaging, a ×20 water immersion objective was used. The excitation and emission wavelengths of the GFP signal were 488 nm and 493–570 nm; for the mCherry signal, they were 561 nm and 600–650 nm for the chlorophyll autofluorescence, they were 488 nm and 650–700 nm.To study the localization of proteins in the cell, Nucleus) . Briefly2) ½ MS agar medium, with or without 60 mM NaCl. The ½ MS agar medium comprised the vital components of major salts , a vitamin solution (Sigma), 0.5% sucrose (Sigma), and 0.6% agar (Sigma), with the calcium content increased to 0.2 mM CaCl2. Following a growth period of 12 days, we evaluated the seedling root lengths as part of our analysis.For the plant physiology assay, we sterilized WT and OE seeds with PPM and maintained them in darkness at 4 °C for three days. Immediately after this period, the seeds were placed in a low-calcium (0.2 mM CaCl2) ½ MS agar medium, with or without 60 mM NaCl. Following a growth period of 8 days, we evaluated seedling root lengths as part of our analysis. The seeds were also placed in a normal-calcium (2.5 mM CaCl


Flaxseed oil (FO) and vitamin E (VE) both have antioxidant effects on sperm. The present study investigated the effects of dietary supplementation with FO and/or VE on semen quality.16 fertile Simmental bulls were selected and randomly divided into 4 groups (n = 4): the control group (control diet), FO group (control diet containing 24 g/kg FO), VE group (control diet containing 150 mg/kg VE) and FOVE group (control diet containing 150 mg/kg VE and 24 g/kg FO), and the trial lasted 10 weeks.The results showed that the addition of FO independently can increase sperm motion parameters, the levels of catalase (CAT), glutathione peroxidase (GSH-Px), testosterone (T) and estradiol (E2), while reduce oxidative stress in seminal plasma (P < 0.05). Supplement of VE independently can increased the motility, motility parameters, CAT and superoxide dismutase (SOD) levels, and reduce oxidative stress in seminal plasma (P < 0.05). There was an interaction effect of FO × VE on motility and reactive oxygen species (ROS), while GSH-Px and ROS were affected by week × VE 2-way interaction, levels of T and E2 were also affected by the dietary FO × week interaction (P < 0.05). The triple interaction effects of FO, VE and week were significant for malondialdehyde (MDA) (P < 0.05). Compared with the control group, sperm from the FOVE group had a significantly higher in vitro fertilization (IVF) rate, and subsequent embryos had increased developmental ability with reduced ROS levels at the eight-cell stage, then increased adenosine triphosphate (ATP) content and gene expression levels of CAT, CDX2, Nanog, and SOD at the blastocyst stage (P < 0.05). Metabolomic and transcriptomic results indicated that dietary supplementation of FO and VE increased the expression of the metabolite aconitic acid, as well as the expression of ABAT and AHDHA genes.With in-silico analysis, it can be concluded that the effects of dietary FO and VE on improving semen quality and embryo development may be related to increased aconitic acid via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway. However, most sperm are damaged by oxidative stress during cryopreservation. This damage causes a loss and decline in motility, viability and fertilization ability. Therefore, improving the antioxidant capacity of frozen-thawed sperm and reducing the level of oxidative stress is particularly important for maintaining semen quality .ω-3 Polyunsaturated fatty acids (ω-3 PUFAs) play important roles in the regulation of reproductive processes such as testosterone synthesis, sperm acrosome integrity and membrane fluidity . RuminanVitamin E (VE), one of the major antioxidants against reactive oxygen species (ROS) and lipid peroxidation, has been shown to be a major component of the antioxidant system of sperm , 7. VE din vitro fertilization (IVF) technology has made significant progress in animal reproduction  were selected from Changchun Xinmu Sciences & Technologies Co., Ltd, and divided randomly into four groups (n = 4). They were all kept under similar management. Ingredients and chemical composition of the diets are shown in 2.2®  extender to a concentration of 1.28 × 108, then loaded into thin tubes (IMV Technologies), and the semen was equilibrated at 4°C for 2 h, and subsequently cooled at approximately 8 min from 4°C to − 140°C by a turbofreezer  as previously described by Memon et al.  artificial vagina connected to a disposable tube, and quickly transferred to an adjacent laboratory where the physiological parameters were assessed after semen collection. Semen samples were expanded in a BioXcelln et al. . After t2.32, MM-0023O1, 4 pmol/L-120 pmol/L) were determined using specific enzyme-linked immunoassay kits , according to the manufacturer’s instructions. Briefly, semen was centrifuged at 3000 rpm for 15 min, and the seminal plasma was extracted before analysis. Results were measured at 450 nm on a spectrophotometer .The oxidative stress index and hormones of embryo culture medium and seminal plasma included superoxide dismutase , catalase , glutathione peroxidase , ROS , malondialdehyde , testosterone  and estradiol (E2.42 (10 μL/mL), cysteamine (10 