Cytochrome Content Research Articles

Molecular Surface Quantification of Multifunctionalized Gold Nanoparticles Using UV-Visible Absorption Spectroscopy Deconvolution.

Multifunctional gold nanoparticles (AuNPs) are of great interest, owing to their vast potential for use in many areas including sensing, imaging, delivery, and medicine. A key factor in determining the biological activity of multifunctional AuNPs is the quantification of surface conjugated molecules. There has been a lack of accurate methods to determine this for multifunctionalized AuNPs. We address this limitation by using a new method based on the deconvolution and Levenberg-Marquardt algorithm fitting of UV-visible absorption spectrum to calculate the precise concentration and number of cytochrome C (Cyt C) and zinc porphyrin (Zn Porph) bound to each multifunctional AuNP. Dynamic light scattering (DLS) and zeta potential measurements were used to confirm the functionalization of AuNPs with Cyt C and Zn Porph. Transmission electron microscopy (TEM) was used in conjunction with UV-visible absorption spectroscopy and DLS to identify the AuNP size and confirm that no aggregation had taken place after functionalization. Despite the overlapping absorption bands of Cyt C and Zn Porph, this method was able to reveal a precise concentration and number of Cyt C and Zn Porph molecules attached per AuNP. Furthermore, using this method, we were able to identify unconjugated molecules, suggesting the need for further purification of the sample. This guide provides a simple and effective method to quickly quantify molecules bound to AuNPs, giving users valuable information, especially for applications in drug delivery and biosensors.

Identification of blood cell changes in pediatric oncological patients through Raman spectroscopy

AbstractThis study aimed to use the Raman spectroscopy technique to identify biochemical changes in blood cell samples of healthy, leukemic, and non‐leukemic subjects by principal component analysis (PCA) and to discriminate the groups by partial least squares discriminant analysis (PLS‐DA). For the study, 121 blood samples were collected from healthy children and adolescents among those with leukemia and other types of cancer (non‐leukemics). A volume of 80 μl of blood cells was placed in an aluminum sample holder and submitted to dispersive Raman spectroscopy (laser: 830 nm and 250 mW), with an exposure time of 30 s. A total of 308 Raman spectra were analyzed and indicated significant differences in the bands referred to nucleic acids (671 and 756 cm−1), amino acids (671, 1,215, and 1,544 cm−1), proteins (756, 1,215, 1,430, 1,531, and 1,548 cm−1), β‐carotene (1,531 cm−1), and cytochrome c (756 and 1,371 cm−1), showing higher intensity in normal samples. The analysis of the PCA variables (PCs) showed that there was a higher concentration of cytochrome c (633, 749, 855, 1,004, 1,127, 1,212, 1,370, 1,433, 1,531, and 1,614 cm−1), in the normal samples, and a higher concentration of phenylalanine (1,004 cm−1) was observed in the leukemic samples with statistically significant (ANOVA, p < 0.05). Discriminant analysis by PLS‐DA model classified the spectra into the groups normal (healthy), leukemic, and non‐leukemic with accuracy of 67.5%; when considering normal versus cancer, the accuracy increased to 98.3%, with sensitivity of 100% and specificity of 87.8%. Results demonstrated the applicability of Raman spectroscopy for analyzing biochemical compounds in blood cells, thus identifying the spectral differences between the groups normal, leukemic, and non‐leukemic (with other types of cancer). However, it presented a low accuracy due to classification errors when discriminating between the groups leukemic and non‐leukemic.

Quantitative extraction and concentration of cytochrome c by liquid–liquid phase separation of aqueous ionic liquids under microflow

In this study, phase separation of aqueous solutions of tetrabutylphosphonium trimethylbenzensulfonate ([P4444][TMBS]) under microflow is utilized for simultaneous target-selective extraction and separation. A microchannel with well structures at the bottom is fabricated in a poly(dimethysiloxane) (PDMS) device. A mixture of protein samples containing [P4444][TMBS] is flowed through the device while it is heated. A protein favorable to be distributed to [P4444][TMBS]-rich phase is selectively extracted and trapped in the well structure. As the separated [P4444][TMBS]-rich phase has a smaller volume, the target analyte is thus enriched. Herein, the selective extraction and concentration of cytochrome c from an aqueous mixture of bovine serum albumin, horseradish peroxidase, and azurin is examined. Quantitative concentrations are confirmed based on the volume ratio of each phase determined by color-information-based colorimetry. Precise selection and control of phase-separable materials enhance the versatility of liquid–liquid phase separation for the highly-sensitive detection of biomaterials and metal ions in small amounts of samples.

