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This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 µM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

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Nowadays, there is limited research focusing on the biosorption of Pb2+ through microbial process, particularly at the level of gene expression. To overcome this knowledge gap, we studied the adsorption capacity of Stenotrophomonas rhizophila JC1 to Pb2+, and investigated the physiological mechanism by means of SEM, EDS, FTIR, membrane permeability detection, and investigated the molecular mechanism through comparative transcriptomics. The results showed that after 16 h of cultivation, the biosorption capacity of JC1 for 100 mg/L of Pb2+ reached at 79.8%. The main mechanism of JC1 adsorb Pb2+ is via intracellular accumulation, accounting for more than 90% of the total adsorption. At the physiological level, Pb2+ can precipitate with anion functional groups (e.g., -OH, -NH) on the bacterial cell wall or undergo replacement reaction with cell component elements (e.g., Si, Ca) to adsorb Pb2+ outside of the cell wall, thus accomplishing extracellular adsorption of Pb2+ by strains. Furthermore, the cell membrane acts as a "switch" that inhibits the entry of metal ions into the cell from the plasma membrane. At the molecular level, the gene pbt specificity is responsible for the adsorption of Pb2+ by JC1. In addition, phosphate permease is a major member of the ABC transporter family involved in Pb2+, and czcA/cusA or Co2+/Mg2+ efflux protein plays an important role in the efflux of Pb2+ in JC1. Further, cellular macromolecule biosynthesis, inorganic cation transmembrane transport, citrate cycle (TCA) and carbon metabolism pathways all play crucial roles in the response of strain JC1 to Pb2+ stress.

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The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

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Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

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Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.

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Mitochondria are fundamental for human spermatozoa motility and fertilizing ability. Mitochondria participate not only in ATP production, but also in reactive oxygen species production, redox equilibrium, and calcium regulation, all of which are central for human spermatozoa motility, capacitation, acrosome reaction, and ultimately, oocyte fertilization. Mitochondrial membrane potential is a key indicator of mitochondrial health and activity. Most commonly used methods for the study of mitochondrial membrane potential, however, cannot be applied to human spermatozoa due to their unique characteristics, including high motility and time-dependent decay of quality, limiting the study of this important parameter in these cells. Here, we describe an easy, fast, and cheap protocol for the quantitative evaluation of human spermatozoa mitochondrial membrane potential, using the fluorescent cationic dye 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). JC-1 is a sensitive marker for mitochondrial membrane potential, exhibiting a potential-dependent accumulation in the mitochondria. At high mitochondrial membrane potential, JC-1 forms J-aggregates, which emit red fluorescence, whereas at low mitochondrial membrane potential, JC-1 remains at its monomer state, which emits green fluorescence. We first describe how to evaluate human spermatozoa mitochondrial membrane potential using JC-1 and a fluorescence plate reader, for high-throughput studies. The calculation of the JC-1 ratio (indicative of the J-aggregates/monomers ratio) is then used to quantitatively evaluate mitochondrial health and activity. In addition, we describe an imaging protocol for the qualitative evaluation of human spermatozoa mitochondrial membrane potential using a fluorescence microscope. This allows for a visual analysis of the results that can complement the quantitative data. These protocols can be used to study the effects of spermatozoa exposure to compounds of interest, and alterations due to diseases or different conditions. While these protocols are illustrated with human spermatozoa, they can be adapted and used on spermatozoa of different species. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Quantitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and a fluorescence plate reader Basic Protocol 2: Qualitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and fluorescence microscopy Support Protocol: Preparation of the JC-1 working solution.