μL/mL), and 10% FBS, incubated for 22 h at 38.5°C, 5% CO2 in air and high humidity.Oocyte collection and IVM were performed as described previously . Briefly2.52PO4, 0.1 mg/mL MgCl2·6H2O, 0.25 mg/mL CaCl2·6H2O, 0.758 mg/mL sodium lactate, 2.1 mg/mL NaHCO3, 6.25 mg/mL BSA, 10.417 μL/mL MEM non-essential amino acids, 10 μL/mL fructose, 10 μL/mL heparin and 10 μL/mL sodium pyruvate). Frozen-thawed pooled semen was washed through 45% and 90% Percoll gradients  were used to wash the pellet . Sperm concentration was calculated with a hemocytometer and adjusted to 106/mL with SOF-IVF medium.After 22 h, cumulus–oocyte complexes were added to SOF-IVF medium . Briefly, embryos were incubated with 10 mM DCFH-DA for 20 min at 37°C. After washing three times with 1% phosphate-buffered saline/PVP360 (PBS-PVP), the embryos were examined using a fluorescence microscope  with a 460 nm excitation filter. Fluorescence images of all embryos were recorded using a digital camera , and analyzed using the NIH ImageJ by Wayne Rasband from the National Institute of Health . Fluorescence intensities of the embryos were compared with that of the control after deducting the background value.2.8Adenosine triphosphate (ATP) of embryos was detected using an ATP assay kit . Briefly, embryos were collected, washed twice in PBS-PVP and then fixed in formaldehyde-PVP for 0.5 h at room temperature. Afterwards, the embryos were washed three times with PBS-PVP, 5 min each time, and then incubated with PBS-PVA containing 0.1% Triton X-100 for 1 h for membrane permeabilization. After thoroughly washing with PBS-PVP, the fluorescence signal was captured using an epifluorescence microscope, and the fluorescence intensity of embryos was analyzed using NIH ImageJ software.2.9OCT-4), caudal type homeobox 2 (CDX-2), Nanog, SOD, CAT, B-cell lymphoma 2 (Bcl2), BCL2 Associated X (Bax) genes and 18s rRNA are listed in −△△CT method.Total RNA from bovine blastocysts was extracted using the RNeasy Mini kit . A total of 18 blastocysts were used to extract RNA. The One-Step gDNA Removal and cDNA Synthesis SuperMix  was used to synthesize cDNA. The real-time PCR mix (25 μL) consisted of 2 μL of cDNA, 12.5 μL of SYBR green master mix, 9.5 μL of RNase-free water, and 0.5 μL each of primers (10 pmol). The primers used for the octamer-binding transcription factor 4  sample. Then, 300 μL of cold methanol was added, shaken and mixed, and placed at −20°C for 2 h. After centrifugation at 25,000 ×g for 10 min at 4°C, 350 μL supernatant was placed in a new Eppendorf tube, and the centrifugation step was repeated. Then 25 μL supernatant was added to 225 μL 50% methanol, and 50 μL of supernatant from each sample was taken and mixed with a QC sample, and the remaining transferred to a new 1.5 mL Eppendorf tube for testing. An Acquity UPLC HSS T3 column  was used for reversed-phase separations. The column temperature was maintained at 40°C and the metabolites were eluted at a flow rate of 0.5 mL/min. The injection volume for each sample was 5 μL. A high-resolution tandem mass spectrometer Xevo G2 XS QTOF  was used to detect small molecules eluted from the column. A quality control sample was acquired after every 10 samples to evaluate the stability of the LC-MS.2.11Peak extraction was performed primarily through the commercial software Progenesis QI (version 2.2). The resulting LC-MS data were inputted to the R software package for principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to evaluate differential ions, identify differentially expressed genes, and analyze metabolite and metabolic pathways.2.12Total RNA was extracted from sperm using the RNeasy Micro kit  according to manufacturer’s instruction. Briefly, samples were transferred to tubes containing Buffer RL and one volume of 70% ethanol and then centrifuged. Buffer RW1, DNase I, Buffer RPE and 80% ethanol were added then samples centrifuged. High quality RNA was used for mRNA library construction.2.13High-quality RNA was amplified (more than 200 pg) with oligo-dT and dNTPs to generate full-length cDNA by PCR. The Agilent 2100 bioanalyzer  was used to determine the average molecular length of PCR products. The DNA products were purified by an Agencourt AMPure XP-Medium kit . Single-stranded circular DNA was formatted as the final library, which used phi29  to prepare a DNA nanosphere. The DNA nanosphere was loaded into a patterned nanoarray, with single end 50 base reads generated on a BGISEQ500 platform .2.142 and the quality of semen were analyzed for the main effects of FO and VE. The FO, VE and time interactions used the General Linear Model procedure of the SPSS Institute (19.0) with a repeated measurement analysis. Differences among least square means were determined by the Tukey test and P < 0.05 was considered a significant difference. Differences in embryo development, CAT, SOD and ROS levels in embryo culture medium, and relative levels of gene expression, ROS and ATP in embryos, between the control and FOVE groups were compared by the T-test (SPSS 19.0), and P < 0.05 was considered a significant difference.All data are presented as least squares means ± SD. Each individual bull was considered an experimental unit in all statistical analysis. Data on oxidative stress