Activity of respiratory chain cytochrome complexes and cytochromes content in the rat kidney mitochondria under different nutrients content in a diet

An important role in ensuring the functioning of the respiratory chain belongs to the cytochrome part, which includes complexes III (ubiquinol-cytochrome c oxidoreductase) and IV (cytochrome c oxidase). The key components of these enzymatic complexes are heme-containing cytochromes, the number of which depends on the balance of heme synthesis and catabolism. δ-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway, while heme oxygenase is the key enzyme of heme degradation. It is known that nutritional imbalances drive many risk factors for chronic kidney disease. That is why our research aimed to study the activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes, the level of cytochromes a+a3, b, c, and c1, and the activity of key enzymes of heme metabolism in the mitochondria of rat kidneys under conditions of different content of protein and sucrose in animal diet. The obtained results showed a decreased activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes and reduced levels of mitochondria cytochromes a+a3, b, c, and c1 in the kidney mitochondria under the conditions of nutrient imbalance, with the most pronounced changes found in animals kept on a low-protein/high-sucrose diet. A decrease in δ-aminolevulinate synthase activity with a simultaneous 2-fold increase in heme oxygenase activity was found in kidney mitochondria of animals kept on a low-protein/high-sucrose diet compared to those kept on full-value diet indicating an intensification of heme catabolism along with inhibition of its synthesis. The obtained results testify the energy imbalance under the conditions of low-protein/high-sucrose which in turn can lead to the progression of kidney injury. Keywords: cytochrome oxidase, cytochromes, heme oxygenase, nutrients, ubiquinol-cytochrome c oxidoreductase, δ-aminolevulinate synthase

Individual and combined toxicity of sulfamethoxazole and desethylatrazine to Chlorella vulgaris: growth inhibition, photosynthetic activity and oxidative stress

ABSTRACT The influence of emerging contaminants on the composition of aquatic ecosystems, including antibiotics and pesticide metabolites, is receiving increasing attention. In this study, the acute and chronic toxic effects of single and combined exposure to sulfamethoxazole (SMX) and desethylatrazine (DEA) on Chlorella vulgaris (C. vulgaris) were evaluated in terms of growth, pigment content and antioxidant systems. The findings demonstrated that cytochrome content and cell growth followed a similar trend for both single and combined exposures, with C. vulgaris being affected in a dose-dependent manner, and chlorophyll content decreasing at high exposures. For SMX and DEA, the maximum inhibition rates at a single exposure were 95.14% and 87.31%, respectively. Cell membrane permeability, superoxide dismutase content and catalase release were significantly increased in the early stages of cultivation. Additionally, cell density was used to calculate the single exposure half effect concentration (EC50), which was then used as part of the toxic unit (TU) method for evaluating mixture interactions. TU analysis revealed that the interaction between SMX and DEA shifted from being synergistic to being antagonistic after 7 d of incubation. These results suggest that SMX and DEA may play a significant role in aquatic ecosystems.

Cytochrome-c-oxidase and ATPsynthase content increases in the endometrium of the patients with adenomyosis.

Adenomyosis is characterized by overgrowth of endometrial glands and stroma in the myometrium and is associated with reduced apoptosis. One of the key participants in one of the pathways of apoptosis is cytochrome c, whose expression can be regulated by actin-binding proteins involved in the formation of structures that provide cell motility. The aim of the study was to determine the content of actin-binding proteins, cytochrome c, and terminal members of the mitochondrial respiratory chain in endometrial biopsies of patients with adenomyosis and the control group. The content of all studied proteins was determined by Western blotting, and the mRNA content of the corresponding genes was determined by quantitative RT-PCR. The relative content of alpha-actinin1 and mRNA of the gene encoding it in biopsy specimens from patients with adenomyosis was higher than in controls by 86 and 84% (p < 0.05), respectively. The relative content of alpha-actinin4 did not change, as did cytochrome c. The content of cytochrome-c-oxidase and ATPsynthase in the group with adenomyosis exceeded the control level by 270 and 121% (p < 0.05), respectively, but the relative content of mRNA of these genes did not change, which may indicate a change in regulation at the level of protein synthesis. The results may indicate a local increase in the synthesis of ATP in pathological endometrial cells, which indicates the possible effectiveness of local application of H+-ATP synthase inhibitors (for example, macrolide antibiotic) to reduce the severity of clinical symptoms of adenomyosis.