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Polyhydroxyalkanoates (PHA) are microbial polymers that have received widespread attention in recent decades as potential alternatives to some petrochemical-based plastics. However, widespread use of PHA is often impeded by its cost of production. Therefore, the search for and systematic investigation of versatile microbial PHA producers capable of using various carbon sources, even in the form of animal fats, for PHA biosynthesis is desirable. This study highlights the PHA production capacity, monomer composition and molecular weight synthesized by Burkholderia cepacia JC-1, a locally isolated strain from soil, from various carbon sources. In the category of simple sugars and plant oils, the use of glucose and palm oil at C:N ratio of 40 resulted in the highest accumulation of 52 wt% and 36 wt% poly(3-hydroxybutyrate) [P(3HB)] homopolymer and dry cell weight of 2.56 g/L and 3.17 g/L, respectively. Interestingly, B. cepacia JC-1 was able to directly utilize animal-derived lipid in the form of crude and extracted chicken fat, resulting in appreciable dry cell weight and PHA contents of up to 3.19 g/L and 47 wt% respectively, surpassing even that of palm oil in the group of triglycerides as substrates. The presence of antibiotics (streptomycin) in cultivation medium did not significantly affect cell growth and polymer production. The supply of sodium pentanoate as a co-substrate resulted in the incorporation of 3-hydroxyvalerate (3HV) monomer at fractions up to 37 mol%. The molecular weight of polymers produced from glucose, palm oil and chicken fat were in the range of 991-2118 kDa, higher than some reported studies involving native strains. The results from this study form an important basis for possible improvements in using B. cepacia JC-1 and crude chicken fats in solid form for PHA production in the future.

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To better understand the function of transporter in heavy metal detoxification of bacteria, the transporters associated with heavy metal detoxification in S. rhizophila JC1 were analyzed, among which four members were verified by RT-qPCR. In addition, the removal rates of four single metal ions (Cr6+, Cu2+, Zn2+, Pb2+) and polymetallic ions by strain JC1 were studied, respectively. We also researched the physiological response of strain JC1 to different metal stress via morphological observation, elemental composition, functional group and membrane permeability analysis. The results showed that in the single metal ion solution, removal capacities of Cu2+ (120 mg/L) and Cr6+ (80 mg/L) of S. rhizophila JC1 reached to 79.9% and 89.3%, respectively, while in polymetallic ions solution, the removal capacity of each metal ion all decreased, and in detail, the adsorption capacity was determined Cr6+>Cu2+>Zn2+>Pb2+ under the same condition. The physiological response analyses results showed that extracellular adsorption phenomena occurred, and the change of membrane permeability hindered the uptake of metal ions by bacteria. The analysis of transporters in strain JC1 genome illustrated that a total of 323 transporters were predicted. Among them, two, six and five proteins of the cation diffusion facilitator, resistance-nodulation-division efflux and P-type ATPase families were, respectively, predicted. The expression of corresponding genes showed that the synergistic action of correlative transporters played important roles in the process of adsorption. The comparative genomics analysis revealed that S. rhizophila JC1 has long-distance evolutionary relationships with other strains, but the efflux system of S. rhizophila JC1 contained the same types of metal transporters as other metal-resistant bacteria.

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The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by 1H NMR and GC-MS to know the metabolites in each extract. In GC-MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC50 values of 45.9 µg/mL and the dichloromethane extract of the leaves (DLE) showed IC50 values of 47.3 µg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.

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Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.

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I summarize here the history of the use of abbreviations, mostly in subject areas related to dyes, stains and fluorescent probes used in biology and medicine. The dozen most popular abbreviations in these fields are identified and their salient characteristics noted. The pros and cons of each abbreviation are discussed with relevant citations. Certain abbreviations that are not in the list, e.g., AZAN and LN, are mentioned because they have an unusual origin; while others, i.e., INEPT and INADEQUATE are presented because they are bizarre. A related topic is abbreviations used for citations, which require further efforts to decipher. In the past, brevity helped conserve materials, such as ink and paper, and promoted more rapid publishing. I suggest that the use of many abbreviations in the current era of electronic publishing may not be necessary.

