Acacia species  by examining their chemical composition, antioxidant, and antimicrobial properties. Using high-resolution mass spectrometry, a comprehensive analysis successfully identified targeted compounds, including flavonoids (flavonols/flavones) and phenolic acids, such as 4-hydroxybenzoic acid, p-coumaric acid, and ellagic acid. Additionally, p-coumaric acid was specifically identified and quantified within the hydroxycinnamic aldehydes. This comprehensive characterization provides valuable insights into the chemical profiles of the studied species. Among the studied species, A. pycnantha exhibited a higher concentration of total phenolic compounds, including catechin, myricetin, quercetin, and coniferaldehyde. Furthermore, A. pycnantha displayed notable antibacterial activity against K. pneumoniae, E. coli, S. Typhimurium, and B. cereus. The identified compounds in Acacia pods and their shown antibacterial activities exhibit promising potential for future applications. Moreover, vibrational spectroscopy was a reliable method for distinguishing between species. These significant findings enhance our understanding of Acacia species and their potential for various industrial applications.This study intended to evaluate the potential industrial applications of various  Acacia spp. is one of those species that colonizes open spaces more quickly than native species, being then able to spread to other locations. This motivated the interest in valorizing the different parts of this invasive species to promote their harvest. Due to the potential of generated biomass, Da Costa et al. -, 229.0144 (indicating loss of both CO2 and CO), and 185.0254 (indicating loss of two CO2 molecules and one CO molecule)  × 100. In this equation, Abs0 represents the absorbance value of the control, while Abs1 represents the absorbance value obtained when the test sample was present at various concentrations.The radical scavenging activity was calculated using the formula: I% = [ [The quantification of antioxidant activity is expressed by the Antioxidant Activity Index (AAI), which was determined through the following calculation: AAI = /(IC50) . Various50) . All tesβ-carotene solution (500 mg/mL in chloroform) was added to 40 µL of linoleic acid, 400 µL of Tween 40, and 1 mL of chloroform. This mixture was evaporated at 45 °C in rotary evaporation for 5 min to remove chloroform and diluted with oxygenated distilled water (100 mL). Then, (5 mL) were pipetted into test tubes containing the extracts at different concentrations (300 μL) of the previous emulsion. The control consisted of 5 mL of the emulsion, 300 µL of methanol, and standard butylated hydroxytoluene (BHT) in methanol at the same concentration as samples and was used as a reference. Finally, the tubes were shaken and placed at 50 °C in a water bath for 1 h. The samples’ absorbances were measured at 470 nm, using a spectrophotometer, against a blank consisting of an emulsion without β-carotene. The measurements were carried out at the initial time (t = 0 h) and at the final time (t = 1 h). The antioxidant activity was measured in terms of the percentage of inhibition of β-carotene’s oxidation by % Inhibition = (Abst=1 sample − Abst=1 control)/(Abst=0 control − Abst=1 control). Where Abst=1 was the absorbance of the sample or control at the final time of incubation, and Abst=0 was the absorbance in control at the initial time of incubation [After preparation, a volume of 500 µL of a Klebsiella pneumoniae ATCC 13883, Salmonella Typhimurium ATCC 13311, Escherichia coli ATCC 2592), Gram-positive bacteria  and one yeast . The microbial strains’ stock cultures were maintained in 20% glycerol at −80 °C. The yeast strain was sub-cultured in Sabouraud Dextrose Agar (SDA) and the bacterial strains in Brain-Heart Infusion agar (BHI) 24 h before the antimicrobial testing.The antimicrobial activity of the extracts was evaluated against several microbial strains: Gram-negative bacteria  standard protocols (M2-A8 for bacteria and M44-A2 for yeasts). The inoculums were prepared by suspending several microbial colonies in a sterile saline solution, which adjusted turbidity to 0.5 McFarland. Sterile cellulose discs (6 mm diameter) were saturated with 20 μL of the extracts dissolved in DMSO at 200 mg/mL (4 mg/disc), which were then placed on the inoculated agar plates. DMSO (20 μL/disc) was used as a negative control, and tetracycline (30 µg/disc) or amphotericin B (25 µg/disc) were used as positive controls. The bacterial plates were incubated for 24 h at 37 °C, and those inoculated with the yeast were incubated for 48 h at 37 °C. Using a digital pachymeter, the inhibition zone diameters were measured millimeters after the incubation period. This assay was conducted three independent times .v/v, DMSO) was added to the first row of a 96-multiwell plate. Then, 50 µL of MHB was added to the other wells. Following, serial two-fold dilutions were completed with a multichannel pipette, the tips discarded with the last 50 µL; 10 µL of the resazurin indicator solution  and 30 µL of MHB were added to all the plate wells. Ultimately, 10 µL of the inoculums ascertained at 0.5 McFarland were also added to the wells. Several controls were considered: an antibiotic used a positive control, a column with all solutions except the extracts, and a column with all solutions except the inoculums. The plates were incubated for 18 h at 37 °C and were prepared in independent triplicates. The resazurin microtiter method was employed to determine the extracts’ Minimum Inhibitory Concentration (MIC). Initially, for bacterial strains, 100 µL of each extract (20 mg/mL in Müeller-Hinton Broth (MHB), and 10%, N-morpholino)propane sulfonic acid (MOPS). Microdilution susceptibility testing was also conducted, changing the final volume in the wells to 200 µL.In the case of the yeast, the working suspension (inoculum diluted 1:1000 in culture medium) was supplemented with resazurin solution . The culture medium used for yeasts was RPMI-1640, supplemented with glutamine and phenol red, without bicarbonate, and buffered with 3-(Positive observations were made when the color transitioned from purple to pink or became colorless. The minimum inhibitory concentration (MIC) value was determined as the concentration at which the first observable change occurred .m/z 50–1000 range by data-dependent MS/MS mode with a 0.5 isolation window, a 3 Hz acquisition rate, and a fixed cycle length of 3 s. At an absolute threshold of 153, precursor ions were chosen for auto MS/MS, with the active exclusion mode set to three spectra and released after 1 min. However, precursor ions with intensities up to five times higher than previous intensities were examined.Representative phenolic compounds were subsequently identified by a previously developed methodology using LC-ESI-HRMS/MS . The ethp-Coumaric acid, coniferaldehyde, (+)-catechin, rutin, myricitrin, myricetin, quercetin, kaempferol and naringenin) were diluted with ethanol to the final concentration of 1 mg/mL and stored at 4 °C in the dark.A previously validated methodology  was usedFor sample analysis, a high-performance liquid chromatography system with a diode array detector (HPLC-DAD) by Agilent Technologies was employed. The dried samples were dissolved in 500 µL of ethanol and filtered through a 0.22 µm filter before being injected into the chromatographic system. Chromatographic separation occurred on a YMC-Triart PFP  with pre-column . The mobile phase consisted of acetonitrile (A) and 0.1% trifluoroacetic acid in water (B)in a gradient mode: 10% A (0–3 min), 10- 15% A (3–15 min), 15% A (15–20 min), 15–18% A (20–25 min), 18–30% A (25–40 min), 30–50% A (40–45 min), 50–100% A (45–50 min); and returning to, 10% A (50–55 min). The flow rate was 1 mL/min, with a 50 μL injection volume. The column and sampler temperatures were 35 and 4 °C, respectively. Detected analytes have absorbance within the 255 to 360 nm wavelength range.For the isolation of rutin and myricitrin, the mobile phase comprised (A) 0% acetonitrile and (B) 100% orthophosphoric acid, using a gradient mode: 0% A (0–2 min), 9% A (2–14 min), 13% A (14–22 min), 33% A (22–38 min), and 43% A (38–44 min), maintained 43% (44–55) min, and returning to 0% A (55–65 min). The flow rate was 0.8 mL/min, with a 50 µL injection volume. The column and sampler temperatures were set at 24 °C and 4 °C, respectively. Rutin and myricitrin were detected at 255 and 263 nm, respectively. The identification of the target compounds was accomplished by comparing their retention times with those obtained from analytical standards. The identified compounds were quantified by comparing their peak areas observed in the extract chromatograms with calibration curves constructed using the respective standard solutions.−1, scanner velocity of 5 kHz, and wavenumber range from 4000 to 200 cm−1. Spectra were acquired on the powder for each species before and after extraction. The measurements were duplicated in an 8 mm optic space quartz cell with the opposite face mirrored.The methodology described in a previous study,  was emplThe statistical technique employed in this study was a one-way analysis of variance (ANOVA) to assess the presence of any statistically significant disparities among the measured parameters in the various samples. The least significant difference (LSD) was used in the context of the ANOVA to determine that each individual means differed from the others. STATISTICA 7 software  was used to perform the analysis above.