THE ACTIVITY OF HEME OXYGENASE AND δ-AMINOLEVULINATE SYNTHASE IN THE RAT KIDNEY UNDER CONDITIONS OF TOXIC INJURY WITH ACETAMINOPHEN

The aim of this work was to study the activity of δ-aminolevulinate synthase, heme oxygenase and the content of mitochondrial cytochromes in the rat kidney conditions of toxic damage with acetaminophen against the background of protein deficiency. δ-Aminolevulinate synthase activity was determined spectrophotometrically taking into account the molar extinction coefficient of 0.023·103 mol-1·cm-1, heme oxygenase activity – by the amount of bilirubin formed and expressed in nanomoles per 1 mg of protein per 1 minute. The determination of the contents of cytochromes a+a3, b, c and c1 in the suspension of isolated mitochondria was carried out by the difference spectra between the reduced and oxidized states in the region of their spectral maxima. The study was conducted on 4 groups of animals: Group I - control animals (K); II – rats kept on a low-protein diet (LPD); ІІІ – animals with acetaminophen-induced liver injury receiving complete ration (AII); IV – animals with acetaminophen-induced liver injury that were previously maintained on semi-synthetic low-protein ration (LPD/AII). It was established that under the conditions of consumption of a low-protein diet, there are no significant changes in the activities of heme oxygenase and δ-aminolevulinate synthase, but a decrease in the content of mitochondrial cytochromes a+a3, b, c, c1 is observed, which probably indicates the critical role of providing protein in the diet for the synthesis of cytochromes. At the same time, under the conditions of toxic damage by acetaminophen, regardless of whether the ration is provided with protein, there is a decrease in the activity of δ-aminolevulinate synthase by an average of 1.6 times with a simultaneous increase in the activity of heme oxygenase by approximately 1.5 times compared to the control values, which indicates an increase in the processes of heme catabolism on against the background of slowing down of its synthesis under the studied conditions. The increase in the activity of heme oxygenase against the background of the decrease in the activity of δ-aminolevulinate synthase can be considered as one of the possible mechanisms of the established decrease in the content of mitochondrial cytochromes under the conditions of acetaminophen intoxication, which in turn may further lead to a violation of the activity of the enzymes of the cytochrome part of the respiratory chain. The obtained results regarding the content of cytochromes, the activities of key enzymes of heme metabolism in the kidneys can be considered as prerequisites for deepening the energy imbalance under the conditions of toxic damage to the kidneys by acetaminophen.

Unveiling the Mechanism of Enhanced Extracellular Electron Transfer by Polarity Inversion of Bioelectrodes

Polarity inversion from the bioanode to the biocathode enables a rapid formation of efficient biofilms for microbial electrochemical reduction. Though this approach has been widely adopted for various applications, the mechanism behind the enhanced extracellular electron transfer (EET) during the polarity inversion process remains unclear. Therefore, in this study, the succession pattern and electron transfer mechanism of the inverted denitrifying biocathode were investigated by continuously monitoring the electrochemical properties, denitrification dynamics, and biofilm characteristics. The results indicated that the highest current density of the biocathode after inversion from bioanode was twice that of the steady current density, and the dominant flora of the electrode biofilm shifted from Geobacter sp. to denitrifying bacteria (e.g., Shinella and Comamonas). The reversal polarity induced a decreasing content of cytochrome C, while iron-containing compounds were found to be responsible for an enhanced EET within the denitrification process. This study provides a deep understanding of the EET mechanism behind the electrode polarity inversion and is of great significance for the future development of biocathode applications.

Evidence and Mechanisms of Selenate Reduction to Extracellular Elemental Selenium Nanoparticles on the Biocathode.