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In this study, a higher metal ions-resistant bacterium, Stenotrophomonas rhizophila JC1 was isolated from contaminated soil in Jinchang city, Gansu Province, China. The Pb2+ (120 mg/L) and Cu2+ (80 mg/L) removal rate of the strain reached at 76.9% and 83.4%, respectively. The genome comprises 4268161 bp in a circular chromosome with 67.52% G + C content and encodes 3719 proteins. The genome function analysis showed czc operon, mer operon, cop operon, arsenic detoxification system in strain JC1 were contributed to the removal of heavy metals. Three efflux systems (i.e., RND, CDF, and P-ATPase) on strain JC1 genome could trigger the removal of divalent cations from cells. cAMP pathway and ABC transporter pathway might be involved in the transport and metabolism of heavy metals. The homology analysis exhibited multi-gene families such as ABC transporters, heavy metal-associated domain, copper resistance protein, carbohydrate-binding domain were distributed across 410 orthologous groups. In addition, heavy metal-responsive transcription regulator, thioredoxin, heavy metal transport/detoxification protein, divalent-cation resistance protein CutA, arsenate reductase also played important roles in the heavy metals adsorption and detoxification process. The complete genome data provides insight into the exploration of the interaction mechanism between microorganisms and heavy metals.

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Nanotechnology can be applied to design antibacterial agents to combat antibiotic resistance. The aim of the present study was to assess the antimicrobial effects and cytotoxicity of GdYVO4:Eu3+ nanoparticles (NPs). Biofilm inhibition activity, antimicrobial activity, bacterial viability inhibition and DNA cleavage activity of GdYVO4:Eu3+ NPs were studied. In addition, the impact of GdYVO4:Eu3+ NPs on the mitochondrial membrane potential (ΔΨM) of host immune cells and, hence, their apoptosis was analyzed by JC-1 staining using flow cytometry. GdYVO4:Eu3+ NPs demonstrated good antimicrobial, cell viability inhibition and DNA cleavage activities. In addition, GdYVO4:Eu3+ NPs showed good biofilm inhibition activity against S. aureus and P. aeruginosa and inhibition percentages were 89.15% and 79.54%, respectively. However, GdYVO4:Eu3+ NPs promoted mitochondrial depolarization and apoptosis of leukocytes at high concentrations. GdYVO4:Eu3+ nanoparticles are promising antibacterial agents. However, more efforts should be exerted to ensure their safety.

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The proton electrochemical gradient generated by respiratory chain activity accounts for over 90% of all available ATP and, as such, its evaluation and accurate measurements regarding its total values and fluctuations is an invaluable component in the understanding of mitochondrial functions. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulations and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. Here we discuss the advantages and disadvantages of several of these methods, ranging from one that is dependent on the movement of a particular ion (tetraphenylphosphonium (TPP+) with a selective electrode) to the selection of a fluorescent dye from various types to achieve the same goal. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane, or the fluorescence of accumulated dye particles, is a sensitive and accurate method of evaluating the Δψ in respiring mitochondria (either isolated or still inside the cell).

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The evaluation of conventional and biofunctional sperm parameters is of fundamental importance for assessing male reproductive function. Among these, sperm motility is one of the most important parameters. Indeed, asthenozoospermia is a frequent cause of male infertility. Sperm motility depends on mitochondrial function and the measurement of mitochondrial membrane potential (MMP) better accounts for the function of this intracellular organelle. On the basis of these premises, the present study assessed whether the MMP predicts sperm motility at 4 h in patients with low or normal MMP. To accomplish this, 31 men were enrolled. Sperm analysis was conducted according to the WHO 2010 criteria. Particular attention was paid to the evaluation of MMP after liquefaction (T0) using JC-1 staining by flow cytometry. Sperm total and progressive motility were measured at T0 and after 4 h from seminal fluid collection (T4). Patients were divided into two groups based on their sperm mitochondrial function at T0. Group A (n = 18) was composed of men with normal mitochondrial function since they had a percentage of spermatozoa with low MMP (L-MMP) below the normal reference value of our laboratory (<36.5%). In contrast, group B (n = 13) was made up of men with impaired sperm mitochondrial function (L-MMP > 36.5%). Group A had a slight but not significant reduction in total and progressive sperm motility at T4 compared with the values recorded at T0. In contrast, patients in group B showed a significant decline in both total and progressive sperm motility at T4 compared with T0 (p < 0.05). The results of this study showed that worse mitochondrial function, assessed by staining with JC1, is associated with a significant decline in sperm motility over time. These findings may be of clinical relevance in programs of assisted reproduction techniques. Based on our knowledge, there is no other evidence in the literature that has shown this relationship in healthy men with low MMP of idiopathic etiology, but normozoospermics according to the WHO 2010 criteria.