®, version 7.5.18  and UnscramblerX 10.5  were used.For spectral data analysis, OPUSp-coumaric acid, (+)-catechin, rutin, myricitrin (myricetin 3-O-rhamnoside), myricetin, quercetin, kaempferol, and naringenin in the tandem mass spectra. The discrepancy in mass between the identified metabolites and their accurate mass was consistently within a range of less than 5 ppm for precursor ions and less than 10 ppm for product ions. In the The acquired data by LC-ESI-HRMS/MS were processed by Data Analysis 4.1 software . Extracted ion chromatograms (EIC) were conducted with a mass window of ±5 ppm to identify characteristic fragment ions associated with 4-hydroxybenzoic acid, ellagic acid, p-coumaric acid, 4-hydroxybenzoic acid, and ellagic acid, were employed to differentiate between the various species of Acacia pods under investigation. Even though there are some differences between each analyzed Acacia spp., A. pcynantha has more total phenolic compounds, catechin, myricetin, quercetin, coniferaldehyde, and antibacterial activities against E. coli, K. pneumoniae, S. Typhimurium, and B. cereus. Less undoubtedly, these more prevalent results were also found in A. dealbata. The total phenolic content of pods ranged from 0.12 to 1.75 mg GAE/g extract, with considerable variances between species. Whereas A. pycnantha had the highest concentration and A. longifolia had the lowest. The total flavonoid content varied from 4.48 to 6.55 mg QE/g extract, with the maximum value obtained for A. longifolia and the lowest value for A. cyclops. The low MIC values obtained, notably for A. dealbata and A. pycnantha extracts, demonstrate the Acacia pods extracts’ antibacterial efficacy against K. pneumoniae ATCC 13883 and B. cereus ATCC 11778. The compounds identified in Acacia pods show potential for future applications, and vibrational spectroscopy was found to be a reliable method for distinguishing between species. These findings contribute to understanding Acacia species and their potential industrial uses.With the ultimate aim of assessing the suitability of Acacia species for industrial applications, this study successfully examined the phenolic composition, as well as the antioxidant and antimicrobial properties of extracts obtained from seven Acacia species. Using LC-ESI-HRMS/MS, even compounds belonging to different organic families were unequivocally identified and subsequently quantified using HPLC-DAD. Flavonoids (flavonols/flavones) and phenolic acids contents, namely


Oxidative stress degrades oocytes during in vitro maturation (IVM). Catalpol, a well-known iridoid glycoside, exhibits antioxidant, anti-inflammatory, and antihyperglycemic effects. In this study, catalpol supplementation was tested on porcine oocyte IVM and its mechanisms. Corticalgranule (GC) distribution, mitochondrial function, antioxidant capacity, DNA damage degree, and real-time quantitative polymerase chain reaction were used to confirm the effects of 10 μmol/L catalpol in the maturation medium during IVM. Catalpol treatment significantly increased the first-pole rate and cytoplasmic maturation in mature oocytes. It also increased oocyte glutathione (GSH), mitochondrial membrane potential and blastocyst cell number. However, DNA damage as well as reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Mitochondrial membrane potential and blastocyst cell number were also increased. Thus, the supplementation of 10 μmol/L catalpol in the IVM medium improves porcine oocyte maturation and embryonic development. Oocyte in vitro maturation (IVM) is a process of oocyte meiosis, in an in vitro environment, from the first meiotic prophase  to the second meiotic intermediate phase (metaphase II (MII) stage). Oocyte IVM is a key step in in vitro embryo production and plays a significant role in fertilization and subsequent embryonic development ,2. IVM cCurrently, a large gap between in vivo and in vitro maturation of oocytes exists, due to developmental arrest caused by an unstable IVM environment  . OxidatiCatalpol, a highly active cyclic enol ether terpenoid, is primarily extracted from groundnuts and has anti-inflammatory, anti-apoptotic, hypoglycemic, antiviral, and antioxidant effects . In diabThe main aim of this study was to investigate the effect of catalpol on porcine cumulus-oocyte complex (COCs) IVM, and subsequent embryonic developmental competence in parthenogenetic activation (PA).Catalpol was purchased from the Chengdu Manstead  and dissolved in tissue culture medium 199 (TCM199). The catalpol-supplemented medium was stored at −20 °C. TCM199 and fetal bovine serum (FBS) were obtained from Gibco . The ROS, glutathione (GSH), and mitochondrial membrane potential assay kits  were purchased from Beyotime Biotechnology Inc. . Unless otherwise stated, all chemicals and reagents used in this study were purchased from Sigma-Aldrich .2, and 100% humidity. Catalpol  was added to the maturation medium at different amounts. The extrusion of the first polar body after IVM was used to evaluate oocyte maturity under a stereomicroscope.Porcine ovaries were obtained from a nearby slaughterhouse , deposited in a saline solution pre-heated to 37 °C, and immediately transported to the laboratory. Alcohol and saline were used to clean the ovaries three times each. COCs were extracted from 2 to 6 mm follicles. We selected COCs with a homogeneous cytoplasm and three or more layers of compact cumulus cells (CCs), using a stereomicroscope . Selected COCs were washed two to three times with 2% FBS-supplemented TCM199. Finally, the COCs were incubated in a maturation medium (a modified bicarbonate-buffered TCM-199 medium) for 44 h at 38.5 °C, 5% CO2. The oocyte cleavage and number of blastocysts were measured at 48 h and 168 h, respectively.After IVM, COCs were placed in a 0.1% hyaluronidase solution. Oocytes were observed under a microscope and after gentle blowing with a 100 μL pipette, oocytes with intact zona pellucida, normal cytoplasm, and discharged first polar body were collected. Mature oocytes were placed in ionomycin for 5 min, washed two to three times with NCSU-23, and transferred to an N6-dimethyladenine incubator for 3.5 to 4 h. Ten to 20 oocytes were transferred into 35 μL NCSU-23 drops contained on culture plates covered with 3 mL paraffin oil The plates were incubated at 38.5 °C and 5% COSeven MII-stage oocytes were washed three times with phosphate-buffered saline (PBS) and placed in a 200 μL centrifuge tube containing 10 μL lysate. The tubes were placed in a polymerase chain reaction (PCR) instrument for 15 min at 75 °C. Subsequently, 1 μL DNase I (Fermentas) (1 IU) and 1.3 μL 10×RDP buffer was added and the centrifuge tubes were placed in a PCR instrument for 30 min at 37 °C. Then, 1 μL EDTA (50 mM), 4 μM random primer , and 1 μL mixture deoxynucleotides  were added and the centrifuge tubes were placed in a PCR instrument for 10 min at 65 °C. Finally, 4 μL 5×First-Strand Buffer, 2 μL dithiothreitols (0.1 M), 0.25 μL SuperScriptTM II Reverse Transcriptase , and 0.5 μL RNase Inhibitor (TaKaRa Bio) was added and the centrifuge tubes were placed in a PCR instrument for 10 min at 25 °C, 90 min at 42 °C, 10 min at 95 °C, and cooled to 4 °C. The procedures were performed on ice.Extraction of CC RNA was performed using the TRIZOL reagent , according to the manufacturer’s instructions. Using the PrimeScript™ reagent kit , 500 ng of the template was reverse transcribed into cDNA. The SYBR green assay system was used for quantitative real-time PCR (qRT-PCR) on an ABI 7500 Fast RT-PCR System . CCs and MII-stage oocytes normalized each mRNA to β-actin.During IVM, different concentrations of catalpol  were utilized, and CC expansion was measured. CC expansion was calculated using a previously published rating method (grade 1–3) . Grade 1Mitochondrial membrane potential was examined using a mitochondrial membrane Potential Kit , according to the manufacturer’s instructions. Briefly, denuded oocytes (DOs) were cultured with 0.05% JC-1 fluorescent dye for 30 min at 38.5 °C. After washing several times with 0.1% buffered saline—Professional Video Assistant phosphate (PBS-PVA), images were captured using a fluorescence microscope . The fluorescence intensity of the oocytes was calculated using ImageJ-win64 software .2. Oocytes were placed on glass slides and imaged using a confocal microscope  after repeated washes with 0.1% PBS-PVA. ImageJ computed oocyte fluorescence intensity .Oocytes were incubated in PBS with 3% BSA and 180 nM Mito Tracker Red CMX Ros (Beyotime Biotechnology) for 30 min at 38.5 °C and 5% COIntracellular oocyte ROS levels were measured using a ROS test kit (Beyotime Biotechnology Inc.). MII stage oocytes were treated with 10 μmol/L dichlorofluorescein diace-tate  for 30 min at 38.5 °C. After three washes with 0.1% PBS-PVA, pictures were taken with a fluorescence microscope (EVOS FL Auto) at constant settings. The mean optical density values (IntDen/Area) were analyzed using ImageJ software .g for 10 min at 4 °C, collecting supernatant. According to the manufacturer’s instructions, the total GSH was determined using the total GSH assay kit (Beyotime Biotechnology Inc.). Working and 0.5 mg/mL nicotinamide adenine dinucleotide phosphate (NADPH) solutions were prepared. For standard curve preparation, 25, 15, 10, 5, and 2 μM GSH solutions were prepared using protein removal reagent S. Total GSH assay working solution (150 μL) was mixed with 10 μL of the collected supernatant or standard in a 96-well plate. The plate was incubated for 5 min at room temperature (25 °C), 50 μL of 0.5 mg/mL NADPH solution was added, and the solution was mixed well. After 25 min, the absorbance was measured at 412 nm, using an enzyme marker .The oocytes were washed three times with PBS-PVA, and three times the volume of the protein removal reagent S. The oocytes were freeze-thawed three times in liquid nitrogen and a 37 °C water bath, placed on ice for 5–10 min, and centrifuged at 10,000× g for 10 min to remove the supernatant for analysis. The thiobarbituric acid (TBA) storage solution and MDA assay working solution were prepared according to the lipid oxidation assay kit (Beyotime Biotechnology Inc.) instructions (ratio of TBA dilution: TBA storage solution: antioxidant = 150:50:3). Lysate (100 μL) was used as a blank control and 1, 2, 5, 10, 20, 50 μM dilutions were prepared with distilled water, for the standard curve. The sample (100 μL) and 200 μL of the MDA assay working solution were added to the EP tube, mixed, heated in a boiling water bath for 15 min, cooled to room temperature (25 °C), and centrifuged at 1000× g for 10 min. The supernatant (200 μL) was added to a 96-well plate and quantified at 532 nm with an enzyme marker. To prevent leakage of the boiling liquid, the EP tubes were coated with a sealing film and kept away from the heating surfaces.The oocytes around the MIIstage were blown clean, and 50 oocytes from each group were collected, washed three times with PBS-PVA, lysed on ice with western and immunoprecipitation cell lysis solution (P0013), and centrifuged at 10,000–12,000× The control and optimal catalpol concentration treatment groups were selected for subsequent experiments. Mature oocytes were incubated in 4% paraformaldehyde (PFA) for at least 30 min and washed three times with PBS-PVA. The oocytes were permeabilized overnight in EP tubes containing 