Intracellular selenium nanoparticles (SeNPs) production is a roadblock to the recovery of selenium from biological water treatment processes because it is energy intensive to break microbial cells and then separate SeNPs. This study provided evidence of significantly more extracellular SeNP production on the biocathode (97-99%) compared to the conventional reactors (1-90%) using transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy. The cathodic microbial community analysis showed that relative abundance of Azospira oryzae, Desulfovibrio, Stenotrophomonas, and Rhodocyclaceae was <1% in the inoculum but enriched to 10-21% for each group when the bioelectrochemical reactor reached a steady state. These four groups of microorganisms simultaneously produce intracellular and extracellular SeNPs in conventional biofilm reactors per literature review but prefer to produce extracellular SeNPs on the cathode. This observation may be explained by the cellular energetics: by producing extracellular SeNPs on the biocathode, microbes do not need to transfer selenate and the electrons from the cathode into the cells, thereby saving energy. Extracellular SeNP production on the biocathode is feasible since we found high concentrations of C-type cytochrome, which is well known for its ability to transfer electrons from electrodes to microbial cells and reduce selenate to SeNPs on the cell membrane.

High Immobilization Efficiency of Basic Protein within Heparin-Immobilized Calcium Phosphate Nanoparticles.

Previously, we achieved one-pot fabrication of heparin-immobilized calcium phosphate (CaP) nanoparticles with high dispersibility by a precipitation process in a highly supersaturated reaction solution. In this study, we revealed that the heparin-immobilized CaP nanoparticles have a greater co-immobilizing capacity for basic proteins than for acidic proteins. In this process, heparin acted as not only a particle-dispersing agent but also as an immobilizing agent for basic proteins; it remarkably (approximately three-fold) improved the immobilization efficiency of cytochrome C (a model basic protein) within the CaP nanoparticles. The content of cytochrome C immobilized within the nanoparticles was increased with an increase in cytochrome C concentration in the reaction solution and by aging the nanoparticles. The obtained nanoparticles were dispersed well in water owing to their large negative zeta potentials derived from heparin, irrespective of the content of cytochrome C. Similar results were obtained also for another basic protein, lysozyme, but not for an acidic protein, albumin; the immobilization efficiency of albumin within the nanoparticles was decreased by heparin. These findings provide new insights into the co-immobilization strategy of proteins within heparin-immobilized CaP nanoparticles and will be useful in the design and fabrication of nanocarriers for protein delivery applications.

Insights into the microbial response mechanisms to ciprofloxacin during sulfur-mediated biological wastewater treatment using a metagenomics approach

The fate and removal of ciprofloxacin, a class of fluoroquinolone antibiotic, during sulfur-mediated biological wastewater treatment has been recently well documented. However, little is known regarding the genetic response of microorganisms to ciprofloxacin. Here, a lab-scale anaerobic sulfate-reducing bioreactor was continuously operated over a long term for ciprofloxacin-contaminated wastewater treatment to investigate the response of the microorganisms to ciprofloxacin by adopting a metagenomics approach. It was found that total organic carbon (TOC) removal and sulfate reduction were promoted by approximately 10% under ciprofloxacin stress, along with the enrichment of functional genera (e.g., Desulfobacter, Geobacter) involved in carbon and sulfur metabolism. The metagenomic analytical results demonstrated that ciprofloxacin triggered the microbial SOS response, as demonstrated by the up-regulation of the multidrug efflux pump genes (8–125-fold higher than that of the control) and ciprofloxacin-degrading genes (4–33-fold higher than that of the control). Moreover, the contents of ATP, NADH, and cytochrome C, as well as related functional genes (including genes involved in energy generation, electron transport, carbon metabolism, and sulfur metabolism) were markedly increased under ciprofloxacin stress. This demonstrated that the carbon and sulfur metabolisms were enhanced for energy (ATP) generation and electron transport in response to ciprofloxacin-induced stress. Interestingly, the microbes tended to cooperate while being subjected exposure to exogenous ciprofloxacin according to the reconstructed metabolic network using the NetSeed model. Particularly, the species with higher complementarity indices played more pivotal roles in strengthening microbial metabolism and the SOS response under long-term ciprofloxacin stress. This study characterized the response mechanisms of microorganisms to ciprofloxacin at the genetic level in sulfur-mediated biological wastewater treatment. These new understandings will contribute the scientific basis for improving and optimizing the sulfur-mediated bioprocess for antibiotics-laden wastewater treatment.

