Phosphatidylserine Translocation Research Articles

Evaluating the Protective Effects of MitoQ and Antifreeze Protein III on Cryopreserved Canine Sperm.

Cryopreservation can adversely affect sperm motility, structural integrity, and fertilization ability. This study investigated the effects of MitoQ and antifreeze protein III (AFP III) on frozen-thawed semen from eight adult dogs using a Tris-fructose extender. Ejaculates were divided and diluted with a standard Tris-fructose-egg yolk extender containing MitoQ (200 nM/mL) and AFP III (0.75, 1.0, 2.0 µg/mL), individually or combined. Post-thaw, samples were evaluated for motility, viability, membrane and acrosome integrity, lipid peroxidation, apoptosis indicators, mitochondrial function, and reactive oxygen species (ROS-H2O2). The results showed significant (p < 0.05) improvements in motility rate, progressive motility, VAP, VSL, VCL, ALH, and BCF with MitoQ or AFP alone. AFP III (0.75, 1.0 µg/mL) showed higher values than controls (p > 0.05), while MitoQ alone showed no significant effect. Viability and acrosome integrity improved with AFP III. Membrane integrity and lipid peroxidation were better in 0.75 and 1.0 µg/mL AFP III groups. ROS-H2O2 levels and mitochondrial membrane potential were unaffected except at 1.0 µg/mL AFP III. The phosphatidylserine translocation assay showed no significant differences in dead sperm between controls and individual treatments, but significant differences occurred with combined MitoQ/AFP III. In conclusion, AFP III and MitoQ in diluents protect canine sperm cells from cryodamage.

Insights into the Mode of Action of Novel Morpholinated Curcumin Derivatives Exhibiting Potent Antitumor Activity in Bladder Cancer Cells In Vitro.

Although curcumin is a well-known natural polyphenol with many biological activities, its clinical application has been limited by low aqueous solubility and stability. Therefore, curcumin derivatives have been proposed to overcome these limitations and increase anticancer activity. This study tested curcumin derivatives with modified feruloyl moieties (2a and 2a-B) and the β-diketo moiety (2a-B) to better understand their anticancer mechanism against human bladder cancer cells. The anticancer activity of 2a and 2a-B was determined using MTT (hypoxic conditions) and LDH (normoxic conditions) assays. An ELISA-based protein panel was used to find the potential molecular targets, while flow cytometric, colorimetric, fluorescent, and luminescent assays were used to investigate the cell death mechanism. It was shown that compound 2a exerted a more potent cytotoxic effect under hypoxic conditions, while compound 2a-B demonstrated a comparable effect in normoxic and hypoxic conditions. The potential molecular targets modified by 2a and 2a-B depending on oxygen concentration were also proposed. Both compounds alter cell cycle progression by blocking the cell cycle in the G2/M phase and decreasing the percentage of cells in the G0/G1 phase. Compound 2a-B led to phosphatidylserine translocation, increased caspase 3/7 activity, and decreased mitochondrial membrane potential, suggesting a mitochondrial apoptosis pathway. We found that the Akt signaling pathway may modulate the activity of compound 2a-B, as evidenced by enhanced cytotoxic activity in combination with MK-2206, an Akt 1/2/3 inhibitor. Thus, our results provide new insights into the anticancer activity of compounds 2a and 2a-B; however, further studies are needed to better understand their therapeutic potential.

Influence of antifreeze protein III on canine sperm cryopreservation.