1% Triton X-100, washed three times with BSA, and incubated overnight at 4 °C in peanut lectin lectin-FTTC (1:100). The tubes were washed three times with PBS-PVA under light-proof conditions, pressed, and photographed using a laser confocal microscope.CCs around porcine oocytes were removed after maturation using 0.1% hyaluronidase digestion and 200 μL pipette blowing. Next, 30-min PBS-PVA washes in 4% PFA, were performed three times. Oocytes were extracted and rinsed three times with a blocking solution for 5 min, to complete fixation. Blocking solution washes were performed for 5 min and the oocytes were fully permeabilized for 30 min at room temperature (25 °C) using 1% Triton X-100. The oocytes were closed with 1% BSA for 1 h and washed three times with PBS for 5 min. Next, oocytes were incubated in 1% BSA-diluted primary antibody overnight at 4 °C. Oocytes were washed three times with PBS at room temperature (25 °C). The oocytes were then incubated for 1.5 h at room temperature (25 °C) with a 1% BSA-diluted secondary antibody before being rinsed three times with PBS for 5 min. Hoechst 33,342 oocyte-staining was performed for 15 min. Three PBS-PVA washes were performed for 5-min. Oocytes were sealed with petroleum jelly on clean slides and photographed under a confocal microscope.After PA, we collected the 7-day-old blastocysts and, washed them three times with PBS-PVA. Following this, they were placed in 200 μL EP tubes containing 4% PFA and incubated for 30 min. The stained blastocysts were washed three times with PBS-PVA, transferred to 10 μg/mL Hoechst 33,342 dye, and incubated in the dark for 15 min at 38.5 °C. The stained blastocysts were washed three times, placed on slides, sealed with petroleum jelly, and photographed under a fluorescence microscope. Finally, the ImageJ program tallied cell counts. .p < 0.05, p < 0.01, and p < 0.001 indicated statistical significance.All experiments were tripled. IBM SPSS Statistics 18.0  was used for statistical analyses. To evaluate CC expansion, COC excretion rate, embryonic development, and relative gene expression levels, the one-way analysis of variance (ANOVA) and Duncan’s multiple range test were utilized. Data are presented as mean ± SEM. Different catalpol concentrations  were used during IVM and the effects were estimated using the first polar body rate . The firPTGS2), pentraxin 3 (PTX3), and hyaluronan synthase 2 (HAS2) mRNA expression was significantly higher in the catalpol group  was significantly higher than that in the control group . A. In addol group B.Abnormal CG distribution in the 0 μmol/L catalpol treatment group  was significantly higher than in the 10 μmol/L catalpol treatment group  .p < 0.01; p < 0.05; The 10 μmol/L catalpol treatment group had clustered, homogenous mitochondria A; in addp < 0.001; p < 0.001; p < 0.001; SOD1), catalase from Micrococcus lysodeikticus (CAT), and GSH peroxidase 1 (GPX1).To explore the role of catalpol on ROS production in porcine oocytes, the fluorescent probe DCFH-DA was introduced into oocytes. The 10 μmol/L catalpol treatment group showed considerably lower ROS fluorescence intensity compared to the control group  in oocytes is closely associated with ROS levels [CAT, SOD1 and GPX1 in oocytes. Therefore, we speculate that catalpol enhances oocyte quality by promoting the transcriptional levels of these genes in the porcine oocyte complex of the oocyte thalamus, reducing cytoplasmic ROS content, and enhancing the antioxidant capacity of oocytes.Intracellular GSH production protects cells from oxidative stress and is essential for oocyte cytoplasmic maturation. ,49; 10 μS levels ,51,52,53S levels . MDA is Normally, high levels of ROS in cells inhibit the repair of DNA double-stranded breaks, triggering oxidative DNA damage, . TherefoOocyte quality is critical for embryonic development; however, embryonic development is also an important indicator of mature oocyte quality in vitro . CatalpoIn conclusion, the supplementation of 10 μmol/L catalpol IVM increased CC expansion, as well as nuclear and cytoplasmic maturation. Catalpol supplementation improved DNA damage, COCs antioxidant capacity, and PA-derived porcine embryonic development. Therefore, catalpol is a potent antioxidant supplement for in vitro porcine oocyte maturation.


SMCP expression. Lower concentrations also demonstrated improved viability and reduced apoptosis and ROS levels. In conclusion, myo-inositol treatment during cryopreservation improved sperm quality, reduced apoptosis and ROS levels, and enhanced fertility rates in boar semen.During cryopreservation, sperm undergoes structural and molecular changes such as ice crystal formation, DNA fragmentation, and reactive oxygen species (ROS) production, leading to decreased sperm quality after thawing. Antioxidants play a crucial role in preventing these damages, both in vivo and in vitro. One potent antioxidant is myo-inositol, known for its protective effects on sperm against ROS. This study aimed to investigate the protective effect of myo-inositol on cryopreserved boar semen. The semen was diluted, cooled, and cryopreserved using a BF5 extender. It was then divided into five groups: control and different concentrations of myo-inositol . The post-thaw evaluation included assessments of motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), caspase activity, gene expression, ROS levels, apoptosis, and IVF with treated semen. Results showed that myo-inositol at 0.5 mg/mL improved motility, acrosome integrity, and fertilization ability. It also reduced the expression of pro-apoptotic genes and increased  Cryopreservation is an essential procedure for the long-term preservation of semen. During this process, multiple changes occur, such as fluctuations in pH, osmotic changes, and energy depletion. These changes then lead to chemical, mechanical, and structural changes, as well as excessive reactive oxygen species (ROS) production, and consequently, cryoinjuries occur ,2.2− and for rapid dismutation of O2− to H2O2, but an extremely low level of catalase, which converts H2O2 to water and oxygen. This system is able to protect sperm during cooling but not cryopreservation due to the lack of catalase and exhaustion of other antioxidants. During freezing and thawing, there was an increase in the production of H2O2, and this is probably the primary source of oxidative damage during this process. An increase in ROS production causes extensive cell damage, including morphological defects, lipid peroxidation, DNA fragmentation, decreased fusion with the oocyte, and compromised pregnancy after in vitro fertilization [2+, bicarbonate, and media components into the gamete. This disrupts homeostasis, facilitates the degradation of proteins and mRNAs, and worsens fertilization rates [In a normal state, ROS are produced in small amounts to maintain normal sperm functions, which include facilitating vital intracellular signaling cascades for appropriate sperm functions such as maturation, hyperactivation, and capacitation, as well as the acrosome reaction (AR). This ROS ends up being transferred into harmless by-products by the natural antioxidant system found in semen or seminal plasma . Howeverlization . Sperm tlization . The memon rates ,7. Indeeon rates ,9,10. Toon rates .Lamiaceae that contain rosmarinic acid with known beneficial effects on frozen-thawed boar sperm motility, viability, acrosome integrity, and reduced lipid peroxidation and DNA oxidation. Extract of Camellia sinensis (green tea extract) contains polyphenols that improve frozen-thawed sperm viability, reduce lipid peroxidation, and increase monospermic and blastocyst formation. Grapes contain multiple antioxidants such as myo-inositol and resveratrol, which are known to enhance frozen-thawed sperm parameters including mitochondrial membrane potential, decreasing ROS, and apoptosis [An antioxidant is one of the freezing–thawing semen additives that have ameliorative effects on semen quality by maintaining the balance between reductive and oxidative action, which improves sperm motility, membrane integrity, and fertilization ability ,13. Eithpoptosis ,14,15. Spoptosis .2+ levels, osmoregulation, and ATP production, thus regulating sperm motility and capacitation, improving acrosome reactions, improving zona pellucida binding and fertilization ability, stimulating oxidative metabolism, activating the Akt protein that induces sperm maturation, and increasing phosphorylation of BCL-2 [Myo-inositol is a compound that is naturally extracted from various organisms. It is primarily found in plants and fungi; it is present in many foods such as fruits, seeds, and nuts; and it is also produced within the human body . It playof BCL-2 ,19,20,21of BCL-2 ,23. Multof BCL-2 ,24,25,26All reagents were purchased from Sigma-Aldrich , unless otherwise mentioned within the article. Semen was collected from fertile Duroc boars from the KPG AI Center, Korea; 60 ejaculates from different boars were used in this study. Samples were collected using the gloved-hand method, and sperm-rich fractions of ejaculates with >75% motile and 80% morphologically normal spermatozoa were used in this study. Eligible samples were diluted (2.5 × 109 ± 0.5/90 mL) in BTS. The diluted semen was cooled and maintained at 17 °C and transported to the laboratory within 1 h of collection and dilution. Extended semen was received and distributed in a 15 mL falcon tube and kept at 17 °C for 2 h, then processed. A tris-egg yolk extender was prepared according to a previously described method  as follog, washed with Beltsville thawing solution (BTS), and centrifuged again. The supernatant was removed, and semen was added to extender 1 at a concentration of 2 × 108 sperm/mL and incubated at 4 °C for 1 h. Extender 2 was then added to extender 1 at a ratio of 1:1, and semen was mixed with the extender. The mixture was placed in a 0.5 mL semen straw  and equilibrated at 4 °C for 25 min. The semen straw was placed over a 4 cm liquid nitrogen surface for 20 min and then inserted into LN2 for storage. After at least 24 h, frozen semen was thawed at 37.5 °C for 25 s. Thawed semen was diluted 1:4 (vol:vol) with BTS, and the pertinent parameters were evaluated.Motility was evaluated after 2 h of incubation, and a motility of >80% was used. Semen was cryopreserved according to a modified two-step protocol that was previously described , as foll2 (min–max); static: <10 µm/s; drafting: 0; slow-medium: 