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[Display omitted] • Lanthanide-chelate based smart imprinted cryogel for selective recognition of cytochrome-c. • Superior selective, simple and cheap strategy for efficient recognition of serum cytochrome-c. • Extraction of serum cytochrome-c with 92.78 % recovery. The recognition and detection of proteins has an important role in the fields of biochemistry and medicine in term of diagnosis and prognosis. In this study, molecularly imprinted cryogel (MIP) was synthesized for the selective recognition of Cytochrome-c (Cyt-c) by cryopolymerization techniques with the lanthanide-chelate approach using (MAAP) 2 -Ce(III) as complex functional monomer and Cyt-c as a template protein. The incorporation of template with monomer was characterized by near- infrared (Near-IR) spectroscopy. The binding capacity was optimized in accordance with numerous experimental parameters including pH, initial Cyt-c concentration, flow rate, temperature and ionic strength. As a result, the maximum binding capacity reached to 98.33 mg g−1 in a buffer system with a pH of 6.0. Beside of the excellent reusability performance of prepared MIP, it was successfully applied to separation of template Cyt-c in the existence of hemoglobin (Hb) and myoglobin (Mb) chosen as interfering proteins. The relative selectivity constants of the Cyt-imprinted polymer were determined as 13.84 for the Cyt/Mb pair and 16.05 for the Cyt/Hb pair. Consequently, a high selectivity, simple and cheap strategy for efficient separation of proteins was provided with this novel MIP cryogel. [ABSTRACT FROM AUTHOR]

Cyclosporine‐A (CsA) is a powerful immunosuppressive agent and hepatotoxicity results from CsA treatment. This study aimed to elucidate the effectiveness of tyrosine kinase inhibitor nilotinib against CsA‐induced hepatotoxicity and the underlying molecular mechanisms. Male Sprague‐Dawley rats were allocated into four groups and received drugs for 28 days as follows: Control group: received vehicle, Nilotinib group: received nilotinib (20 mg/kg orally), CsA group: received CsA by subcutaneous injection (20 mg/kg daily), CsA‐nilotinib: received nilotinib and CsA. Serum lactate dehydrogenase (LDH), liver function biomarkers, hepatic levels of oxidative stress biomarkers, nuclear factor erythroid‐2 like‐2 (Nrf2), total antioxidant capacity (TAC), interleukin‐2 (IL‐2), IL‐1β, IL‐6, and cytochrome‐C were assessed. Additionally, the protein levels and mRNA expression of Bcl2 associated X protein (Bax), caspase‐3, nuclear factor‐κB (NF‐κB), hemoxygenase‐1 (HO‐1) were measured. Moreover, liver tissues were assessed histopathologically using hematoxylin–eosin and Masson trichrome stain. Nilotinib treatment decreased serum LDH, alanine aminotransferase, aspartate aminotransferase, and γ‐glutamyltransferase (γ‐GT), hepatic malondialdehyde, and cytochrome‐C. It also increased superoxide dismutase, reduced glutathione, glutathione reductase, glutathione peroxidase, glutathione‐S‐transferase (GST), TAC, and Nrf2 compared to CsA‐injected rats. In addition, nilotinib decreased NF‐κB, IL‐1β, IL‐6, Bax, and caspase‐3, while elevated IL‐2 and immunoexpression of HO‐1. Additionally, mRNA expression of Bax and caspase‐3 was elevated and that of HO‐1 and inhibitory protein κB‐α was reduced in the nilotinib‐treated group. Moreover, nilotinib significantly attenuated CsA‐induced histopathological alterations. Nilotinib may have a promising role as a hepato‐protective through its antiapoptotic, antioxidant, and anti‐inflammatory effects. Research Highlights: Nilotinib has an ameliorative effect against Cyclosporine‐A‐induced liver injury.Nilotinib decreased serum LDH and ALT, AST, and γ‐GT.Nilotinib increased GSH, SOD, GR, GPx, GST, TAC, Nrf2, and HO‐1 levelsNilotinib decreased hepatic MDA, NF‐κB, IL‐1β, IL‐6, Bax, and caspase‐3 levels.Nilotinib attenuated Cyclosporine‐A‐induced histopathological alterations.Nilotinib raised Bax and caspase‐3 mRNA, lowered HO‐1 and NFκBia mRNA expression. [ABSTRACT FROM AUTHOR]