index, T, E33.1P < 0.05) (P < 0.05). There was an interaction effect of FO × VE on motility (P < 0.05).The diet supplemented with VE significantly enhanced motility, straight distance (DSL), straight line velocity (VSL), curvilinear velocity (VCL) at week 8 and increased average path (DAP), DSL, curvilinear distance (DCL), velocity of the average path (VAP), VSL and VCL at week 10 compared with groups with no VE supplementation ( < 0.05) Table 3.3.2P < 0.05). The diet supplemented with FO or VE alone significantly increased CAT and decreased ROS at weeks 8 and week 10 compared with no FO or VE supplementation (P < 0.05). There was an effect of week × VE interaction on GSH-Px and ROS, and ROS was also affected by a dietary FO × VE 2-way interaction (P < 0.05). The triple interaction effects of FO, VE and week were significant for MDA (P < 0.05). Levels of T and E2 in frozen-thawed semen plasma are also shown in 2 at weeks 4, 8 and 10 compared with those from bulls fed the diet without FO. At week 8, elevated T content was observed with supplementation of VE in the diet (P < 0.05). Levels of T and E2 were also affected by the dietary FO × week interaction (P < 0.05).As shown in 3.3P < 0.05).The above results show that adding FO and VE to the diet for 10 weeks can improve the motility and motion parameters of frozen-thawed sperm, and also improve the antioxidant capacity, and the combined addition of FO and VE has a better effect. The control group and FOVE group samples of week 10 were used for further experiments. IVF results Figure 13.4P < 0.05) in the FOVE group compared with the control group (P < 0.05).Intracellular ROS levels were measured by assessing DCFH fluorescence (3.5P < 0.05). In contrast, FO and VE supplementation resulted in decreased MDA levels in embryo culture medium, but the activity of SOD did not differ among the groups (P > 0.05).3.6CDX2, Nanog, SOD and CAT compared with control group (P < 0.05).The effects of dietary FO and VE combined on blastocyst gene expression were examined by q-PCR. The relative abundance of gene expression in blastocysts is presented in 3.7P < 0.05  > 1, fold change ≥ 1.2 and 3.8P-value < 0.01. The results showed that 1336 genes and 987 genes were up-regulated and down-regulated, respectively, in the FOVE group compared with the control group  and Hydroxyacyl-CoA dehydrogenase subunit alpha (HADHA) genes of the propionate metabolic pathway, providing energy for bull sperm and promoting the IVF rate and subsequent embryonic development.In this study, metabolomics and transcriptomics were used to clarify the mechanism of dietary supplementation of FO and VE on semen quality and embryonic development. FO contains up to 50% ω-3 PUFAs, especially α-linolenic acid , which p2  and ABAT genes in sperm. It has been reported that LIPA and ABAT are involved in the TCA cycle and maintain mitochondrial membrane function  is a part of the fatty acid β-oxidation pathway  induces antioxidant defense pathways and increases H2O2 levels, and subsequently promoting the production of antioxidant enzymes (AOX1 genes in this study shows that the antioxidant capacity of sperm was increased.Our results showed that dietary FO and VE increased the expression of lipase A, lysosomal acid type (function , 55. As xpressed . Enoyl-C pathway . Our res enzymes . The eleFinally, we performed a combined analysis of metabolomic and transcriptomic data to identify the pathways by which dietary FO and VE affect sperm. We found that propanoate metabolism played an important role in improving sperm motility. Propionate metabolism is downstream of lipid metabolism and contributes to the tricarboxylic acid cycle and provides energy . A previABAT and HADHA genes in the propionic acid metabolism pathway to increase aconitate, thereby improving semen quality and subsequent embryo development. However, Due to the small number of bulls available for study under actual conditions of the experiment, as a large livestock animal, we cannot obtain a large number of experimental animals to carry out the experiment. In addition, an inherent limitation of this analysis was the in silico platform with results only based on bioinformation instead of direct investigation of biological mechanisms. Therefore, more method validation should be used to validate our hypothesis.Based on transcriptome and metabolome analysis, as shown in 5via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway.In conclusion, combined supplementation of FO and VE in bull diets could improve frozen-thawed sperm motion parameters, IVF rate and subsequent embryonic development by enhancing antioxidant capacity, which may be related to increased aconitic acid The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by Jilin Agricultural University in P.R. China and approved by the Experimental Animal Welfare and Ethics Committee of Jilin Agricultural University. Written informed consent was obtained from the owners for the participation of their animals in this study.CY wrote the paper. KZ and XM performed the experiments. ZW and JZ did the statistical analysis. HL collected the samples and data. WL and JW edited and reviewed the paper. All authors contributed to the article and approved the submitted version.