25 µm/s; rapid: >45 µm/s; progressive STR: >45; connectivity: 11 pixels; VAP points: 5 pixels; head point: 10–100; and frame rate: 25 frames per second. Consecutive parameters were evaluated, including total sperm motility (TM%), progressive motility (PM%), rapid progressive motility (RPM), and medium progressive motility (MPM). Additionally, sperm kinematics were assessed, including curvilinear velocity , average path velocity , straight linear velocity , straightness , and linearity .Thawed semen motility was evaluated using computer-assisted sperm analysis . Briefly, 2 µL of diluted thawed sperm were placed into a chamber  on a warm plate at 38 °C, and five fields for each group were evaluated. The adjustment of parameters is as follows: area: 10–80 µmViability was evaluated using the LIVE/DEAD™ Sperm Viability Kit  according to a previously described method ,30. In bPisum sativum agglutinin (PSA) were combined with fluorescein isothiocyanate (FITC) [According to Yu’s method, a combination of two strains of e (FITC) . A clean®, Sigma-Aldrich, St. Louis, MO, USA) was mixed in, and it was incubated at 37 °C for 20 min in the dark. The billet was centrifuged for 5 min at 860 g, the supernatant was discarded, and then the billet was resuspended in 500 mL of Dulbecco’s phosphate-buffered saline (DPBS). Following this, 10 µL was placed on the slide, cover slipped, and examined under a fluorescence microscope  using a rhodamine filter. A total of 200 sperm cells were counted. The positive cells showed strong red fluorescence in the middle piece and tail, and the negative cells showed red without strong fluorescence [Caspase activity was detected using a CaspGLOW™ Red Active Caspase Staining Kit and a Fluorescein Active Caspase Staining Kit  in accordance with the manufacturer’s instructions. The detailed methodology is as follows: 0.5 mL of thawed semen was kept in a 15 mL falcon tube, diluted with 3.2 mL of DBPS, and 300 µL of the mixture was transferred to an Eppendorf tube. Thereafter, 1 µL of sulforhodamine  and PI . An amou®  in accordance with the manufacturer’s instructions. Briefly, after thawing at 37 °C for 25 s, spermatozoa were washed twice with DPBS (centrifugation at 160× g for 2 min), and the supernatant was removed. The TRIZOL reagent was added, mixed well with the sample, and incubated for 5 min at room temperature. Then chloroform was added at a rate of 1:5 TRIZOL, mixed well, and incubated for another 5 min. Then it was centrifuged at 1600× g at 4 °C for 10 min. Then take 30 µL of supernatant and mix with 30 µL of isopropanol and 2 µL of glycogen . The concentration and purity of RNA were measured . Total RNA from all samples was equalized to the same concentration, and quantitative real-time PCR (qPCR) was conducted to assess transcription abundance  and an ABI 7500 real-time PCR system . The total volume of the reaction mixture was 20 µL, consisting of the following: 2 µL RNA, 10 µL TB buffer, 0.8 µL enzyme mix, 0.8 µL forward primer, 0.8 µL reverse primer, 0.4 µL ROX dye II, and 5.2 µL nuclear-free water. The PCR conditions were as follows: stage 1, reverse transcription: 5 min at 42 °C and then 10 s at 95 °C; stage 2, PCR reaction: 40 cycles of 5 s at 95 °C and 34 s at 56 °C; and stage 3, dissociation. GAPDH  was used as an indigenous control, while the value of gene expression for the control group was set at 1. The gene quantities were determined by using the equation n R = 2 − (∆Ct sample − ∆Ct control). To assess gene expression, the total RNA for the control and myo-inositol-treated groups was prepared using the TRI method  and TRIZ2 for 22 h with hormones and 20 h without hormones [After the ovaries arrived at the lab in physiological saline, the initial temperature was evaluated at 35–37 °C. The medium-sized follicle content was aspirated using 18-gauge hypodermic needles attached to a 10 mL syringe. The collected oocytes were washed twice with Hep-buffered Tyrode’s medium (TLH) and transferred to a petri dish containing TLH, where at least two layers of cumulus and granulated cytoplasmic oocytes were collected. Every 60 oocytes were cultured in IVM medium TCM-199  supplemented with cysteine, porcine follicular fluid, sodium pyruvate, epidermal growth factor (EGF), equine chronic gonadotropin, hCG, kanamycin, and insulin. Oocytes were incubated at 39 °C and 5% COg for 2 min, first with BTS and then with DPBS. The supernatant was discharged, and 200 µL of MTBM was added and mixed. Of this, 10 µL was taken for motility testing, and 10 µL was used for concentration determination. After complete concentration determination, the sperm concentration was adjusted to 5 × 106 sperm/mL for fertilization. Each drop received 5 µL of the prepared sperm. The oocytes and sperm were co-incubated in a 5% CO2 incubator at 39 °C for at least 6 h, then the oocyte was denuded by pipetting to remove extra sperm around the oocytes [For the evaluation of IVF, two groups of cryopreserved semen were assessed: the control and myo-inositol-treated semen groups. In brief, mature oocytes were selected, washed twice with IVF medium (MTBM), and moved into fertilization drops containing 40 µL of MTBM covered with mineral oil , each drop containing 15 oocytes, and each group containing at least 30 oocytes, which were then placed in the incubator. After thawing, the semen was washed twice with centrifugation at 520×  oocytes .2 and 5% O2 at 39 °C). Cleavage was checked on Day 2, and blastocysts were checked on Day 7 [Following IVF, presumed zygotes were rinsed twice with Porcine Zygote Medium 3 (PZM-3) culture medium, and every 10 oocytes were placed in a PZM drop covered with mineral oil and incubated in two gas incubators , 200 µL of stander and sample were added to each well in duplicate and read in a spectrophotometer  at 532 nm. The result of the mean reading is an MDA level of nmol/2 × 107.Lipid peroxidation was assessed via a lipid peroxidation kit (MDA) . Two experiment groups, the control and myo-inositol-treated groups, were tested in brie, and thawed semen was washed two times with PBS, then the pellet was mixed with 300 µL of MDA lysis buffer and 3 µL of BHT, mixed and homogenized in ice for 10 min, then centrifuged at 4 °C and 13,000× g for 3 min, after which the supernatant was discarded, and the belts were suspended in 500 µL of DPBS. This was repeated, and the belt was resuspended into 500 µL of DPBS and mixed well by pipetting. An amount of 50 µL of suspension was added to 950 µL of DBPS in a 5 mL FACS tube (FALCON). Then, 1 µL of 20 mM 2′,7′-dichlorodihydrofluoresceindiacetate  and 1 µL of PI stock (2.4 mM) were added and incubated in the dark for 1 h. Samples were analyzed using a FACSCalibur flow cytometer . All non-sperm events were excluded from further analysis. The mean fluorescence intensity (MFI) and FL1 (DCF) signals were detected using a 530/30 nm bandpass filter. FL2 (PI) signals were detected using a 585/42 nm bandpass filter, and the MFI of DCF was measured to evaluate the intracellular mean H2O2 in the sperm population. Three results were obtained: PI +ve, which indicates dead sperm; PI +ve/H2CFDA +ve, which indicates viable sperm with high H2O2; and PI −ve/H2CFDA −ve, which indicates live sperm with low H2O2. The mean ratio between viable sperm with high H2O2 and total viable sperm evaluated as ROS levels was calculated.To determine ROS levels, 2′,7′-dichlorodihydrofluorescein diacetate  and the following protocol were used . In brieg for 3 min, and the supernatant was discarded. Then the sperm pellet was resuspended in 1×Annexin V binding buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, and 25 mM CaCl2). For a 1X working solution, dilute 1 part of the 10X Annexin V Binding Buffer with 9 parts of distilled water. A total of 20 μL of suspension was added to 980 μL of 1×Annexin V binding buffer in an Eppendorf tube. Next, in a 5 mL FACS tube (FALCON), 5 μL of Annexin V-FITC stock and 5 μL of PI (stock of 1 mg/mL) were added to a 100 μL suspension containing sperm under dark conditions. After incubation for 15 min, 400 μL of 1×Annexin V binding buffer was added, and the samples were assessed using a FACSCalibur® flow cytometer . For this assessment, the labeling patterns in the annexin (AN)/PI analysis were classified as follows: viable (AN−/PI−), viable but phosphatidylserine (PS) translocated (AN+/PI−), dead and PS translocated (AN+/PI+), and dead and late necrotic sperm (AN−/PI+). The apoptosis index was calculated as the ratio of AN+/PI− spermatozoa to total viable (PI−) spermatozoa.Apoptosis was assessed using a combination of FITC annexin and PI stain  in accorThis experiment was designed to study the effects of myo-inositol supplementation prior to freezing on the post-thaw sperm parameters of boars. The experiment was conducted in two trays: first, to evaluate the effect of myo-inositol supplementation on post-thawing sperm parameters, including motility parameters (CASA), viability (SYBR14/PI), acrosome integrity (PSA/FITC), and MMP (Rh123/PI). All parameters were measured for the five experimental groups.In the second part of the experiment, the effects of myo-inositol on sperm parameters including oxidative status, apoptosis, gene expression, and fertilization parameters in cryopreserved boar sperm were evaluated. Six tests were carried out. To evaluate the IVF outcome by cleavage on Day 2 and blastocysts on Day 7, the control and 0.5 mg/mL myo-inositol-treated groups were selected to evaluate fertilization parameters and gene expression levels. qPCR was used to analyze the transcription levels of selected genes.Oxidative status was assessed by measuring ROS levels and lipid peroxidation in two experimental groups: control and 0.5 mg/mL myo-inositol. We used H2DCFDA and PI flow cytometer probes to measure ROS and a spectrophotometer kit to measure lipid peroxidation.Thirdly, we assessed apoptosis in two ways: caspase activity using a spectrophotometric kit  for all treated groups and apoptosis index (FITC-Anexin V and PI staining) by flow cytometry for the control and myo-inositol-treated groups.t-test was conducted to evaluate the data of the remaining experiments . The results are expressed as mean ± SEM, and a probability value of p < 0.05 was statistically significant.Each experiment was repeated at least three times. All experimental data were evaluated using SAS software . Sperm-related data  were compared using one-way analysis of variance, followed by