Estimation of the Effects of Neonicotinoid Insecticides on Wild Raccoon, Procyon lotor, in Hokkaido, Japan: Urinary Concentrations and Hepatic Metabolic Capability of Neonicotinoids.

Toxicological effects of neonicotinoid insecticides (NNIs) have been reported for mammals, such as humans, rats, and mice. However, there are limited reports on their toxic effects on wild mammals. To predict NNI‐induced toxic effects on wild mammals, it is necessary to determine the exposure levels and metabolic ability of these species. We considered that raccoons could be an animal model for evaluating NNI‐induced toxicities on wildlife because they live near agricultural fields and eat crops treated with NNIs. The objective of the present study was to estimate the effects of NNI exposure on wild raccoons. Urinary concentrations of NNI compounds (n = 59) and cytochrome P450‐dependent metabolism of NNIs (n = 3) were evaluated in wild raccoons captured in Hokkaido, Japan, in 2020. We detected either one of the six NNIs or one metabolite, including acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, and desmethyl‐acetamiprid in 90% of raccoons (53/59); the average cumulative concentration of the seven NNI compounds was 3.1 ng/ml. The urinary concentrations were not much different from those reported previously for humans. Furthermore, we performed an in vitro assessment of the ability of raccoons to metabolize NNIs using hepatic microsomes. The amounts of NNI metabolites were measured using liquid chromatography–electrospray ionization–tandem mass spectrometry and compared with those in rats. Raccoons showed much lower metabolic ability; the maximum velocity/Michaelis–Menten constant (V max/K m) values for raccoons were one‐tenth to one‐third of those for rats. For the first time, we show that wild raccoons could be frequently exposed to NNIs in the environment, and that the cytochrome P450‐dependent metabolism of NNIs in the livers of raccoons might be low. Our results contribute to a better understanding of the effects of NNIs on raccoons, leading to better conservation efforts for wild mammals. Environ Toxicol Chem 2022;41:1865–1874. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

No Differences Between 12 Weeks of Block- vs. Traditional-Periodized Training in Performance Adaptations in Trained Cyclists

The purpose of this study was to compare the effects of 12 weeks load-matched block periodization (BP, n = 14), using weekly concentration of high- (HIT), moderate- (MIT), and low- (LIT) intensity training, with traditional periodization (TP, n = 16) using a weekly, cyclic progressive increase in training load of HIT-, MIT-, and LIT-sessions in trained cyclists (peak oxygen uptake: 58 ± 8 ml·kg−1·min−1). Red blood cell volume increased 10 ± 16% (p = 0.029) more in BP compared to TP, while capillaries around type I fibers increased 20 ± 12% (p = 0.002) more in TP compared to BP from Pre to Post12. No other group differences were found in time-trial (TT) performances or muscular-, or hematological adaptations. However, both groups improved 5 and 40-min TT power by 9 ± 9% (p < 0.001) and 8 ± 9% (p < 0.001), maximal aerobic power (Wmax) and power output (PO) at 4 mmol·L−1 blood lactate (W4mmol), by 6 ± 7 (p = 0.001) and 10 ± 12% (p = 0.001), and gross efficiency (GE) in a semi-fatigued state by 0.5 ± 1.1%-points (p = 0.026). In contrast, GE in fresh state and VO2peak were unaltered in both groups. The muscle protein content of β-hydroxyacyl (HAD) increased by 55 ± 58% in TP only, while both TP and BP increased the content of cytochrome c oxidase subunit IV (COXIV) by 72 ± 34%. Muscle enzyme activities of citrate synthase (CS) and phosphofructokinase (PFK) were unaltered. TP increased capillary-to-fiber ratio and capillary around fiber (CAF) type I by 36 ± 15% (p < 0.001) and 17 ± 8% (p = 0.025), respectively, while BP increased capillary density (CD) by 28 ± 24% (p = 0.048) from Pre to Post12. The present study shows no difference in performance between BP and “best practice”-TP of endurance training intensities using a cyclic, progressively increasing training load in trained cyclists. However, hematological and muscle capillary adaptations may differ.