Lilium lancifolium Thunb.) are rich in phytochemicals and have many potential biological activities which could be deep-processed for food or medicine purposes. This study investigated the effects of microwaves combined with hot-air drying on phytochemical profiles and antioxidant activities in lily bulbs. The results showed that six characteristic phytochemicals were identified in lily bulbs. They also showed that with an increase in microwave power and treatment time, regaloside A, regaloside B, regaloside E, and chlorogenic acid increased dramatically in lily bulbs. The 900 W (2 min) and the 500 W (5 min) groups could significantly suppress the browning of lily bulbs, with total color difference values of 28.97 ± 4.05 and 28.58 ± 3.31, respectively, and increase the content of detected phytochemicals. The highest oxygen radical absorbance activity was found in the 500 W, 5 min group, a 1.6-fold increase as compared with the control (57.16 ± 1.07 μmol TE/g DW), which was significantly relevant to the group’s phytochemical composition. Microwaves enhanced the phytochemicals and antioxidant capacity of lily bulbs, which could be an efficient and environmentally friendly strategy for improving the nutrition quality of lily bulbs during dehydration processing.Lily bulbs ( Lilium lancifolium Thunb.) is a perennial herbaceous plant of the Liliaceae family whose bulbs are white and almost spherical. It is widely cultivated for its edible bulbs, which have been used for many centuries in East Asia as a food and traditional herb with a high nutritional and medicinal value. The bulbs are not only rich in nutrients such as carbohydrates, amino acids, dietary fibers, and mineral elements but also have abundant phytochemicals, such as phenolics, flavonoids, polysaccharides, and alkaloids [Lily  were cultivated in the same field at Tibet Agricultural and Animal Husbandry University’s Research Base, Linzhi, Tibet, China. Fresh lily bulbs were collected in the early spring before budding and washed with tap water to remove the residual dirt and soil on the surface. Then, the lily bulbs’ surfaces were dried, and the lily bulbs were peeled to small scales. The small scales of a hundred lily bulbs (approx. 5 kg) were evenly divided into ten groups (approx. 500 g per group) for the subsequent microwave treatment and hot-air drying experiments. An inverter microwave oven was used for fresh lily bulb pretreatments. The microwave pretreatment conditions were set at 2 min with changed powers as 100 wattages (W), 300 W, 500 W, 700 W, and 900 W and rated power at 500 W with changed times as 1 min, 3 min, 4 min, and 5 min. Fresh lily bulbs were used as a non-microwave pretreatment control group (CK). After microwave pretreating, all ten lily-bulb groups were dried at 60 °C with an air-dry oven until the moisture level dropped below 1% for subsequent analysis. The lily bulbs . The color difference values of three parameters were measured to characterize the color of lily bulbs: the brightness value L*, with a larger L* meaning a higher brightness ; the redness–greenness value a*, with −a* meaning green and +a* meaning red; and the yellowness–blueness value b*, with −b* indicating blue and +b* indicating yellow. The total color difference values were calculated using the following formula: Total phenolics of lily bulbs were extracted in accordance with the previous method  with somThe determination of the antioxidant activities in lily bulbs included 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity, 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical scavenging capacity, and oxygen radical absorbance capacity (ORAC). DPPH method was conducted as reported previously , the abs2 in William’s Medium E (WME) supplemented with 5% FBS , 10 mM HEPES, 100 μg/mL gentamicin, 0.05 μg/mL hydrocortisone, 2 mM L-glutamine, 5 μg/mL insulin, 50 units/mL penicillin, and 50 μg/mL streptomycin. HepG2 cells in WME were inoculated on 96-well microplates at densities of 2.5 × 104 cells/well (cytotoxicity) or 1.5 × 104 cells/well (antiproliferative activity) and cultured at 37 °C for 4 h or 24 h. The medium was replaced by a growth medium which contained lily bulbs extract at different concentrations, and then the cells were cultured at 37 °C for 24 h (cytotoxicity) and 72 h (antiproliferative activity). The absorbance of the reaction solution in each well was measured at 595 nm using Microplate Reader , and the final number of living cells per well was calculated using the methylene blue colorimetric method. The antiproliferative activity was calculated using IC50 values, and the cytotoxicity was calculated using CC10 values and expressed as mg per mL dry weight (mg/mL DW) with three replicates.The antiproliferation and cytotoxicity assays were performed using the methylene blue colorimetric method as previously reported . The Hepp < 0.05) by using IBM SPSS 25.0 . Pearson correlation was analyzed by Origin 2018 . Results were expressed as mean ± SD for triplicates.Significance analysis was conducted through one-way analysis of variance (ANOVA) and Tukey multiple comparison test  and the 500 W, 5 min group (58.08 ± 4.36 μg/g DW), with no significant difference. Compared to the CK group (432.8 ± 13.5 μg/g DW), microwave treatment caused a 4.3–33.29% declination of regaloside C, except for 500 W, 5 min group (488.7 ± 6.0 μg/g DW). The increase of microwave power induced an increase (100–500 W) and then a decline (500–900 W) in regaloside C content, reaching a maximum value in the 500 W, 2 min group (400.4 ± 10.1 μg/g DW). Regaloside C tended to increase with increasing microwave time, except for the 500 W, 4 min group, and reached a maximum in the 