Duncan’s multiple range test. An unpaired two-tailed Student’s p < 0.05). The rapid and medium-progressive motility of the myo-inositol-treated groups were not significantly different from that of the control group (p > 0.05). Between different myo-inositol concentrations, 0.5 mg/mL reveals a significant increase in TM, PM, and RPM, and 2 mg/mL reveals a significant decrease in all motion parameters compared to other myo-inositol groups.The effects of myo-inositol supplementation in semen extenders on boar sperm motility are shown in p < 0.05). The other treated groups had higher viability than the control group, but there was no statistical difference between them and the control group (p ≥ 0.05).p ≥ 0.05). BAX, a pro-apoptotic gene, showed a significant decrease in expression in the myo-inositol-treated group compared to the control group. The expression of the Bcl-2 apoptosis regulator gene (Bcl-2), an anti-apoptotic gene, did not differ significantly from that in the control group. ROMO1 gene expression levels were significantly lower in the myo-inositol group than in the control group; conversely, the SMCP gene, which is related to sperm motility, showed higher expression in the myo-inositol-treated group than in the control group. The NOX5 and SMOX genes were not significantly different between the two groups.Based on the results of the prior experiments, the optimal treatment concentration was set at 0.5 mg/mL, and subsequent experiments were conducted with only the control and 0.5 mg/mL treatment groups. Real-time PCR was conducted for the control and 0.5 mg/mL myo-inositol-treated groups. p < 0.05), as shown in The effects of myo-inositol-treated semen were evaluated as previously described. For this test, the control and the myo-inositol-treated (0.5 mg/mL) groups were compared. The cleavage percentage was evaluated on Day 2, and blastocyst percentages were calculated on Day 7. Although there was no significant difference in cell number between the two groups, the myo-inositol-treated group demonstrated a significant increase in cleavage and blastocyst rates compared to the control group (p ≥ 0.05).Oxidative stress was evaluated using the reactive oxygen levels shown in Flow cytometry revealed that myo-inositol positively affected the decrease in apoptosis levels after the cryopreservation. Boar sperm have weak defenses against ROS because of the low antioxidant concentration in seminal plasma. In addition to this, it contains a high concentration of polyunsaturated fatty acids (PUFA), which inhibit oxidative damage. During cryopreservation, an increase in free radicals leads to oxidative stress, which leads to structural and functional changes and finally apoptosis. Myo-inositol improves sperm quality, reduces ROS production, protects acrosome integrity and mitochondrial function, protects against cryodamage, and reduces apoptosis ,41,42,43Sperm motility and viability are greatly reduced after thawing; this reduction is related to the factors mentioned previously ,45. ThisMyo-inositol plays a role in protecting sperm acrosome integrity through osmoregulation and reducing ROS production . In the Our observation reveals negative effects of the highest myo-inositol concentration (2 mg/mL) on some boar sperm parameters. These results were not surprising. There was a previous study on dogs that revealed a significant decrease in motility, viability, and some kinematics in dogs, and the author refers to negative effects due to either disturbance in osmolarity or toxicity . On the 2O2 in sperm leads to a reduction in embryonic production and blastocyst number [2O2, which may explain the low blastocyst numbers in the control group.Fertilization success is highly related to semen quality, which translates to the oxidative status of sperm and directly affects fertilization and blastocyst percentage ,57. Henct number . In our The freeze–thaw process is stressful to sperm, and during this process, sperm confront prolonged stress and increase ROS production, which leads to DNA fragmentation, changes in membranes, and cell apoptosis. Multiple studies report strong beneficial effects of myo-inositol on protecting sperm DNA and reducing ROS and apoptosis during cryopreservation ,65,66. OReactive oxygen species (ROS) damage is a well-known occurrence in boar sperm cells since the presence of polyunsaturated fatty acids (PUFA) renders the sperm membranes more susceptible to oxidative damage. Lipid peroxidation caused by ROS leads to increased apoptosis ,69. Our In conclusion, supplementation of the semen extender with myo-inositol improved boar sperm parameters after the freeze–thaw process, including motility, viability, acrosome integrity, decreased apoptosis, oxidative state-related genes, reduced ROS levels, and improved fertilization ability and blastocyst rate. Our study recommendation is to use 0.5 mg/mL of myo-inositol as a supplement to the boar semen extender used for AI. To fully understand the benefits of myo-inositol on boar semen, further studies are needed to investigate its impact on embryo quality (both in vivo and in vitro) and lipid peroxidation.


A global fittinganalysis led to an analytical equation describing quantitatively theprotective effects of aminosterols as a function of their concentrationand relevant membrane effects. The analysis correlates aminosterol-mediatedprotection with well-defined chemical moieties, including the polyaminegroup inducing a partial membrane-neutralizing effect (79 ± 7%)and the cholestane-like tail causing lipid redistribution and bilayermechanical resistance (21 ± 7%), linking quantitatively theirchemistry to their protective effects on biological membranes.Natural aminosterols are promising drug candidates againstneurodegenerativediseases, like Alzheimer and Parkinson, and one relevant protectivemechanism occurs via their binding to biological membranes and displacementor binding inhibition of amyloidogenic proteins and their cytotoxicoligomers. We compared three chemically different aminosterols, findingthat they exhibited different (i) binding affinities, (ii) chargeneutralizations, (iii) mechanical reinforcements, and (iv) key lipidredistributions within membranes of reconstituted liposomes. Theyalso had different potencies (EC These transient aggregates form both during the process ofamyloid fibril formation and upon their release from mature fibrils,and oligomers are able to interact with a number of molecular targets,including biological membranes, giving rise to a cascade of dysfunctionalevents.3Many of the most severe neurodegenerativediseases originate fromthe conversion of specific polypeptide chains from their native solublestates into amyloid aggregates, forming various types of extracellulardeposits or intracellular filaments, including amyloid plaques bythe amyloid-β (Aβ) peptide, neurofibrillary tangles bythe tau protein, Lewy bodies by α-synuclein (αS), amyloidplaques by the prion protein  isolated from thedogfish shark 13 SQ  has just completeda multicenter, randomized, double-blind, placebo-controlled phase-2bclinical trial in patients with PD-related constipation  and shown improvement in constipation as well as hallucinationsand dementia.14 A previous multicenter,open label phase 2 study in patients with Parkinson’s disease had also shown improvement in constipation,sleep, REM-behavior disorder, hallucinations, and dementia.15 Both TRO and SQ have passed phase 1 clinicaltrials showing good safety and tolerability .These compounds are known as squalamine(SQ) and trodusquemine(TRO), which are also known as ENT-01 and MSI-1436, respectively 1A. In mo11 TRO was reportedto stably insert into the hydrophilic portion of the first upper layerof the membrane, down to the interface between the hydrophilic andhydrophobic portions, exposing both sulfate and spermine groups tothe aqueous phase  as a model of thelipid bilayer of the cell membrane.us phase 1B.11 Theer plane 1B. The ier plane 1C: (i) aInspite of these recent advances, it is not yet clear which ofthe three aforementioned changes of the membrane bilayer induced byAMs are the most effective in mediating their protective role andif all of them are relevant. Moreover, it remains to be elucidatedwhich of the chemical moieties of the AMs are mainly involved in mediatingtheir protection. These drawbacks limit our ability to predict thepotency of other natural AMs and to anticipate the potency of otherrelated or newly designed molecules. More generally, they limit ourunderstanding of the relationship between the chemistry of small molecules,the physicochemical status of the cell membrane, and the toxicityof misfolded oligomers, thereby hindering the search of generic strategiesto protect cell membranes from the action of deleterious protein oligomers.Mus musculus,16 whereas TRO and SQ were originallyisolated from the dogfish shark Squalus acanthias.18 The three AMs share a sterol group, an alkylmoiety of the cholestane-type fused to the sterol at C-17, and analkyl polyamine tail fused to the sterol at C-3 and replacing thehydroxyl group  values of 10 μM BODIPY TMR-X-labeled AM (AM-BODIPY)and Alexa Fluor 594-labeled AM (AM-A594) were measured in the absenceand presence of 0.5 mg/mL LUVs incubated with the three AMs for 15min. BODIPY and A594 labeled the distal primary amino group of thepolyamine chain, which is known to stick out of the membrane fromprevious studies on TRO11 and is not expected,therefore, to affect the AM-membrane binding affinity significantly.In addition, the two probes have different chemical properties. BODIPYhas a lower molecular weight, is hydrophobic, and has a neutral netcharge, whereas A594 has a higher molecular weight, is hydrophilic,and has a negative net charge. These differences enable the assessmentof whether the probe chemistry affects dramatically the LUV-AM bindingaffinity.To investigate whether TRO,SQ, and ENT-03 bind to LUVs, the fluorescence anisotropy  when incubatedwith LUVs (0.5 mg/mL) for 15 min (p < 0.001). Notably,SQ labeled with both dyes showed a greater increase compared to theother two AMs, as noticed also for the r value ofSQ-A594 (p < 0.001). This can be attributed tothe shorter polyamine group of SQ relative to TRO and ENT-03, whichreduces the distance of the dye probe from the membrane. Fluorescenceof 10 μM L-Arg-BODIPY and L-Arg-A594 did not change significantlyupon LUV addition , indicating a higher affinity for LUVs comparedto the other two