Introduction. Worldwide, nanoparticles especially gold-nanoparticles (Au-NPs) are widely used in medicine, cancer treatment and cosmetic industry. They are easily conjugated with different biomedical and biological agents and effortlessly absorbed with few side effects. The pars distalis of the pituitary gland is considered as the maestro of the endocrine peripheral glands since it secrets trophic hormones that controls their functions. 5-10% of the non-granular pars distalis cells are folliculo-stellate cells (FSCs) that support the granular cells' functions. The aim of the study was to explore the histological and the biochemical effects of repeated exposure to Au-NPs on the pars distalis in adult male albino rats with highlighting the impact on FSCs. Material and methods. Thirty-six adult male albino rats were divided equally into control group and Au-NPs group (received 40 μg/kg/day of 11 ± 2 nm spherical Au-NPs orally for 2 weeks). Then, rats were euthanized and deposition of Au-NPs in pars distalis was investigated. Biochemical investigations and histological studies including hematoxylin and eosin staining, periodic acid Schiff's reaction, immunohistochemistry (IHC) for S-100, connexin 43 (Cx43) and Cytochrome-C (Cyt-C) as well as electron-microscopic and morphometric studies were carried out. Results. The Au-NPs group demonstrated structural disorganization in the pars distalis, inflammation, congestion and increased extracellular PAS-positive colloid deposition due to the accumulation of Au-NPs. A significant increase in the immunoreactivity of S-100, Cx43 and Cyt-c, along with a significant increase in TNF-a, MDA, and bFGF content in the pituitary homogenates, was noted as compared to the control group. Ultrastructurally, degenerative changes were observed in the secretory cells. FSCs showed proliferation and increased phagocytic activity. Conclusions. Repetitive exposure of adult male albino rats to Au-NPs prompted the accumulation of these nanoparticles in the pars distalis that was accompanied by cellular degeneration and dysfunction of the secretory cell and proliferation of FSCs. Thus, monitoring of the pars distalis hormonal levels might be useful for early detection of some hazardous effects possibly associated with the use of gold-nanoparticles. [ABSTRACT FROM AUTHOR]

The programmed cell death (PCD) occurs when the targeted cells have fulfilled their task or under conditions as oxidative stress generated by ROS species. Thus, plants have to deal with the singlet oxygen 1O2 produced in chloroplasts. 1O2 is unlikely to act as a primary retrograde signal owing to its high reactivity and short half-life. In addition to its high toxicity, the 1O2 generated under an excess or low excitation energy might also act as a highly versatile signal triggering chloroplast-to-nucleus retrograde signaling (ChNRS) and nuclear reprogramming or cell death. Molecular and biochemical studies with the flu mutant, which accumulates protochlorophyllide in the dark, demonstrated that chloroplastic 1O2-driven EXECUTER-1 (EX1) and EX2 proteins are involved in the 1O2-dependent response. Both EX1 and EX2 are necessary for full suppression of 1O2-induced gene expression. That is, EXECUTER proteolysis via the ATP-dependent zinc protease (FtsH) is an integral part of 1O2-triggered retrograde signaling. The existence of at least two independent ChNRS involving EX1 and β-cyclocitral, and dihydroactinidiolide and OXI1, respectively, seem clear. Besides, this update also focuses on plant PCD and its relation with mitochondrial cytochrome-c (Cytc) release to cytosol. Changes in the dynamics and morphology of mitochondria were shown during the onset of cell death. The mitochondrial damage and translocation of Cytc may be one of the major causes of PCD triggering. Together, this current overview illustrates the complexity of the cellular response to oxidative stress development. A puzzle with the majority of its pieces still not placed. [ABSTRACT FROM AUTHOR]

Rapid, efficient, and robust quantitative analyses of dynamic apoptotic events are essential in a high‐throughput screening workflow. Currently used methods have several bottlenecks, specifically, limitations in available fluorophores for downstream assays and misinterpretation of statistical image data analysis. In this study, we developed cytochrome‐C (Cyt‐C) and caspase‐3/‐8 reporter cell lines using lung (PC9) and breast (T47D) cancer cells, and characterized them from the response to apoptotic stimuli. In these two reporter cell lines, the spatial fluorescent signal translocation patterns served as reporters of activations of apoptotic events, such as Cyt‐C release and caspase‐3/‐8 activation. We also developed a vision‐based, tunable, automated algorithm in MATLAB to implement the robust and accurate analysis of signal translocation in single or multiple cells. Construction of the reporter cell lines allows live monitoring of apoptotic events without the need for any other dyes or fixatives. Our algorithmic implementation forgoes the use of simple image statistics for more robust analytics. Our optimized algorithm can achieve a precision greater than 90% and a sensitivity higher than 85%. Combining our automated algorithm with reporter cells bearing a single‐color dye/fluorophore, we expect our approach to become an integral component in the high‐throughput drug screening workflow. [ABSTRACT FROM AUTHOR]