Sperm cryopreservation is crucial in reproductive biotechnology; however, the longevity of frozen and thawed semen is limited by the deterioration of sperm cell integrity. This study aimed to examine the effects of adding antifreeze protein III (AFP III) to the diluent, using samples from eight healthy mature dogs. The ejaculates were divided into aliquots and diluted with a standard Tris-fructose-egg yolk extender containing AFP III at concentrations of 0, 0.75, 1.0, and 2.0μg/ml. After thawing, the samples were analyzed for kinematic parameters, membrane Integrity, lipid peroxidation, viability, acrosome integrity, intracellular hydrogen peroxide, mitochondrial membrane potential and apoptotic metrics. The results show that while motility and velocity were not significantly different between the treated and control groups (p>0.05), the treated groups generally performed better. Specifically, the 0.75 and 1.0μg/ml groups exhibited better movement compared to the 2.0μg/ml group. Additionally, there was a significant difference (p<0.05) in membrane integrity between the control and treated groups, though no differences were observed among the treated groups. Significant differences (p<0.05) were also observed in viability and acrosome integrity, with the 0.75 and 1.0μg/ml groups outperforming the control and 2.0μg/ml groups. There were no significant variations (p>0.05) in phosphatidylserine translocation, lipid peroxidation, mitochondrial membrane potential, or hydrogen peroxide levels. However, the 0.75 and 1.0μg/ml groups demonstrated superior effects compared to both the control and the 2.0μg/ml groups. These results suggest that the addition of antifreeze proteins, specifically AFP III, markedly improves the protection of canine sperm during cryopreservation. This enhancement is evident in various parameters, underscoring the beneficial effects of AFP III in maintaining sperm quality.

The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds. We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro. Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays. Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells. In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death. In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.

Design and discovery of carboxamide-based pyrazole conjugates with multifaceted potential against Triple-Negative Breast cancer MDA-MB-231 cells

We report the design, synthesis, and validation of carboxamide-based pyrazole and isoxazole conjugates with a multifaceted activity against Breast Cancer Cell Line MDA-MB-231. The study established that amongst the series, N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (5g) exhibits the highest potency in inhibiting Breast Cancer Cell Line MDA-MB-231 with an IC50 value of 15.08 ± 0.04 µM. The MDA‐MB‐231 cells, upon treatment with compound 5g, exhibited characteristic apoptotic specific activities such as nuclear fragmentation, phosphatidylserine translocation to the outer plasma membrane, release of lactate dehydrogenase (LDH), and upregulation of caspase 3 and caspase 9 activities. Also, the modulation of pro and antiapoptotic proteins in 5g treated MDA-MB-231 cells was revealed by membrane array analysis. More importantly, the combination of paclitaxel and compound 5g has exhibited improved activity by several folds via their synergistic effects.

Photothermal induction of pyroptosis in malignant glioma spheroids using (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods

Plasmonic photothermal therapy (PPTT) employing plasmonic gold nanorods (GNRs) presents a potent strategy for eradication of tumors including aggressive brain gliomas. Despite its promise, there is a pressing need for a more comprehensive evaluation of PPTT using sophisticated in vitro models that closely resemble tumor tissues, thereby facilitating the elucidation of therapeutic mechanisms. In this study, we exposed 3D glioma spheroids (tumoroids) to (16-mercaptohexadecyl)trimethylammonium bromide-functionalized gold nanorods (MTAB-GNRs) and a near-infrared (NIR) laser. We demonstrate that the photothermal effect can be fine-tuned by adjusting the nanoparticle concentration and laser power. Depending on the selected parameters, the laser can trigger either regulated or non-regulated cell death (necrosis) in both mouse GL261 and human U-87 MG glioma cell lines, accompanied by translocation of phosphatidylserine in the membrane. Our investigation into the mechanism of regulated cell death induced by PPTT revealed an absence of markers associated with classical apoptosis pathways, such as cleaved caspase 3. Instead, we observed the presence of cleaved caspase 1, gasdermin D, and elevated levels of NLRP3 in NIR-irradiated tumoroids, indicating the activation of pyroptosis. This finding correlates with previous observations of lysosomal accumulation of MTAB-GNRs and the known lysosomal pathway of pyroptosis activation. We further confirmed the absence of toxic breakdown products of GNRs using electron microscopy, which showed no melting or fragmentation of gold nanoparticles under the conditions causing regulated cell death. In conclusion, PPTT using coated gold nanorods offers significant potential for glioma cell elimination occurring through the activation of pyroptosis rather than classical apoptosis pathways.

Cinnamaldehyde potentiates cytotoxic and apoptogenic effects of doxorubicin in prostate cancer cell line.