500 W, 5 min group.Regaloside A, regaloside B, regaloside C, regaloside E, Chlorogenic acid as well as The antioxidant activities in lily bulbs are presented in 10, CC20, and CC50 (mg/mL). The data indicated that there were positive relationships between the concentration of the extracts of lily bulbs and the cytotoxicity effect. Particularly, the 100 W, 2 min group and the 500 W, 1 min group showed maximum cytotoxicity to HepG2 cells than others. It was found that the CC50 value of the extracts under different treatment conditions is greater than the IC50 value, which means that all the lily bulb extracts have potential antiproliferative activity in concentrations that are nontoxic to HepG2 cells. The half-maximal inhibitory concentration (IC50) was used to express the cell antiproliferation of lily bulb extracts. The IC50 value of the lily bulb extracts decreases considerably with an increase in microwave power from 300 to 900 W and an increase in time from 3 to 5 min. For different microwave power, it was found that microwave pretreatment at 100 W for 2 min had the strongest inhibitory effect and the lowest IC50 (21.17 ± 0.36 mg/mL), followed by 900 W (30.16 ± 1.28 mg/mL). From different microwave times, the IC50 value of the 5 min and 500 W treatment is the lowest.The changes in antiproliferative activities and the cytotoxicity of lily bulb extracts with different microwave pretreatments were shown in The antiproliferative effects on HepG2 cells of lily bulb extracts were shown in p < 0.05), regaloside A (p < 0.01), regaloside B (p < 0.01), regaloside E (p < 0.01), Chlorogenic acid (p < 0.05) and p-coumaric acid (p < 0.01) whereas the DPPH value was significantly negatively correlated with them. It was also found that there was no significant correlation between the antioxidant activities assays.In order to determine the effect of the phytochemical composition of the lily bulb on the color and antioxidant activity studied, a simple correlation analysis was carried out. p-coumaric acid during microwave pretreatment, as the L* values are positively correlated with them , which is significantly relevant to phytochemical composition). However, the 900 W, 2 min group showed better antiproliferation activity than other groups without cytotoxicity with an IC


AF4 was found to have broad-spectrum antifungal activity against more than 110 fungal isolates. In this study, we found that clinical isolates of Candida tropicalis and Candidaauris exposed to AF4 exhibited low MICs of 4 to 8 mg/L. Time-kill assays indicated the in vitro pharmacodynamic potential of AF4. Biocompatibility assays demonstrated ~75% cell viability at 8 mg/L of AF4, indicating the lipopeptide’s minimally cytotoxic nature. In lipopeptide-treated C. tropicalis and C. auris cells, scanning electron microscopy revealed damage to the cell surface, while confocal microscopy with acridine orange(AO)/propidium iodide (PI) and FUN-1 indicated permeabilization of the cell membrane, and DNA damage upon DAPI  staining. These observations were corroborated using flow cytometry (FC) in which propidium iodide, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123 (Rh123) staining of cells treated with AF4 revealed loss of membrane integrity, increased reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction, respectively. Membrane perturbation was also observed in the 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence study and the interaction with ergosterol was observed by an ergosterol binding assay. Decreased membrane dipole potential also indicated the probable binding of lipopeptide to the cell membrane. Collectively, these findings describe the mode of action of AF4 against fungal isolates by membrane disruption and ROS generation, demonstrating its antifungal potency.Limited treatment options, recalcitrance, and resistance to existing therapeutics encourage the discovery of novel antifungal leads for alternative therapeutics. Antifungal lipopeptides have emerged as potential candidates for developing new and alternative antifungal therapies. In our previous studies, we isolated and identified the lipopeptide variant AFIMPORTANCEC. tropicalis is a major concern for candidiasis in India and C. auris has emerged as a resistant yeast causing difficult-to-treat infections. Currently, amphotericin B (AMB) and 5-flucytosine (5-FC) are the main therapeutics for systemic fungal infections; however, the nephrotoxicity of AMB and resistance to 5-FC is a serious concern. Antifungal lead molecules with low adverse effects are the need of the hour. In this study, we briefly describe the antifungal potential of the AF4 lipopeptide and its mode of action using microscopy, flow cytometry, and fluorescence-based assays. Our investigation reveals the basic mode of action of the investigated lipopeptide. This lipopeptide with broad-spectrum antifungal potency is apparently membrane-active, and there is a smaller chance that organisms exposed to such a compound will develop drug resistance. It could potentially act as a lead molecule for the development of an alternative antifungal agent to combat candidiasis. Candida albicans, a dimorphic fungus which causes either superficial  or deep-seated  candidiasis, is one of the leading opportunistic fungal pathogens in immunocompromised patients  can cause infections sporadically, often complicating the management of candidiasis, because candidiasis can be recalcitrant to treatment or become resistant , we identified three antifungal lead molecules with broad spectrum activity against 81 fungal isolates of Candida and Cryptococcus species and 11 isolates of filamentous fungi   , 14 was 4 against the clinical isolates