AMs. This observation can be attributed to the shorterhydrophilic spermidine moiety of SQ, as opposed to the longer hydrophilicspermine moiety of TRO and ENT-03, which increases the overall hydrophobicityof SQ and explains its higher affinity for LUVs. By contrast, TROand ENT-03 displayed similar KD valuesin both analyses with BODIPY and A594 .Since all three labeled AMs increase their fluorescence upon LUVbinding, we exploited this spectroscopic property to obtain quantitativemeasurements of the affinity of the three labeled AMs for LUVs. Theywere therefore incubated for 15 min (10 μM) with increasingconcentrations of unlabeled LUVs and we observed in all cases a significantincrease in fluorescence emission 3. By fiteled AMs 1. With bKD value determinedwith BODIPYfor a given AM was about 4-fold lower than that determined for thesame AM with A594, indicating that the chemistry of the probe affectsthe AM-LUV affinity to some extent. However, the rankings and relativedifferences of the KD values determinedfor the three AMs are similar when determined with either probe, indicatingin both cases that TRO and ENT-03 have similar affinities for theLUV membrane, within experimental error, and that SQ has a ca. 2-foldhigher affinity. Since the two probes add a hydrophilic and hydrophobiccomponent to the AMs, it is likely that the binding constant of agiven unlabeled AM is intermediate between these two values. Negativecontrols with labeled L-Arg and increasing concentrations of LUVsshowed the lack of variation in fluorescence, confirming that thebinding abilities of labeled AMs to LUVs were mediated by the AM ratherthan the fluorescent probe bound to them . Two kineticphases were observed, occurring on the time scales of ca. 500 ms and10 s, respectively, therefore indicating that the binding was veryrapid and that, after a time of 15 min explored here, binding hasattained equilibrium. This holds true even at the lowest LUV concentration(0.12 mg/mL) tested . Thesetwo phases may either represent the signature of a two-step bindingmechanism or reflect two ligand subpopulations that bind LUVs withdifferent kinetics. Assignment of the two phases to well defined molecularevents is beyond the scope of the present analysis.To explore the kinetics of the AM-LUVbinding, TRO-A594 as a representativeAM and LUVs were rapidly mixed using a stopped-flow apparatus to finalconcentrations of 10 μM and 0.5 mg/mL, respectively, and theTRO-A594 fluorescence change during the binding process was monitoredin real time . Since light scattering intensity isproportional to the second power of the mass of the light scatteringparticles, by incubating LUVs with increasing concentrations of thethree AMs, it was possible to obtain a measure of the increase ofLUV mass due to AM incorporation in these three cases 4.p < 0.05), whereas TRO and ENT-03 displayedsimilar saturating concentrations (p > 0.05). The saturatingconcentrations were found to be 35 ± 7, 12 ± 4, and 43 ±5 μM for TRO, SQ, and ENT-03, respectively. These values canalso be obtained from the LUV mass increase at saturation, which yieldsthe mass of incorporated AMs, which were found to be 35 ± 4,15 ± 2, and 43 ± 4 μM for TRO, SQ, and ENT-03, respectively,in very good agreement with those estimated above. All AMs did notshow a significant increment of LUV diameter , ruling out that the observed increase of lightscattering intensity upon AM addition was due to an increase in LUVsize. This analysis indicates that AMs bind to LUVs even without fluorescentlabeling and allows the maximum AM occupancy to be estimated for allthree AMs.All AMs induced an increase in light scatteringintensity, andthus LUV mass, until a saturating concentration, after which theyexhibited a plateau phase, where no more AMs were incorporated. SQreached the plateau at a significantly lower concentration comparedto the other AMs . Weobtained a value of ca. 28–35 μM TRO at saturation, inagreement with the value of 35 ± 4 μM TRO estimated withlight scattering .To obtain an independent estimate of the AM occupancyat LUVs atsaturation, we employed a microfluidic technique using LUVs and TROas a representative AM  measurements, the resistanceof the bilayer to a breakthrough force (BTF) perpendicular to thebilayer plane, monitored with supported lipid bilayers (SLBs) andatomic force microscopy (AFM), and the distribution of lipids in themembrane, monitored with lipid–lipid fluorescence resonanceenergy transfer (FRET).Since 5 μMAM and 1.0 mg/mL LUVs are concentrations at which binding is complete, we carriedout the following analyses at these concentrations, in all cases aftercovesiculating AMs with the lipids of LUVs to rule out incompletebinding. In particular, we evaluated the effect of the three AMs onthree physicochemical properties of the LUV lipid bilayer previouslyfound to be altered by TRO and thought to represent important factorsfor the vulnerability of the lipid plasma membrane to misfolded proteinoligomers:11 whereas values of −18.7 ± 0.3, −21.0± 0.5, and −18.7 ± 0.3 mV (mean ± SEM) wereobtained for LUVs covesiculated with TRO, SQ, and ENT-03, respectively,at the same temperature . SQ induced a smaller decrease compared to TRO and ENT-03(p < 0.01), which showed by contrast comparabledecreases (p > 0.05). This is in agreement withthechemical properties of the three AMs, with SQ carrying a spermidinegroup, which is shorter and less positively charged than the sperminegroup of TRO and ENT-03.The ζ was measured using nakedLUVs and LUVs covesiculated with TRO, SQ, or ENT-03 2. For naperature 2. These Tm) of LUVs in the absence and presenceof each AM (Tm of this LUV system is dominated by SM (Tm of 35–40 °C), since DOPC (Tm of −17 °C), which is the most abundant lipid,is already in a fluid-like phase in the examined temperature range.20 For naked LUVs, the Tm was observedat 24.5 ± 2 °C. All AMs induced an increase of the transitiontemperature of LUVs, up to values of 46 ± 2 °C (p < 0.01), 35 ± 2 °C (p <0.05), and 46 ± 2 °C (p < 0.01) withLUVs covesiculated with TRO, SQ, and ENT-03, respectively, indicatingtrend variations similarly to those measured with ζ at 20 °C.The transition width is about 6 °C for the naked LUVs, but becomeslarger with AM, up to 16 °C in the case of TRO-containing LUVs,confirming that AMs modify the packing of the bilayer and system disorder.21ζ measurements versus temperature were thenused to determinethe phospholipid gel to liquid-crystalline phase transition temperaturerequired to penetrate the bilayer with the AFM tip. Measurements wereperformed on SLBs in the absence and presence of each AM 6. As pre11 To investigate whether SQ and ENT-03 could havea similar impact, we carried out a series of lipid–lipid FRETexperiments in the absence and presence of each AM. In these experiments,0.0625% of a given lipid was labeled with a donor (D) fluorescent probe and the same fractionof CHOL was labeled with an acceptor (A) fluorescent probe, namely,BODIPY FL and BODIPY 542/563, respectively. Four different combinationsof FRET pairs were then analyzed in the presence of SQ and ENT-03 and then comparedto data obtained with LUVs without AMs and with LUVs with TRO (E) in the CHOL-D/CHOL-A pair (p < 0.001) and a reduction in the GM1-D/CHOL-A pair (p < 0.01), indicating a reduction of the mean shortestdistance between CHOL molecules or GM1 molecules and an increase ofthe distance between CHOL and GM1 molecules (p < 0.001 relative to absence of AM), but wasfound to occur to a significantly lesser extent (E for the SM-D/CHOL-A and DOPC-D/CHOL-Apairs (p > 0.05), in agreement with the effectofTRO  fromsmall unilamellar vesicles (SUVs) composed of DMPS,t 198 nm 8A–C.This conformational change was compared for thethree AMs as aprogressive increase of mean residue ellipticity at 222 nm, showingthat SQ is slightly less effective, as it requires higher concentrationsto displace the protein from the membrane relative to TRO and ENT-03,which had similar displacement activities 8D. This We then passed from LUVs to cultured cells (human neuroblastoma SH-SY5Ycells) to investigate whether all three AMs bind to the plasma membraneof cells and protect them from misfolded protein oligomers. All threeAMs were labeled with BODIPY TMR, which is a neutral and hydrophobicprobe that does not alter the positive net charge of the AM. SH-SY5Ycells were treated with 5 μM TRO-BODIPY, SQ-BODIPY, or ENT-03-BODIPYfor 30 min at room temperature and analyzed with confocal scanningmicroscopy 9A. All t42 as sampleoligomers and evaluating the influx of calcium ions (Ca2+) from the extracellular space to the cytosol of cultured SH-SY5Ycells, which is thought to be the earliest insult following the oligomer-membraneinteraction.30 SH-SY5Y cells were treated for 15 min with ADDLs  in the absence or presence of different concentrationsof AMs and then their Ca2+ levels were evaluated with aspecific fluorescent probe that enters inside the cells and producesgreen fluorescence (F) only when bound to Ca2+ . TRO and ENT-03 wereboth more effective than SQ (p < 0.001 in bothcases). At the highest AM concentration tested (5 μM), all threeAMs were able to completely suppress the ADDL-induced Ca2+ influx down to the levels of untreated cells  composed of Aβ to Ca2+ 9B,C. ADDed cells 9C.2+-derived fluorescence (F) shown in R) valuesranging from 0% (no effect) to 100% (full effect) using:F0 and Funtreated are the F values with and without ADDLs, respectively, both withoutAMs .The obtained R values were plotted versus AM concentrationin one single semi-log plot to obtain the typical dose–responsecurve used in pharmacology . A fairly good agreement was foundbetween theoretical R values redetermined with R values . Nevertheless, the agreement was not entirely satisfactorydue to differences among the three AMs.All data of Camacology 9D. The rR values at corresponding AMconcentrations, we recognize two different contributions to the EC50 of the AMs: the change in charge and the change in packing,which add to an offset EC50 value in the absence of thesetwo changes (EC′50):To improve the agreementand identify the AM-induced membrane alterations responsible for theobserved changes of 32 This leads to aphenomenological Hill equation, where all R valueswere analyzed in a multivariable and multiparameter global fittingprocedure:p is the experimentally determined and normalized changeof lipid distribution and packing observed upon AM addition  are used to express one