Nowadays, herbal medicine has been utilized to treat various diseases such as cancer, which showed successful therapeutic efficacy in previous studies. This study for the first time evaluated the cytotoxic potential of cinnamaldehyde (CIN) alone and in combination with doxorubicin (DOX), a well-known potent anti-tumor agent, on the proliferation of prostatic cancer cell line (PC3). The cytotoxicity and apoptotic activities of CIN and DOX, either separately or together, were determined on PC3 cells by the MTT test and Annexin V/PI assay, respectively. To further investigate which apoptotic pathway participated in cell death a collection of prominent markers of apoptosis induction including caspase-3/7 activations, mitochondrial membrane potential (MMP), and phosphatidyl serine translocation were detected. The different concentrations of CIN and DOX significantly inhibited the proliferation of PC3 cells in a concentration-dependent way within a 24-h treatment. In addition, the induction of apoptosis by CIN was accompanied by an increase in the activation of caspase-3/7 in PC3 cells with IC50 concentrations of 12.5 and 10 μg/mL for CIN and DOX, respectively. Moreover, the morphological observations obtained from flow cytometry MMP and caspase-3/7 activity assays, altogether, revealed the potential effect of CIN on apoptosis induced in PC3 cells by DOX. Taken together, the current study concluded that the combination of CIN and DOX could lead to the production of a potential therapeutic agent for prostate cancer. However, further in vivo and clinical studies are still needed to validate this combination in prostate cancer therapy.

Escin induces cell death in human skin melanoma cells through apoptotic mechanisms.

Natural products based on their significant anti-cancer potencies have been used in cancer treatment. A natural blend of triterpenoid saponins derived from the horse chestnut (Aesculus hippocastanum L.), has been investigated in various diseases based on its main active ingredient escin. Herein, we examined the potential antiproliferative, proapoptotic, and cytotoxic activities of escin on human skin melanoma (CHL-1) cells. Cytotoxicity of escin was determined by MTT assay. Morphological changes were detected by confocal microscopy and ultrastructural changes by transmission electron microscopy studies. Phosphatidylserine translocation assay, Bcl-2 activation assessment, and oxidative stress analysis were used to determine the cell death mode of the cells. The results showed that escin reduced cell viability in a dose-dependent manner within 24h of exposure and was highly cytotoxic at lower concentrations (IC50 value 6μg/mL). Escin inactivated Bcl-2 signaling and triggered apoptosis by increasing the reactive oxygen species and by causing morphological and ultrastructural changes that implicate to the proapoptotic activity. Escin has been found to exert high potential for an anti-cancer drug following further in vitro and in vivo investigations.

Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

Polystyrene nanoplastics as an ecotoxicological hazard: cellular and transcriptomic evidences on marine and freshwater in vitro teleost models

The contamination of marine and freshwater environments by nanoplastics is considered a global threat for aquatic biota. Taking into account the most recent concentration range estimates reported globally and recognizing a knowledge gap in polystyrene nanoplastics (PS-NPs) ecotoxicology, the present work investigated the harmful effects of 20 nm and 80 nm PS-NPs, at increasing biological complexity, on the rainbow trout Oncorhynchus mykiss RTG-2 and gilthead seabream Sparus aurata SAF-1 cell lines. Twenty nm PS-NPs exerted a greater cytotoxicity than 80 nm ones and SAF-1 were approximately 4-fold more vulnerable to PS-NPs than RTG-2. The engagement of PS-NPs with plasma membranes was accompanied by discernible uptake patterns and morphological alterations along with a nuclear translocation already within a 30-min exposure. Cells were structurally damaged only by the 20 nm PS-NPs in a time-dependent manner as indicated by distinctive features of the execution phase of the apoptotic cell death mechanism such as cell shrinkage, plasma membrane blebbing, translocation of phosphatidylserine to the outer leaflet of the cell membrane and DNA fragmentation. At last, functional analyses unveiled marked transcriptional impairment at both sublethal and lethal doses of 20 nm PS-NPs, with the latter impacting the “Steroid biosynthesis”, “TGF-beta signaling pathway”, “ECM-receptor interaction”, “Focal adhesion”, “Regulation of actin cytoskeleton” and “Protein processing in endoplasmic reticulum” pathways. Overall, a distinct ecotoxicological hazard of PS-NPs at environmentally relevant concentrations was thoroughly characterized on two piscine cell lines. The effects were demonstrated to depend on size, exposure time and model, emphasizing the need for a comparative evaluation of endpoints between freshwater and marine ecosystems.