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A water extraction and alcohol precipitation method was applied to extract polysaccharides from Codonopsis pilosula (CPP), response surface methodology was used to optimize the extraction conditions and synthesis of C. pilosula polysaccharide iron (CPPI), and the properties of CPPI were evaluated. The optimum extraction conditions for CPP were as follows: liquid-solid ratio of 29.39 mL/g, time of 1.25 h and temperature of 62.84 °C. The optimum synthesis conditions for CPPI were pH 8.9, temperature 70.30 °C and the ratio of citric acid to CPP1 of 2.95. An HPSEC-MALLS-RID system, UV spectroscopy, FT-IR spectroscopy and NMR were used for characterization of the polysaccharide. CPPI exhibited antioxidant activity in vitro and a relatively strong inhibitory effect on A2780 cells growth. After CPPI treatment, the reactive oxygen species increased, the mitochondrial membrane potential decreased, and DNA damage was observed in A2780 cells. Therefore, CPPI should be explored as a potential antioxidant and an antitumor drug in a clinical setting.

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AIMS: In hepatocytes, the retinoic acid receptor-related orphan receptor α (RORα) regulates the transcription of diverse genes encoding lipogenic enzymes, antioxidant enzymes, and mitochondrial factors via the regulation of the transcriptional activity of their promoters. The coordination of the expression of RORα by driving its transcription would provide better aspects for managing liver homeostasis. MAIN METHODS: The transcriptional expression of RORα was measured after treatment of RORα agonists on primary hepatocytes and liver. The histone status of Rora gene bodies was examined by analyzing ChIP-seq database. To elucidate molecular mechanism for RORα autoregulation, broad ChIP assays for promoters and enhancers with histone and RORα antibodies were performed. KEY FINDINGS: We report that natural and synthetic RORα agonists, cholesterol sulfate and JC1-40, respectively, increased the transcriptional expression of RORα in primary hepatocytes. An analysis of histone status around the Rora gene body identified promoter and enhancer regions of RORα. We found that RORα indirectly increased histone acetylation of H3K9 at the promoter region and directly enhanced histone monomethylation of H3K4 by binding to enhancer regions. Interestingly, disturbance of mixed-lineage leukemia 4 (MLL4), a histone methyltransferase for enhancers, abolished the JC1-40-induced activation of RORα via a decrease in H3K4me1. Finally, we observed that the MLL4-mediated autoregulation of RORα also occurred in human liver cancer cell lines. SIGNIFICANCE: The ability of RORα to modulate its own transcription is crucial for liver homeostasis, and ligand-dependent autoregulation could amplify the therapeutic effects of RORα in fatty liver diseases.

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Retinoblastoma is common primary intraocular malignancy of infants and childhood. Neferine is a major bisbenzylisoquinoline alkaloid derived from the lotus plumule in Nelumbo nucifera. This study evaluated the mitigation role of Neferine on retinoblastoma in vitro and in vivo. Xenotransplantation model was established by injecting WERI-Rb-1 cells subcutaneously. Upon induction of retinoblastoma , mice were intraperitoneally injected with Neferine (0, 0.5, 1, 2 mg/kg) or ethanol every 3 days for 30 days. Tumor weight and tumor volume were measured every three days and compared between four groups. Then, mice were sacrificed and immunohistochemical examination was performed to compare Ki67, VEGF content between groups. WERI-Rb-1 cells were used for in vitro experiments and the anti-angiogenic role of Neferine was assessed by analyzing nodes/HPF number. In WERI-Rb-1 xenotransplantation model, compared with control group, 1 mg/kg Neferine treatment significantly inhibited tumor weight (0.39 ± 0.04 g vs. 0.25 ± 0.03 g, P< 0.05) and tumor volume (2163 ± 165 mm3 vs. 1276 ± 108 mm3, P< 0.05) after 30 days. Compared with ethanol-injected mice, 2 µM Neferine treatment significantly enhanced apoptosis rate (2.1 ± 0.6% vs. 14.6 ± 2.6%, P< 0.05), accompany downregulation of Ki67 (0.09 ± 0.02% vs. 0.01 ± 0.004%, P< 0.05) and VEGF (0.28 ± 0.04% vs. 0.05 ± 0.03%, P< 0.05) expression. Additionally, 2 µM Neferine treatment significantly decreased JC-1 red/green percentage. High-dose Neferine could decrease retinoblastoma angiogenesis in association with a significant inhibition on tumor growth and invasion. These findings suggested that Neferine could be a new treatment or adjuvant against retinoblastoma.