dependent variable (R),upon the global fitting of the four constants  using all the available data. Fitting all R values to –7 M, −3.99 ± 0.82 × 10–7, −1.03± 0.22 × 10–7, and 0.795 ± 0.015for EC′50, kcharge, kpacking, and n, respectively.The equation can describe well the behavior of the three AMs plottedseparately in three independent graphs .Thisequation represents a Hill equation integrated with the t graphs 9F–H, wheR value was leftout from the analysis, one by one, to redetermine, through the globalfitting, the four constants of R valuecorresponding to the left-out experimental R value.A good agreement was found between redetermined theoretical versusexperimental R values , indicating theability of R values that are not present in the analysis.The model and resulting kcharge and kpacking values areboth negative, indicating that both the partial charge neutralizationand compaction of the membrane contribute to the increase of AM potency. Their relativecontributions amount to 79 ± 7 and 21 ± 7%, respectively,indicating that the charge effect is predominant. A hypothetical nonnaturalAM that does not affect ζ and p  wouldhave a potency 6–7-fold lower than that of TRO. TRO appearsthe most effective AM because it produces the highest effects in termsof both membrane neutralization and compaction. ENT-03 is marginally,albeit significantly, less effective because it has a much lower effecton membrane compaction. This feature accounts for only 21 ± 7%of the effect, therein explaining why its potency is only slightlylower. By contrast, SQ appears markedly less effective because itleads to a much lower change in charge than TRO and ENT-03, despitea packing effect similar to that of ENT-03.What can we learn from this analysis? The KD)of the AMs for LUVs as a parameter in the global fitting,but this has not resulted in any improvement, particularly becausethe least protective SQ has also the highest binding affinity forthe lipid bilayer, suggesting that binding affinity is a less relevantfactor. This can be rationalized by the fact that the lipid concentrationis very high in the two-dimensional carpets of cells, making the AMswork in a saturation regime similar to that of high LUV concentrationin the conditions explored in We also includedthe different binding affinities  rather than experimental observables (Δζand Δp):R are the independent and dependent variables, respectively; EC′50, apolyamine, and aSO3/COO are constants free to float in the global fittinganalysis using all the available data. apolyamine and aSO3/COO are parameters accountingfor the change of the EC′50 parameter when a spermine,rather than a spermidine, and a −SO3–, rather than a −COO– group, are presentin the AM, respectively. The two parameters were constrained to 0when the spermine and −SO3– groupswere absent and replaced by spermidine and carboxylate, respectively.Fitting all R values to –7, −2.36 ± 0.47 × 10–7, and −0.55 ± 0.12 × 10–7 M forEC′50, apolyamine, and aSO3/COO, respectively .To attribute the potency as well as the apolyamine and aSO3/COO values were again foundto be negative, indicating that the longer spermine and SO3–-containing tail cause a potency increase (orEC50 decrease) relative to a hypothetical nonnatural AMhaving a spermidine and carboxylate group. They account for 81 ±7 and 19 ± 7% of the effect, respectively, in good agreementwith the values obtained with the experimental analysis and confirmingthat the charge effect of the polyamine is predominant over the chemistryof the tail on the other side of the molecule that causes a redistributionof lipids and increased packing. The net positive charge of AMs allowsa larger decrease of the negative charge of the cell membrane providedby GM1, which is a physicochemical change reported to be crucial inprotecting biological membranes from Aβ oligomer binding andresulting cell toxicity also using other compounds, such as europiumpositive ions.33 The EC′50 value obtained for the hypothetical nonnatural AM with spermidineand carboxylate (3.83 ± 0.39 × 10–7 M),which is higher than that of any AM analyzed here having at leastthe spermine (triamine) or SO3– group,is lower than the hypothetical nonnatural AM of the previous analysishaving a monoamine group and shorter tail on the other side (5.94± 0.59 × 10–7 M). Since all natural andnonnatural AMs studied here have a steroid scaffold, their EC50 values remain in the 10–8–10–7 M range.The r). Importantly,their binding to the membranes of cultured SH-SY5Y cells results intodifferent degrees of protection against the action of misfolded proteinoligomers of the Aβ42 peptide to Ca2+ influx.The protection depends on AM concentration, level of charge neutralizationof the membrane and change of packing resulting from lipid redistribution.The three natural AMs studied here havea different chemistry andcombination of specific, relevant functional groups. Consequently,their binding to the lipid bilayer of a liposome membrane resultsin different perturbations of the physico-chemical properties of theliposome bilayer, such as their surface charge (ζ), resistanceto a mechanical breakthrough force (BTF) perpendicular to its planeand distribution of CHOL, and GM1 lipids  for 180 min. The mixture was hydrated ata total lipid concentration of 1.0 mg/mL with distilled water to formmultilamellar vesicles (MLVs), left to swell for 1 h at 60 °C,and then extruded 17 times through a polycarbonate membrane with 100nm pores using a miniextruder (Avanti Polar Lipids) at the same temperature,to form large unilamellar vesicles (LUVs). After cooling to room temperature,LUVs were stored at 4 °C for a maximum of 1 week. For the measurementof the ζ potential and BTF, and for the lipid-lipid FRET experiments,5 μM of each AM was added during the hydration phase of LUVspreparation.Liposomeswere produced with a lipid mixture composed of 1,2-dioleoyl-sn-glycero-3-phosphocoline and sphingomyelin in a molar ratio of 2:1 (mol/mol), 1% (mol) cholesterol ,and 1% (mol) monosialotetrahexosylganglioside 1 . The lipids were dissolved in chloroform/methanol (2:1), andthe organic solvent was removed by evaporation R)-3-keto-7-hydroxycholestan-24-olsulfate steroid intermediate as previously described.39 The synthesis of ENT-03 was carried out similarly to the other AMs,by coupling a polyamine tail to a steroidal skeleton;16 the step by step procedure is reported in a deposited patentand will be published in a separate paper .The >95% chemical purities of all AMs were assessed by HPLC-ELSD and 1H-NMR . All AMswere stored aspowders until use. For the labeling procedure, AMs were dissolvedin distilled water to obtain a 100 mM stock solution and stored at4 °C. BODIPY TMR-X NHS Ester and Alexa Fluor 594 NHS Ester  were both dissolvedin anhydrous DMSO to obtain 15 and 10 mM stock solutions, respectively,and stored at −20 °C. For labeling, 5 mM AM, 0.5 mM dye,0.1 M sodium bicarbonate buffer, pH 8.3 for BODIPY and pH 7.0 forA594, were incubated in a final volume of 20 μL at 25 °Cfor 2 h under mild orbital shaking. During labeling with BODIPY theAM precipitates, therefore, after the incubation, the solution wascentrifuged at 18,000g for 15 min; the pellet wasdried with a nitrogen flow and resuspended in 20 μL DMSO tomaintain the initial concentrations. During labeling with A594, TROremains in solution, whereas SQ and ENT-03 precipitate. Hence, thesolution with TRO labeled with A594 was directly used after incubation,while those with SQ and ENT-03 were centrifuged and resuspended inDMSO as described for the BODIPY labeling. With these procedures,the labeled:total AM was 1:10 in all cases. No unreacted dye was detectedusing mass spectrometry, following a previously described procedure.11 As a negative control, L-Arg was labeled withboth BODIPY and A594 under the same conditions used for AM labelingand no precipitate was observed.SQ and TRO were synthetized by coupling spermidine and spermine,respectively, to the  values were then acquired at 570 nm after excitationat 544 nm and at 617 nm after excitation at 590 nm, respectively,in the absence and presence of 0.5 mg/mL unlabeled LUVs incubatedfor 15 min in the dark, using a 3 × 3 mm black walls quartz cellat 25 °C on an Agilent Cary Eclipse spectrofluorometer (AgilentTechnologies) equipped with a thermostatted cell holder attached toan Agilent PCB 1500 water Peltier system.BODIPY or A594-labeled AMs and L-Arg (negative control) were dilutedwith distilled water to 10 μM. The fluorescence anisotropy (BODIPY or A594-labeled AMs and L-Arg (negative control) were dilutedwith distilled water to 10 μM. Fluorescence emission spectraof AMs and L-Arg labeled with BODIPY and A594 were acquired from 550to 650 nm (excitation at 544 nm) and from 600 to 700 nm (excitationat 590 nm), respectively, in the absence and presence of 0.5 mg/mLunlabeled LUVs incubated for 15 min in the dark, using the cell andspectrofluorometer described above.F isthe fluorescence intensity at a given LUV concentration, F0 is fluorescence intensity at 0.0 mg/mL LUVs, A is the difference between the fluorescence emission ofunbound and bound AMs, and KD is the dissociationconstant of the LUV-AM complex.BODIPY or A594-labeled AMs and L-Arg (negative control) were dilutedwith distilled water to 10 μM and incubated with increasingconcentrations of unlabeled LUVs (from 0.0 to 1.0 mg/mL) for 15 minat 25 °C in the dark. Fluorescence emission of BODIPY and A594-labeledAMs and L-Arg were then acquired at 572 nm (excitation at 535 nm),and at 612 nm (excitation at 590 nm), respectively, using the celland spectrofluorometer described above. The weak fluorescence contributionof unlabeled LUVs was subtracted from fluorescence emission spectra,and resulting values were then normalized to the value obtained inthe absence of LUVs (taken as 100%). The fluorescence emission intensitywas then plotted versus LUV concentration, and data points were thenfitted with:2O. We used a Bio-logicSFM-3 stopped-flow device  attached to a fluorescencedetection system, an FC-08 cuvette (path length 0.08 cm), an excitationat 380 nm, and a band pass filter to collect emission above 475 nm.The flow rate was 2.19 mL/s. The injection time, total volume, anddead-time were 160 ms, 350 μL, and 14 ms, respectively. Thefinal conditions after dilution were 10 μM TRO-A594, with LUVconcentrations ranging from 0.12 to 1.00 mg/mL, 25 °C. Each tracewas averaged over 2–7 experiments, normalized to the maximumfluorescence, and then analyzed