Atomic force microscopy correlates mechanical and electrical properties of HepG2 cells with curcumin concentration

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with a significant impact on human health. Curcumin, a natural compound, induces cytoskeletal changes in liver cancer cells and modifies the distribution of lipids, proteins, and polysaccharides on plasma membranes, affecting their mechanical and electrical properties. In this study, we used nanomechanical indentation techniques and Kelvin probe force microscopy (KPFM) based on atomic force microscopy (AFM) to investigate the changes in surface nanomechanical and electrical properties of nuclear and cytoplasmic regions of HepG2 cells in response to increasing curcumin concentrations. CCK-8 assays and flow cytometry results demonstrated time- and concentration-dependent inhibition of HepG2 cell proliferation by curcumin. Increasing curcumin concentration led to an initial increase and then decrease in the mechanical properties of nuclear and cytoplasmic regions of HepG2 cells, represented by the Young's modulus (E), as observed through nanoindentation. KPFM measurements indicated decreasing trends in both cell surface potential and height. Fluorescence microscopy results indicated a positive correlation between curcumin concentration and phosphatidylserine translocation from the inner to the outer membrane, which influenced the electrical properties of HepG2 cells. This study provides valuable insights into curcumin's mechanisms against cancer cells and aids nanoscale evaluation of therapeutic efficacy and drug screening.

Effect of Trehalose Supplementation in Egg-Yolk-Free Extender on Conventional Parameters and Gene Expression Related to Reactive Oxygen Species, Apoptosis, and Motility of Frozen Dog Spermatozoa.

The present study was conducted to evaluate the effects of trehalose supplementation in egg-yolk (EY)-free tris extender on dog spermatozoa. Pooled spermatozoa were diluted with extender 1 (EY-free tris extender supplemented with 0, 10, 15, 20, or 30 mM trehalose) and cooled (2 × 108 sperm/mL) for 1 hour at 4°C. After that, extender 2 (extender 1 containing 1 M glycerol) was added (v:v) to the diluted sperm, loaded in 0.5-mL straws (1 × 108 sperm/mL), and incubated at 4°C for 30 minutes. The sperm straws were frozen over liquid nitrogen (LN2) vapor for 20 minutes and then plunged directly into LN2. After thawing at 37°C for 25 seconds, sperm progressive motility (CASA), viability (SYBR-14/PI), apoptosis (Annexin V/PI), and reactive oxygen species (ROS; H2DCFDA/PI) were evaluated. Thereafter, the optimal concentrations of trehalose were selected, and the gene expression of BAX, BCL2, NOX5, SMOX, OGG1, and ROMO1 was evaluated after freeze-thawing. Supplementation with 20 and 30 mM trehalose significantly increased sperm progressive motility and viability compared to the control. However, trehalose had no significant effect on sperm ROS or phosphatidylserine translocation index. There were minor numerical increases and decreases in gene expression when the selected optimal concentrations of trehalose (20 and 30 mM) were compared to the control. However, there were no significant differences. We conclude that the addition of trehalose (20 and 30 mM) in EY-free extender could improve sperm motility and viability without significant effects on ROS, apoptosis, or gene expression.

Dog sperm cryopreservation using cryovials and different dilution steps

BACKGROUND: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time. OBJECTIVE: To develop a more efficient freezing method using cryovials. MATERIALS AND METHODS: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 × 108 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4°C for 50 min and then frozen over LN2 for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (−80°C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4°C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN2 vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN2 after freezing and their functional performance and gene expression determined. RESULTS: Progressive motility and acrosome integrity were highest (P &lt; 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P &lt; 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P &lt; 0.05) in Group 4. CONCLUSION: The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm.