of Candida tropicalis ATCC 750 and Candida auris IL-3331 ranged between 4 and 8 mg/L (Table S1). The MICs of C. tropicalis and C. auris were 4 mg/L (Table S1). The MICs for amphotericin B (AMB) were 1 and 2 mg/L for C. tropicalis and C. auris, respectively (Table S1).The MICs and MFCs  of RP-HPLC-purified antifungal variant AF4 at concentrations of 16 and 32 mg/L produced a significant decrease in CFU/mL over a period of 24 h. The log reduction in CFU/mL  values -2,5-diphenyltetrazolium bromide (MTT) assay, we tested the biocompatibility of the antifungal variant AFmg/L AF4 . In all opeptide . AF4 onl) values  of all cC. tropicalis and C. auris revealed that treatment with a standard antifungal and the novel lipopeptide AF4 significantly damaged and altered the ultrastructure of the cells. Untreated cells maintained their rounded or oval shape with a smooth surface and bud-scars, indicating that they were healthy, whereas cells treated with antifungal drugs showed characteristic aggregation and membrane damage, discerned as surface deformities, dimples, or cavities, leading to loss of cellular architecture  also exhibited occasional membrane blebs on the cell surface in addition to complete ultrastructural collapse with loss of shape.Scanning electron micrographs  of C. tr4 exhibited cell membrane damage and PI uptake, visible as orange-red or orange-yellow fluorescence (The cell membrane permeability was assessed using acridine orange (AO), a cell-permeable stain that stains both live and dead cells and is uniformly visible as green fluorescence. The membrane-impermeant fluorescent dye propidium iodide (PI) stains only cells with compromised membrane integrity and is visualized as red fluorescence. The absence of red fluorescence in the control panels  and C inrescence  and C.C. tropicalis and C. auris cells, we used a FUN-1 viability stain. FUN-1 is a two-color fluorescent probe which fluoresces diffused green and is transported to vacuoles to form red fluorescent, cylindrical intravacuolar structures (CIVS) in fungal cells which retain their membrane integrity and metabolic activity, whereas dead cells fluoresce bright yellow-green with no discernible red fluorescent CIVS  fluorescence is used as an indicator of stable membrane dynamics. Untreated cells showed high fluorescence intensity, whereas cells treated with AMB at 1× MIC and AFurbation . C. troped cells .Candida cells was assessed by changes in the membrane potential of protoplasts labeled with 4-(2-[6-(dioctylamino)-2-naphthalenyl] ethenyl)-1-(3-sulfopropyl) pyridinium inner salt (di-8-ANEPPS)  were found to be 5.046 ± 4.7 mg/L for C. tropicalis protoplasts and 6.095 ± 2.05 mg/L for C. auris protoplasts, respectively.The interaction of lipopeptide with the plasma membrane of -ANEPPS) . The var4 and AMB. When C. tropicalis and C. auris cells were treated with the antifungal variant AF4, cell membrane damage was inferred from the increased fluorescence resulting from the uptake of PI. In comparison, untreated cells showed absence or negligible PI fluorescence. The increased fluorescence in cells treated with AMB, AF4, and 70% ethanol is seen as a clear shift of peak along the x axis . All event FC dot plots are shown in Fig. S3A and B.Flow cytometry-based detection of altered permeability of the cell membrane was performed using the vital stain PI  followine x axis . In all 4 treatments showed good congruence. The percentage reduction observed in plate counts was highly comparable to the percentage of PI uptake recorded in flow cytometry, indicating an excellent correlation between these two, as summarized in 4-treated C. tropicalis showed 90.5% PI-positive cells  and side scatter (SSC)  values. It was observed that upon treatment with AF4, cell size and granularity/complexity increased by an average of 17.9% in C. tropicalis and an average of 45% in C. auris across the two concentrations tested . Cells treated with AMB showed a decrease in size and an increase in complexity for both strains used, and those treated with ethanol showed a drastic decrease in size .Morphological changes due to antifungal treatment were observed by flow cytometric analysis of the forward scatter (FSC) , resulting in enhanced fluorescence. As shown in 4-treated yeasts displayed elevated levels of ROS generation inferred from the increased fluorescence. C. tropicalis showed 64% and 77% ROS-positive cells at 8 and 16 mg/L, respectively. AF4-treated C. auris cells also showed a 97% increase in ROS-positive cells at both 8 and 16 mg/L (Reactive oxygen species (ROS) generation is a key marker of antifungal activity and of cells undergoing early apoptosis . Using t 16 mg/L . The ROS4 (8 and 16 mg/L), increased fluorescence of Rh123 was seen as a shift of the histogram along the x axis to the right  within the mitochondrial membrane. After 18 h exposure to antifungal AFx axis was also used to observe the extent of cell death using Rh123 as a live/dead probe . The dot (pseudocolor) plots of SSC height versus Rh123 height of treated samples were used to infer cell death by comparing them with the profile obtained for untreated cells. Of the lipopeptide-treated cells at 8 and 16 mg/L, a significant number lay beyond gate P7 compared to that in the control samples. Cells lying within gate P7 (set according to control samples) were considered live cells and those in gate P8 were considered to have significantly damaged membranes and were therefore recorded as dead cells .The shift of peaks indicating fluorescence along the 4 at different concentrations showed increased fluorescence with damaged abnormal nuclei, including tube-like, teardrop-shaped, or distorted nuclei, as shown in DNA damage is considered a probable indicator of late apoptosis , 23. 