A time-course study of the effects of a high-carbohydrate diet on the growth performance, glycolipid metabolism and mitochondrial biogenesis and function of blunt snout bream (Megalobrama amblycephala)

This study investigated the dynamic effects of feeding a high-carbohydrate diet on the growth performance, glycolipid metabolism and mitochondrial biogenesis and function of Megalobrama amblycephala. Fish were fed diets containing two dietary carbohydrate levels (30% and 43%) for 16 weeks, and were sampled at 2, 4, 8 and 16 weeks. The results showed that weight gain, protein efficiency, tissue glycogen and lipid contents, plasma levels of glucose, TC, TG and AGES, hepatic contents of AMP, ATP and AMPK protein, mitochondrial Na+-K+-ATPase and CS activities, as well as the transcriptions of GLUT2, GK, PK, GS, G6PDH, FAS, SREBP1 and ACCα all increased significantly with increasing sampling times. However, the opposite was true for nitrogen and energy retention, hepatic PGC-1β protein content, the AMP/ATP and the p-AMPK/t-AMPK ratios, as well as mitochondrial respiratory chain complex II, III and IV activities. In terms of diets, tissue glycogen and lipid contents, plasma levels of glucose, TG, TC and AGES, hepatic AMPK content as well as the transcriptions of GLUT2, GK, PK, GS, G6PDH, SREBP1, FAS and ACCα all increased significantly with increasing dietary carbohydrate levels, while the opposite was true for the contents of cytochrome C, PGC-1β and NRF-1, the p-AMPK/t-AMPK ratio, the activities of Na+-K+-ATPase, SDH, CS and complex II, III and IV, as well as the transcriptions of FBPase, CPT1 and ACO. Furthermore, the glycolipid metabolism and mitochondrial biogenesis and function were also significantly affected by the interaction between sampling times and diets. In conclusion, feeding a high-carbohydrate diet for more than 8 weeks could enhance the glucose transport, glycogenesis and lipogenesis of blunt snout bream, but down-regulate gluconeogenesis and fatty acid β-oxidation coupled with the impaired mitochondrial biogenesis and function.

Voriconazole-Induced Hepatotoxicity Concise up-to-date review

Voriconazole is a wide spectrum antifungal used primarily for invasive aspergillosis, an invasive mold infection occurs mostly in immunocompromised patients. Hepatotoxicity is the most common voriconazole-related adverse reaction that leads to treatment discontinuation. Even though reported incidence of hepatic adverse reactions during phase 2 and 3 clinical trials were less than 10%, observational studies in post marketing phase revealed much higher incidence reaching up to 69%. Therefore, the burden caused by hepatotoxicity and interruption of antifungal therapy put immunocompromised patients at serious risk.&#x0D; Currently, there is no biomarker in routine clinical use that can clearly predict susceptibility to voriconazole-induced hepatotoxicity. In effort to identify a predictor, plasma concentrations of voriconazole and cytochrome (CYP) 2C19 genotype/phenotype, which is responsible from substantial inter-individual changes in voriconazole pharmacokinetics, are the most studied subjects. Hepatotoxicity tends to occur at higher concentrations (&gt;4 mg/L), but so far, no significant association has identified in this matter. Although CYP2C19 genotype is strongly associated with voriconazole plasma concentration, current data is insufficient to define a causal relationship between CYP2C19 genotype and voriconazole-induced hepatotoxicity.&#x0D; This article reviews the epidemiology, mechanism, laboratory features of voriconazole-induced hepatotoxicity and current literature investigating the influence of voriconazole plasma concentration and CYP2C19 genetics on voriconazole-induced hepatotoxicity.

Iridium(III) complexes entrapped in liposomes trigger mitochondria-mediated apoptosis and GSDME-mediated pyroptosis

In this report, a new ligand TFBIP (TFBIP = 2-(4′-trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three iridium (III) complexes [Ir(ppy)2(TFBIP)](PF6) (Ir1, ppy = 2-phenylpyridine), [Ir(bzq)2(TFBIP)](PF6) (Ir2, bzq = benzo[h]quinolone) and [Ir(piq)2(TFBIP)](PF6) (Ir3, piq = 1-phenylisoquinoline) were synthesized and characterized. The cytotoxicity in vitro of the complexes toward several cancer cells was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) methods. The complexes show no cytotoxicity (IC50 > 100 μM) against these cancer cells. To enhance anticancer activity, these complexes were trapped in liposomes to form Ir1Lipo, Ir2Lipo and Ir3Lipo. The liposomes Ir1Lipo, Ir2Lipo and Ir3Lipo exhibit high or moderate cytotoxic activity. In particular, Ir1Lipo can effectively inhibit the cell growth with a low IC50 value (< 10 μM) toward A549, HepG2, BEL-7402, B16, HeLa and SGC-7901 cells. Surprisingly, Ir1Lipo has no cytotoxic activity against the normal cell LO2 (IC50 > 100 μM). The apoptosis and pyroptosis were investigated. Ir3Lipo induces apoptosis with a high early apoptotic number of 37%. The reactive oxygen species (ROS) levels, mitochondrial permeability transition pore open and mitochondrial membrane potential were detected. The intracellular Ca2+ concentration and release of cytochrome c were investigated. The expression of Bcl-2 (B-cell lymphoma-2) family proteins was explored by western blot. The antitumor activity in vivo of Ir1Lipo was evaluated with an inhibitory rate of 53%.