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AIMS: ß-Estradiol (ß-E), one of the chemical forms of female gonad hormone exhibited antioxidant efficacy in biochemical system, in vitro. The aim of the study was to investigate whether any other mechanism of protection by ß-E to hepatic mitochondria in presence of stressor agent i.e.,a combination of Cu2+ and ascorbic acid is involved. MAIN METHODS: Freshly prepared goat liver mitochondria was incubated with stressors and 1 µM ß-E and post incubated with the same concentration at 37 °C at pH 7.4. Mitochondrial viability, biomarkers of oxidative stress, activities of Krebs cycle enzymes, mitochondrial membrane potential, Ca2+ permeability were measured. Mitochondrial morphology and binding pattern of ß-E with stressors were also studied. KEY FINDINGS: Upon incubation of mitochondria with Cu, ascorbic acid and their combination there is a significant decline in activities of four of Krebs cycle enzymes in an uncompetitive manner with a concomitant increase in Ca2+ permeability and membrane potential of inner mitochondrial membrane, which is withdrawn during co-incubation with ß-E, but was not reversed during post incubation with the ß-E. The final studies on mitochondrial membrane morphology using scanning electron microscope also exhibited damage. Isothermal titration calorimetry data also showed the negative heat change in the mixture of ß-E with ascorbic acid and also its combination with Cu2+. SIGNIFICANCE: Our results for the first time demonstrated that ß-E protects againstCu2+-ascorbate induced oxidative stress by binding with ascorbic acid. The new mechanism of binding of ß-E with stress agents may have a future therapeutic relevance.

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BACKGROUND: Multiple sclerosis (MS) is a multifactorial disease of the central nervous system in young adults. Mitochondrial respiration provides fuel necessary for cellular function and is especially important in cells with large energy demand including neurons. Various studies suggest that the pathogenesis of MS may be associated with mitochondrial dysfunction. METHODS: We examined 145 volunteers including 62 MS patients and healthy controls. MS patients were divided into two groups according to their disease severity: those with mild disability (EDSS=0-3.0) and those with moderate-severe MS (EDSS=3.5-8). After signing an informed consent, blood was taken and was separated to platelets and lymphocytes. Mitochondria activity was monitored as mitochondrial transmembrane potential following staining with JC1 dye in platelets and lymphocytes utilizing flow cytometry. RESULTS: We examined mitochondria activity as JC1 values from all separated lymphocyte samples and found significantly higher levels of mitochondrial activity in lymphocytes separated from healthy controls vs. MS patients (mean of 87.9% vs. 75.6%, p = 0.001). Significant differences in mitochondrial activity were also found when comparing means of groups divided according to MS disease severity. Interestingly, there were no significant differences in mitochondrial activity between patients treated with diverse medications or untreated patients. Mitochondrial activity was also examined in platelets, but no significant differences were found between groups. CONCLUSIONS: Results obtained here show that mitochondrial activity was significantly lower in MS patients in comparison to healthy controls. In addition, there was a significant difference in mitochondrial activity depending on MS degree of disability. These initial findings in a peripheral examination hold potential for new diagnostic biomarkers to be considered in the future.

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