4’,B. subtilis. These antifungal lipopeptide variants demonstrated potent antifungal activity against many isolates of C. albicans, Candida non-albicans, Cryptococcus species, and filamentous fungi , with RAW264.7 and NIH 3T3 cells showing ~75% viability. Significant cytotoxicity has been observed only at high concentrations of 16 mg/L and greater, which is twice the MFC value (8 mg/L) and at which strong fungicidal activity has been observed against all target pathogens (Table S1). AMB showed significant cytotoxicity when tested at 8 mg/L against NIH 3T3 and RAW264.7 and low cytotoxicity for Vero cell lines. The IC50 values obtained for all three cell lines were found to be more than the MFC (8 mg/L), and a significant difference was observed between the IC50s of AMB and AF4, indicating that AF4 is biocompatible and shows reduced toxicity to cultured cells, encouraging consideration of AF4 as an antifungal lead molecule.The MTT assay showed that AFs tested  24). Ce. Ce4 exh4 showed membrane perturbations and inward collapse of the cell membrane. Compared to untreated cells, cells treated with AF4 and AMB showed two types of damage: inward curving of the cell membrane and surface deformities  caused a reduced proliferation of cells, evidenced by the lower recovery of ergosterol in treated cells compared to that in untreated cells.Because membrane-impermeable dyes were able to cross the membrane, further studies on fungal membrane dynamics were performed to understand the effect of AFfluidity . The modfluidity . The ergdynamics , 32, 33.rescence  in comparescence –36. Addi4, we used the membrane probe di-8-ANEPPS. Di-8-ANEPPS, a potentiometric probe, is incorporated into the outer leaflet of the plasma membrane and is used to observe membrane depolarization. Cells labeled with di-8-ANEPPS are expected to show a decrease in dipole potential when the peptides bind by adsorption or insertion  observed in flow cytometry suggests that AF4 may create surface pores without affecting cell size due to the lipopeptide’s interaction with membrane sterols, as discussed above . AF4 primarily targets the fungal cell membrane, causing severe membrane perturbation and permeabilization by potentially binding the ergosterol present in the plasma membrane. The ergosterol binding and membrane dipole potential experiments indicate that the binding of the lipopeptide to the cell membrane, either by adsorption or insertion, causes changes in the membrane structural properties  and a broad-spectrum antifungal candidate.This lipopeptide has demonstrated highly promising antifungal activity against C. tropicalis and C. auris that were used in this study were obtained from the National Culture Collection of Pathogenic Fungi (NCCPF), Post Graduate Institute of Medical Education and Research  and maintained as 20% glycerol stock at −80°C.All the isolates  and strains of B. subtilis RLID 12.1 followed by an optimized three-step purification process   enabled the purification process    was used as a positive control.The MICs and MFCs of the purified antifungal AF(M27-A3) . C. albi4 fractions were tested for time-dependent activity against C. tropicalis ATCC 750 and C. auris IL-3331 at concentrations of 16 and 32 mg/L as described by Ramya et al.  was used to determine the percentage of metabolically active cells, which indicated the percentage of viability and biocompatibility of drug-treated cells. Approximately 5,000 cells were seeded in 96-well plates into a growth medium supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotic  solution. After 24 h of incubation at 37°C in a 5% CO2-supplemented incubator, spent medium was aspirated and fresh medium with AF4 and AMB at a range of concentrations was added. After 24 h, 2 mg/mL MTT was added to the media in each well and incubated for 3 h, and formazan crystals formed were visualized. Formazan crystals were solubilized by incubating for 15 min with dimethyl sulfoxide (DMSO). Absorbance at 570 nm was read using a Multiskan Go plate reader  and 50% inhibitory concentration (IC50) values were calculated for each cell line , Pune, India, to assess the viability of cells in the presence of the antifungal lipopeptide AFell line .4 and AMB on C. tropicalis ATCC 750 and C. auris IL-3331 membranes. The cells were grown in drug-free medium, medium with the antifungal drug AF4 (8 mg/L) overnight, and medium with AMB (2× MIC) for 3 h at 37°C under shaking conditions. After incubation, cells were centrifuged at 10,000 rpm, washed with 1× phosphate-buffered saline (PBS)  and fixed onto coverslips using 2.5% glutaraldehyde  in 0.1 M cacodylate buffer (Sigma-Aldrich) for 3 h at 4°C. Post-fixation, the sample was washed with 0.1 M sodium cacodylate buffer and sterile distilled water to remove traces of fixative. After washing, 20 μL of 1% osmium tetroxide was added, and the sample was incubated for 1 h and dehydrated using a graded ethanol series (Scanning electron microscopy (SEM) was used to visualize the effect of AFl series , 62. The4. For both staining methods, an inoculum with ~5 × 106 CFU/mL was prepared and incubated with the antifungal drug at a concentration of 8 mg/L for 18 h, respectively, at 37°C under shaking condition. Cells were incubated with AMB at 2 mg/L  and 4 mg/L (C. auris) for 3 h at 37°C under shaking condition, and cells grown without any drug treatment were used as controls. Cells were harvested at 10,000 rpm and AO was added to the resuspended cell pellet at a concentration of 20 μM and PI was added at 5 μg/mL  study using two different staining techniques—a combination of AO  and PI  and the two-color stain