Mechanisms of Antioxidant Resistance in Different Wheat Genotypes under Salt Stress and Hypoxia

Various stressors lead to an increase in ROS and damage to plant tissues. Plants have a powerful antioxidant system (AOS), which allows them to neutralize excess ROS. We detected an intense fluorescent glow of ROS in the cells of the cap, meristem, and elongation zones in the roots of wheat Triticum aestivum (Orenburgskaya 22 variety) and Triticum durum (Zolotaya variety). An increase in ROS was accompanied by DNA breaks in the nuclei of wheat root cells, the release of cytochrome c from mitochondria into the cytoplasm, and the translocation of phosphatidylserine into the outer layer of the plasma membrane under salt stress and hypoxia. The different resistances of the two wheat varieties to different abiotic stresses were revealed. The soft wheat variety Orenburgskaya 22 showed high resistance to salt stress but sensitivity to hypoxia, and the durum wheat variety Zolotaya showed tolerance to hypoxia but high sensitivity to salt stress. Different activations of AOS components (GSH, MnSOD, Cu/ZnSOD, CAT, PX, GPX, and GST) were revealed in different wheat genotypes. The basis for the tolerance of the Zolotaya variety to hypoxia is the high content of glutathione (GSH) and the activation of glutathione-dependent enzymes. One of the mechanisms of high resistance to salt stress in the Orenburgskaya 22 variety is a decrease in the level of ROS as a result of the increased activity of the MnSOD and Cu/ZnSOD genes. Identifying the mechanisms of plant tolerance to abiotic stress is the most important task for improving breeding varieties of agricultural plants and increasing their yield.

Synthesis, Characterization and Apoptosis Capabilities of a Novel Naphthoquinone‐Derived Compound in Triple‐Negative Breast Cancer

AbstractBreast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer‐related deaths. Because triple‐negative breast cancer (TNBC) is highly resistant to clinically employed drugs, developing new drugs and treatment approaches is imperative. Naphthoquinone compounds potentially induce cancer cell death, and their clinical derivatives have been widely used as medicines. This study presents the synthesis and structure of a newly substituted naphthoquinone compound, as determined by spectroscopic techniques, then assesses the compound‘s ability to induce cell death on the MDA‐MB‐231 cell line using SRB viability testing, flow cytometry, and Western Blotting. The study also examines the compound‘s inhibition of cell migration through a scratch assay. The study discovered that the compound demonstrated an antigrowth/cytotoxic impact at concentrations of 3.12 μM and beyond. The cytotoxicity may have been caused by elevated reactive oxygen species, which resulted in DNA damage. The mode of cell death was apoptotic, confirmed by the translocation of phosphatidylserine, the activation of caspases, and the disruption of the mitochondrial membrane potential. The compound seems promising as a new anti‐malignant agent to be used for the treatment of breast cancer and deserves further attention for proof‐of‐concept in animal studies.

Ginger exosome-like nanoparticles (GELNs) induced apoptosis, cell cycle arrest, and anti-metastatic effects in triple-negative breast cancer MDA-MB-231 cells

Ginger exosome-like nanoparticles (GELNs) have been extensively implicated in alleviating inflammation, maintaining intestinal microbiome and are considered competent drug delivery vehicles. Despite this, the current knowledge of the GELN interaction with cancer cells is limited. Triple-negative breast cancer (TNBC), an aggressive variant lacking efficient therapeutics, necessitates novel natural counterparts with minimal side effects. This study investigates the action of GELNs isolated from ginger rhizomes against TNBC cells. GELNs were isolated by ultracentrifugation and characterized physicochemically. The interaction of GELNs with TNBC cells (MDA-MB-231) was studied in detail. The GELNs induced a concentration-dependent decrease in cell viability in MDA-MB-231 cells without affecting the normal cell lines tested. GELNs induced apoptosis as indicated by morphological changes, nuclear fragmentation, membrane damage, phosphatidyl serine translocation, ROS generation, drop in mitochondrial membrane potential, expression of apoptotic specific proteins, and increased caspase activity. GELNs also instigated cell cycle arrest, retarded cell migration and colony formation in TNBC cells. These findings report a novel action of GELNs against TNBC cells and a closer look at the underlying molecular mechanism of this interspecies communication. This opens newer prospects for using dietary ELNs to target therapeutically challenging cancers.