with a double exponential equation:f(t) is the fluorescence recorded at time t, m and q are the slope and interceptof the plateau signal, respectively, A1 and A2 the amplitudes of the exponentialphases, and k1 and k2 are their apparent rate constants. Plots of k1 and k2 versus LUV concentrationwere fitted to straight lines:40TRO-A594(50 μM) was diluted 5-fold into solutions containing differentconcentrations of LUVs dissolved in HI isthe total intensity of light scattered by LUVs in kcps, n is the number of LUVs, m is the mass of a singleLUV, and I0 is the intensity of lightscattered by a single unitary mass of LUV in kcps. LUV hydrodynamicdiameter did not change with addition of any of the AMs, indicatingthat n and I0 remainconstant. By contrast, when the AM is incorporated into the LUVs, m increases. The relative increase of LUV mass determinedby the addition of AMs was then calculated with:IAM and INO are the intensitiesof light scattered by LUVs in the presence and absence of a givenconcentration of AM and mAM and mNO are the LUV mass concentrations in the presenceand absence of a given concentration of AM. The obtained data werethen plotted versus AM concentration.LUVs were diluted with distilledwater to 0.5 mg/mL and incubated for 15 min at 25 °C with increasingconcentrations of AMs (from 0 to 100 μM). The size distributions(light scattering versus apparent hydrodynamic diameter) and countrate  were then recorded at 25 °C,using a Zetasizer Nano S or APS , thermostatted with a Peltiertemperature controller, measurement position 4.20 mm, attenuator 6,and using disposable low volume (45 μL) plastic cuvettes. Accordingto the laws of light scattering, the following equation holds:Fd/Fund) or as the ratio offluorescence values in the diffused channel versus total fluorescence[Fd/(Fd + Fund)]. This ratio parameter correlated directlywith diffusion rapidity and inversely with size of the fluorescentmolecule or its complex with LUVs. When this value was in the appropriaterange, it was converted automatically by the instrument into a hydrodynamicradius (Rh).LUVs werediluted with distilled water to 0.5 mg/mL and incubated for 15 minat 25 °C with 10–100 μM TRO-A594 or 20 μMTRO-BODIPY (1:10 dye:TRO) or 2 μM CHOL-BODIPY (1:1 dye:CHOL);samples with 10 or 50 μM TRO-A594 without LUVs were also prepared.The diffusion of the fluorescently labeled molecule in the varioussamples was evaluated with the microfluidic technique using a FluidityOne-W instrument  and placing a 5 μL dropof the sample on a disposable microfluidic chip made of cyclic olefincopolymer (COC) manufactured using injection molding .The diffusion was evaluated as the ratio of fluorescence values inthe diffused versus that in the undiffused channels . The ζ potential measurements were also usedto determine the transition temperature (Tm) in LUV systems.42 The Tm values were determined by analyzing the first-orderderivative of ζ with respect to temperature (dζ/dT) as a function of temperature: the Tm corresponds to the minimum of the curve, while the amplitudeof the transition was assumed to correspond to the full width at halfmaximum (FWHM) of the derivative curves.Zeta potential (ζ)measurements were performed with a Zetasizer Pro Red Label .LUVs were covesiculated with 5 μM of each AM at a total lipidconcentration of 1 mg/mL. About 600 μL of each LUV sample wasdiluted to obtain a total lipid concentration of 0.25 mg/mL, withphosphate buffer, 5.57 mM ionic strength, pH 7, 20 °C, and putin a disposable folded capillary cell . Eachζ potential value is the average of three independent runs;for each temperature, the ζ potential was determined as themean of 5–8 measurements. The reported error is the standarddeviation of the measurements. The measurements were performed inthe range 10–60 °C, every 2 °C . The electrophoretic mobility measurementswere converted into ζ values according to the Smoluchowsky model.2 solution onto a 1.0 ×1.0 cm2 freshly cleaved mica substrate. The samples werestored for 10 min at room temperature and then incubated for 15 minat 60 °C in a closed chamber at 100% relative humidity. The sampleswere cooled down at room temperature for 2 h and finally gently rinsedwith Milli-Q water to remove nondeposited vesicles. Prior to AFM imaging,samples were kept again at room temperature in a closed chamber at100% relative humidity. AFM imaging usually started 1.5 h after rinsing.LUVs were prepared ata total lipid concentration of 1.0 mg/mL covesiculated with 5 μMof each AM. Supported lipid bilayers (SLBs) were obtained by depositing40 μL of each LUV suspension (after a ten-fold dilution) and10 μL of a 10 mM CaClin situ using thethermal noise method.43 Force maps consistingof 128 × 128 force distance curves were acquired point-by-pointon scan areas of 5 × 5 μm2 or 2.5 × 2.5μm2. The maximum force load was 15–18 nN.Breakthrough forces were evaluated from the force–distancecurves data sets using a home-built software.Force spectroscopy measurements were performed under a liquid environmentwith a Multimode SPM (Bruker) equipped with “E” scanninghead (maximum scan size 15 μm) and driven by a Nanoscope V controller(Bruker). Triangular silicon nitride cantilevers  were used. The actual spring constantof each cantilever was determined LUVs were prepared at a totallipid concentration of 1.0 mg/mL, as described above. TRO, SQ, andENT-03, when present, were added during the hydration phase to a finalconcentration of 5 μM. BODIPY-FL C5-ganglioside GM1 (GM1-D),BODIPY-FL-cholesterol (CHOL-D), BODIPY-FL-sphingomyelin , and BODIPY-FL-DOPC were usedas donor lipids. Cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate was used as an acceptor lipid. The molar fraction of eachlipid labeled with D or with A was 0.0625% of total lipids in allcases.E) were calculated asFDA is the fluorescence intensity of D in the presence of A,and FD is the fluorescence intensity ofD in the absence of A.44 FRET E calculated with r)using:44R0 is the Forster distance and was previously calculated forthis D/A probe pair.11Fluorescence spectra of LUVs containing only lipid-D,only CHOL-A, and both lipid-D and CHOL-A were acquired using the celland spectrofluorometer described above, at 25 °C, with excitationat 450 nm and emission from 480 to 640 nm. FRET efficiencies  was incubatedwith DMPS LUVs  for 30 min and then with increasing concentrations ofTRO, SQ, or ENT-03  for15 additional min, in 20 mM sodium phosphate buffer, pH 6.5, at 30°C. LUVs were prepared with the same procedure reported above,but using 100% DMPS. Far-UV CD spectra were recorded on a Jasco J-810spectropolarimeter equipped with a thermostated cell holder attachedto a Thermo Haake C25P water bath using a quartz cuvette with pathlength of 1 mm, at 30 °C. CD spectra were recorded from 180 to260 nm by averaging 5 spectra with a data pitch of 0.2 nm, a scanningspeed of 50 nm/min, and a response time of 1 s. All spectra were blanksubtracted and truncated at HT > 700 V, then normalized to meanmolarresidue ellipticity using:42 (Bachem) was dissolved in HFIP to 1.0 mM andincubated for 1 h at room temperature to allow complete peptide monomerization.Aβ42-derived diffusible ligands (ADDLs) were preparedas described previously.45 In particular,the HFIP was evaporated with a gentle flow of N2 and thedried protein was resuspended to 5 mM with DMSO and then diluted withphenol red free F-12 HAM to 100 μM. The sample was then incubatedat 4 °C for 24 h and centrifuged at 12000g for10 min, 4 °C, to collect the supernatant containing the oligomers.LyophilizedAβ3 (1:1)supplemented with 10% fetal bovine serum (FBS), 1 mM glutamine, and1% penicillin and streptomycin solution (Sigma-Aldrich). Cells weremaintained in a 5% CO2 humidified atmosphere at 37 °C, grown until 80% confluence for a maximum of 20 passages, and routinelytested to ensure that they were free form mycoplasma contamination.46 The cell line was authenticated by the EuropeanCollection of Authenticated Cell Cultures using short tandem repeatloci analyses.Authenticated human SH-SY5Y neuroblastomacells were purchased from A.T.C.C. and cultured in Dulbecco’sModified Eagle’s Medium (DMEM), F-12 Ham with 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and NaHCO2 equilibration. Ten min before the incubation ending, theHoechst 33342 dye was added to the culture medium (10 μg/mL).The analysis of AM-derived fluorescence and nuclei-derived fluorescencewere performed on a Nikon Eclipse TE300 C2 confocal laser scanningmicroscope (Nikon) equipped with a Nikon 60x immersion oil objective and with Coherent CUBE (diode 405 nm) and CoherentSapphire (Sapphire 561 nm) lasers. The emission filters for imagingwere 452/45 and 595/60 nm. All settings, including pinhole diameter,detector gain and laser power, were optimized for each analysis.SH-SY5Y cells wereplated in 12-well plates containing coverslips at a density of 50,000cells per well. 24 h after plating, the cells were washed with phosphate-bufferedsaline (PBS) and incubated at room temperature for 30 min with 5 μMTRO-BODIPY, SQ-BODIPY, ENT-03-BODIPY, or L-Arg-BODIPY (1:10 dye:molecule)diluted in the Leibovitz’s L-15 (ThermoFisher Scientific),a medium designed for supporting cell growth in the absence of CO2+ levels were measured in living SH-SY5Y cells afterthe different treatments, by loading the cells with 4 μM Fluo-4AM (Thermo Fisher Scientific) for 10 min, as previously reported.46 Ca2+ levels were detected after excitationat 488 nm and emission at 520–580 nm, by a TCS SP8 scanningconfocal microscopy system (Leica Microsystems), equipped with anargon laser source. A series of 1 μm thick optical sections(1024 × 1024 pixels) were taken through the cell depth for eachsample using a Leica Plan Apo 63× oil immersion objective, andall sections were projected as a single composite image by superimposition.The confocal microscope was set at optimal acquisition conditions,e.g., pinhole diameters, detector gain, and laser powers. Settingswere maintained constant for each analysis. Images were then analyzedusing the ImageJ (NIH) software (Rasband 1997–2018). Fluorescenceintensities were typically expressed as a percentage of that measuredin untreated cells.SH-SY5Ycells were plated in 12-well plates containing coverslips at a densityof 40,000 cells per well. 24 h after plating, the cells were washedwith PBS and incubated at 37 °C for 15 min with ADDLs  in the absence or presence of increasing concentrations of TRO, SQ, or ENT-03. CytosolicCa