Crocetin ameliorates non-alcoholic fatty liver disease by modulating mitochondrial dysfunction in L02 cells and zebrafish model

Ethnopharmacological relevanceTraditional Chinese medicine considers that the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD) are related to liver depression and qi stagnation. Saffron and its active ingredient, crocetin (CCT), are used for the treatment of metabolic diseases owing to their “Liver deobstruent” and “Liver tonic” effects. However, the effect of CCT on NAFLD has not been fully elucidated. In the present study, the effect and potential molecular mechanism of CCT were explored in both in vivo and in vitro models of NAFLD. Materials and methodsCCT was isolated from saffron and purity and structure characterization were performed using HPLC, MS, 1H-NMR, and 13C-NMR. The effect of CCT on the viability of L02 cells and its maximum tolerable concentration (MTC) in zebrafish were investigated. Free fatty acids (FFA) and thioacetamide (TAA) were used to induce lipid accumulation in L02 cells and steatosis in zebrafish, respectively. The effects of CCT on indexes related to lipid metabolism, oxidative stress, and mitochondrial function in NAFLD models were explored using biochemical assay kits, Western blot analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), histopathology analysis, and determination of mitochondrial membrane potential (ΔΨm). Morphological analysis of mitochondria was performed using transmission electron microscopy (TEM). ResultsThe levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), and alanine/aspartate aminotransferases (ALT/AST) activities in FFA treated L02 cells were significantly reduced after CCT treatment. CCT treatment significantly increased ATP concentration, ΔΨm, and activities of superoxide dismutase (SOD), catalase (CAT), and cytochrome c oxidase (COX IV) in FFA treated L02 cells. TEM images showed restoration of mitochondrial morphology. CCT decreased ATP concentration and upregulated expression of B-cell lymphoma-2 (Bcl-2) and COX IV, whereas, CCT downregulated expression of BCL2-Associated X (Bax) and cleaved caspase-3 in TAA treated zebrafish. These findings indicated that mitochondrial dysfunction was alleviated after CCT treatment. Oil Red O staining of L02 cells and zebrafish showed that CCT treatment reversed the accumulation of lipid droplets. ConclusionIn summary, CCT treatment effectively alleviated the symptoms of NAFLD and restored mitochondrial function in L02 cells and zebrafish NAFLD model.

Hybrid organotin compounds — modulators of apoptotic processes in the liver when administered once and repeatedly to Wistar rats