Apoptotic effects of triterpenoids isolated from Pleiocarpa pycnantha leaves in cancer cells and molecular docking study of the interactions of camptothecin and ursolic acid with human caspase 3, caspase 9 and topoisomerase I

The aim of this study was to examine the effects of triterpenes from Pleiocarpa pycnantha leaves on the induction of apoptotic signalling in human cells. The molecular mechanisms of triterpenes isolated from P. pycnantha leaves were investigated in vitro on HeLa, MCF-7, HT-29, and KMST-6 cells. The compounds activated several markers associated with apoptosis, viz., phosphatidylserine translocation, caspase activation, oxidative stress, and topoisomerase I inhibition. Compounds 1 and 5 were non-selective, whereas compounds 2, 3, and 4 showed potential as cancer-specific agents by selectively inducing apoptosis only on cancer cells. Theoretical studies on the interactions of compound 1 with caspases −3 and −9 and topoisomerase I were carried out through a molecular docking study and illustrated that compound 1 had an equal binding affinity with the caspases and topoisomerase I comparable to that of camptothecin. The cellular pathway activated by these compounds was dependent on the compound and the cell type.

Iodixanol supplementation in freezing extender improves the antioxidant capacity of semen.

The aim of this study was to evaluate the effect of supplementing bovine semen freezing extender with different concentrations of iodixanol on post-thaw sperm characteristics. Six ejaculates of three Nellore bulls were pooled and diluted in commercial extender (BotuBov®) and then divided into 4 groups: control group (without adding iodixanol); groups G1.5, G3, or G6 according to the concentration of iodixanol solution (RedCushion®). After dilution, the samples were cooled and frozen. Post-thaw semen evaluation included sperm motility by CASA immediately after thawing and after 60 min of incubation at 37°C, flow cytometry analysis for integrity of plasma and acrosomal membranes, membrane destabilization and translocation of phosphatidylserine, mitochondrial membrane potential, and formation of intracellular anion superoxide ( ), hydrogen peroxide (H2 O2 ), and membrane lipid peroxidation. The group G6 presented significantly higher (p < .05) total and progressive motility, percentage of plasma and acrosomal membrane integrity, and H2 O2 than control and group G1.5. Furthermore, group G6 showed lower (p < .05) lipid peroxidation than control. In addition, regardless of the concentration used, the percentage of spermatozoa without phosphatidylserine translocation was higher (p < .05) in all iodixanol supplemented groups. In conclusion, iodixanol supplementation preserved the motility and integrity of sperm membranes during cryopreservation and protected against lipid peroxidation.

Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests.

The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN2 vapors; automated: using a drop curve of -39.82 °C·min-1 for 113 s from -5 to -80 °C during the critical period; and subsequent immersion in NL2. Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.

The increment of annexin V-positive microvesicles versus annexin V-negative microvesicles in CSF of an animal model of Alzheimer’s disease

ObjectiveExtracellular microvesicles (MVs) as a specific signaling molecule have received much attention in nervous system studies. Alterations in the tissue redox status in pathological conditions, such as Alzheimer’s disease (AD), facilitate the translocation of cell membrane phosphatidylserine to the outer leaflet and lead to the MVs shedding. Annexin V binds with high affinity to phosphatidylserine. Some arguments exist about whether Annexin V-negative MVs should be considered in pathological conditions. Material and methodWe compared the kinetics of two phenotypes of Annexin V-positive and Annexin V-negative MVs in the cerebrospinal fluid (CSF) of amyloid-β (Aβ)-treated male Wistar rats with flow cytometry technique. The Aβ was injected bilaterally into the cerebral ventricles. Thioflavin T staining was used to confirm the presence of hippocampal Aβ fibrils two weeks post-Aβ injection. Levels of hippocampal interleukin-1β were assessed as an inflammatory index. The CSF malondialdehyde (MDA) concentration was determined. The cognitive impairment and anxiety behaviors were assessed by object recognition and elevated plus maze tests, respectively. ResultsElevation of MDA levels and a significant rise in the scoring of IL-1β staining were found in the Aβ group. The Aβ induced anxiogenic behavior, impaired novel object recognition memory, and increased the CSF levels of the total number of MVs. The number of Annexin V-positive MVs was significantly higher than Annexin V-negative MVs in all groups. ConclusionData showed that Annexin V-positive MVs potentially have a significant contribution to the pathophysiology of the Aβ-induced cognitive impairment. To catch a clear image of microvesicle production in pathological conditions, both phenotypes of Annexin V-positive and Annexin V-negative MVs should be analyzed and reported.