FUN-1  to observe the mode of action of purified antifungal variant AF 5 μg/mL , 27. The 5 μg/mL , 27. StaThe FUN-1 stain was prepared in glucose-HEPES (GH) buffer and added at a final concentration of 5 μM. Cells were incubated in the darkness for 30 min at 30°C. GH buffer solution was used to wash off unbound stains before imaging , 28, 63.4 to the ergosterol present in fungal cell membranes, an ergosterol binding assay was performed as described by Escalante et al.  was double-diluted in wells of a 96-well microtiter plate with 100, 200, and 400 μg/mL of ergosterol in RPMI or in RPMI without ergosterol. As a control, the same procedure was performed for AMB (16 mg/L) in separate microtiter plates. Approximately 103 cells of yeast cell suspensions were added to each well and the plates were incubated at 37°C for 48 h. The changes in MICs in the presence and absence of exogenous ergosterol were recorded to infer the ergosterol binding capacity of the drugs used in the study.To determine the binding of AFe et al.  with min4 (8 mg/L) and AMB at 1× MIC for 18 and 3 h, respectively, at 37°C under shaking conditions. Cells were then fixed with 0.37% formaldehyde and incubated for 30 min at 28°C. Cells were then washed with PBS and flash-frozen using liquid nitrogen. Cells were subsequently thawed, resuspended in 1× PBS, labeled with 0.6 mM DPH, and incubated for 45 min at 28°C. Post incubation, cells were washed with PBS and homogenized by sonication on ice . The fluorescence emitted by DPH upon its intercalation with the fungal cell membrane is used to determine changes to the membrane lipid bilayer across treatments. Cells were treated with AFn on ice –36. CellCandida cells was determined using fluorescence intensities of the styryl dye di-8-ANEPPS  as described by de Aguiar et al. (Candida cells were harvested and adjusted to 106 CFU/mL. Protoplasts were prepared by treating Candida cells with lyticase enzyme (250U/mL)  for 70 min  with gentle stirring. Post-incubation, 104 CFU/mL of the same suspension was incubated with 10 μM di-8-ANEPPS for 1 h at 25°C in the darkness with stirring. AF4 was added to this suspension at 4, 8, 16, 32, and 64 mg/L and incubated for 90 min. As controls, cell suspensions without fluorescent probe or lipopeptide treatments were used. The ratio of intensities was obtained using excitation wavelengths of 455 and 525 nm and an emission wavelength of 670 nm, with excitation and emission slits set at 5 nm and 10 nm, respectively, using a spectrofluorometer . The dK values were determined using the equation shown below by fitting the experimental data using MATLAB R2022b:R is the ratio of intensities at 455 nm and 525 nm at different lipopeptide concentrations and dK is the apparent dissociation constant.The change in dipole potential of r et al. . Candidar 70 min . A proto4 on membrane integrity, the antifungal lipopeptide AF4 (8 and 16 mg/L) and AMB (2× and 4× MIC) were added to the cell suspension (5 × 106 CFU/mL) in RPMI 1640 and grown for 18 and 3 h, respectively, at 37°C under shaking conditions. Viable cells and 70% ethanol-treated cells were used as negative and positive controls for PI uptake, respectively. Post-incubation, cells were harvested at 10,000 rpm, washed, and stained with PI (7.5 μg/mL) for 20 min. Cell suspensions were analyzed using a FACScan flow cytometer  using a 488-nm laser line and a 586-nm filter for PI. For sample analysis, forward scatter, side scatter, and the percentage of PI-stained cells were collected. The data were processed using FlowJo version 10.8.1 software. In each experiment, untreated and unstained cells were sampled and analyzed first  for 18 h and with 2× and 4× MIC of AMB for 3 h at 37°C under shaking conditions. As a positive control, cells treated with 10 mM H2O2 for 1 h were used. After incubation, cells were centrifuged at 10,000 rpm, washed, and incubated in PBS with 10 μM DCFH-DA for 30 min in darkness at 37°C  by flow cytometry. Cells at ~5 × 10 at 37°C . The cel4 affected the mitochondrial membrane potential (ΔΨm) of the cells, we analyzed the cells with Rh123 . Generally, changes to membrane potential are observed by a shift in the fluorescence of Rh123 in flow cytometry. Cell suspensions with ~5 × 106 CFU/mL were treated with AF4 (8 and 16 mg/L) for 18 h in RPMI 1640 at 37°C under shaking conditions. Heat-treated  cell suspension, sodium azide-treated (40 mM) samples, untreated cells, and unstained cells were analyzed as controls. After treatment and incubation, cells were harvested at 10,000 rpm and Rh123 was added at a concentration of 1 mg/L for 15 min at 35°C; cells were then washed twice and incubated at 35°C for an additional 30 min and analyzed immediately , a nucleic acid stain. Cell suspensions of C. tropicalis and C. auris were treated with antifungal lipopeptide at 2-, 4-, and 8-mg/L concentrations to observe dose-dependent effects on the nucleic acid content of the cells. Cells (~5 × 106 CFU/mL) were incubated with AF4 for 18 h in RPMI 1640 at 37°C under shaking conditions. Untreated cells were used to observe the intact nuclei. Cells harvested at 10,000 rpm post-treatment were stained with 1 μg/mL DAPI for 20 min at 37°C (Nucleic acid damage such as DNA fragmentation and condensation due to treatment with the antifungal lipopeptide AF at 37°C , 23. CelEvery experiment was conducted in duplicate, and results were represented as mean ± standard deviation. Results were analyzed on GraphPad Prism 9 software using the appropriate statistical tests for each experiment, as detailed in the figure legends.All data have been represented as results in the main and supplementary sections. Raw data will be made available upon request.