Introduction. The aim of the study was to evaluate the effect of hybrid organotin compounds bis(3,5–di– tert–butyl–4–hydroxyphenylthiolate) dimethylol (Me3) and ((3,5–di–tert–butyl–4–hydroxyphenylthiolate) triphenylolol (Me5) on the level of markers of oxidative stress and apoptotic processes in the mitochondria during acute and subchronic intragastric administration to Wistar rats (females) in the maximum tolerated dose. Materials and methods. The objects of study were hybrid organotin compounds, the administration was carried out at the maximum tolerated dose of 2000 mg/kg (Me3) and 750 mg/kg (Me5) with a single and multiple intragastric administration. The study was conducted on 60 Wistar rats (females) weighing 190-210g. The concentration of cytochrome C (ng / g protein), caspase-9(ng / g protein), 8-hydroxy-2' — deoxyguanosine (8-OHdG) (ng/g protein), malondialdehyde (MDA) (nM / g protein)was determined in mitochondrial liver samples using test systems by enzyme immunoassay; by the biochemical method-the amount of protein (mg / ml) — by the biuretic method. Results. Me3 in both series of the experiment showed itself as a more pronounced antioxidant than Me5, which did not show its antioxidant properties. In group I animals, there were no statistically significant differences in the level of MDA and Cit C in relation to the control group, no mitDNA damage was detected, but K9 activity increased by 17%. With the introduction of Me5, the value of the MDA indicator increased by 55.5%, 8 — OHdG by 12.4% and Cit C by 66.2%. In group IV, the amount of MDA as the final product of lipid peroxidation (POL) increased by 13.6%, in group V by 22.5%. With the introduction of Me3, the level of Cit C was reduced by 23.5%, with the introduction of Me5, on the contrary, it was slightly increased. K9 activity was reduced in both experimental groups, by 9.6% and 17.3%, respectively. Discussion. Hybrid OOS containing a fragment of 2,6-di-tert-butylphenol have a dual structure. The tin-containing component is prooxidant, and the radical of the spatially hindered phenol, on the contrary, is antioxidant. It is the different ratio of the described fragments in the molecules of the substances under study, in our opinion, that led to the appearance of different degrees of influence on the metabolism of mitochondria. Conclusion. Both substances that modulate changes in oxidative stress and the activity of apoptotic processes are recommended for further research as antitumor medicinal agents.

Exopolysaccharides matrix affects the process of extracellular electron transfer in electroactive biofilm

The applications of bioelectrochemical systems (BESs) in the field of environment and energy are achieved through the bioelectrocatalytic process of electroactive biofilms. As a primary component of biofilm, the role of exopolysaccharides in electroactive biofilm in BESs is poorly understood. This study constructed an exopolysaccharides-deficient Geobacter sulfurreducens-based BES to explore the role of exopolysaccharides in electroactive biofilm. Compared with the wild type, the mutant biofilm expressing less exopolysaccharides decreased the capacity of current generation. In the mutant biofilm, the content of exopolysaccharides decreased significantly, resulting in a thinner biofilm and lower cell viability compared with the wild-type biofilm. However, the mutant with overexpressed pili developed a mature biofilm with extended time, which indicating the importance of exopolysaccharides for early biofilm formation and the compensatory role of pili in biofilm formation. The mutant biofilm had less content of c-type cytochromes (c-Cyts) and lower electrochemical activity of extracellular polymeric substances than the wild-type biofilm, suggesting a function of exopolysaccharides anchoring extracellular c-Cyts that essential to extracellular electron transfer (EET) in electroactive biofilms. Our findings demonstrated the essential role of exopolysaccharides in the process of EET in electroactive biofilm, which contributed to a better understanding and optimization of the performance of BESs.

PACAP-derived mutant peptide MPAPO protects trigeminal ganglion cell and the retina from hypoxic injury through anti-oxidative stress, anti-apoptosis, and promoting axon regeneration

The purpose of this study was to determine whether the MPAPO, derived peptide of pituitary adenylate cyclase-activating polypeptide (PACAP), would protect trigeminal ganglion cells (TGCs) and the mice retinas from a hypoxic insult. The nerve endings of the ophthalmic nerve of the trigeminal nerve are widely distributed in eye tissues. In TGCs after hypoxia exposure, we discovered that reactive oxygen species level, the contents of cytosolic cytochrome c and cleaved-caspase-3 were significantly increased, in the meanwhile, m-Calpain was activated and cytoskeleton proteins (αII-spectrin and Synapsin) were degraded, neurites of TGCs disappeared, but these effects were reversed in TGCs treated with MPAPO. The structure of the mice retinas after hypoxic exposure was disordered. Increased lipid peroxidation (LPO), decreased glutathione (GSH) levels, and decreased superoxide dismutase (SOD) activity, positive cells of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), the disintegration of nerve fibers was examined in the retinas following a hypoxic insult. Disordered retina was attenuated with MPAPO eye drops, as well as hypoxia-induced apoptosis in the developing retina, increase in LPO, and decrease in GSH levels and SOD activity of the retina. Moreover, the disintegrated retinal nerve fibers were reassembled after MPAPO treatment. These results suggest that hypoxia induces oxidative stress, apoptosis, and neurites disruption, while MPAPO is remarkably protective against these adverse effects of hypoxia in TGCs and the developing retinas by specifically activating PAC1 receptor.