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments. [ABSTRACT FROM AUTHOR]

BackgroundSeveral studies have examined the correlation between iron oxidation and H2O2 degradation. The present study was carried out to examine the protective effects of MRN-100 against stress-induced apoptosis in murine splenic cells in vitro. MRN-100, or HydroFerrate fluid, is an iron-based beverage composed of bivalent and trivalent ferrates.MethodsSplenic lymphocytes from mice were cultured in the presence or absence of MRN-100 for 2 hrs and were subsequently exposed to hydrogen peroxide (H2O2) at a concentration of 25 μM for 14 hrs. Percent cell death was examined by flow cytometry and trypan blue exclusion. The effect of MRN-100 on Bcl-2 and Bax protein levels was determined by Western blot.ResultsResults show, as expected, that culture of splenic cells with H2O2 alone results in a significant increase in cell death (apoptosis) as compared to control (CM) cells. In contrast, pre-treatment of cells with MRN-100 followed by H2O2 treatment results in significantly reduced levels of apoptosis. In addition, MRN-100 partially prevents H2O2-induced down-regulation of the anti-apoptotic molecule Bcl-2 and upregulation of the pro-apoptotic molecule Bax.ConclusionOur findings suggest that MRN-100 may offer a protective effect against oxidative stress-induced apoptosis in lymphocytes.

Mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA). The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479), a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens and pigment deposition) eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS) production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition.

[Display omitted] • ECM stiffness promotes cell spreading and proliferation in breast cancer cells. • Raman spectroscopy revealed distinct peaks in cells grown on varying ECM stiffness. • MCR-ALS showed high nucleic acids and low lipid content in cells grown on stiff ECM. • PC-LDA classifies cells into distinct clusters based on ECM stiffness. • Spectral features resonate with ultrastructural changes linked with mechanoresponse. Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening. [ABSTRACT FROM AUTHOR]

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.

Apoptosis a programmed cell death, is an essential mechanism of eliminating damaged or aged cells and thus to maintain tissue integrity. There are two central pathways that lead to apoptosis: a) the positive induction by ligands (death factors) binding to plasma membrane receptors (death factor receptors) and b) negative induction by the loss of suppressor activity. The common execution mechanisms of apoptosis consist of the activation of cytosolic aspartate-specific proteases (ICE-proteases) termed caspases, which can be activated via various intracellular pathways. In the stomach, mucosal surface epithelial cells are constantly exfoliating to the gastric lumen and completely replaced within 3-5 days under physiological conditions. Apoptosis has been reported to take place in all regions of the stomach with apoptotic cells occurring predominantly in the superficial parts of the gastric glands, at a rate of 2-3% for all cells. Following mucosal injury (e.g. ulcer development), apoptosis rapidly increases and remains elevated for 2-3 months. In a 3-month old ulcer scar, the apoptosis rate of mucous, parietal, chief and endocrine cells was found to be similar to that of normal gastric mucosa. Helicobacter pylori (H. pylori) infection induces apoptosis in the gastric mucosa and this action appears to be independent of VacA cytotoxin of H. pylori strains. Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) inhibitors are potent inductors of gastric epithelial cell apoptosis. However, they can abrogate apoptosis or proliferation effects induced by H. pylori. Many details of the exact intracellular and molecular mechanisms regulating apoptosis in gastric mucosa remain to be elucidated.

Multi-walled carbon nanotubes (MWCNTs) were incorporated into the active layer of mesoporous TiO2 films resulting in MWCNTs-TiO2 nanocomposites with improved electrical conductivity. These MWCNTs-TiO2 nanocomposite films were prepared by a direct mixing method and the “doctor blade” technique. The films were sintered at various annealing temperatures (300, 350, 400, and 450 °C) in order to examine the effect of annealing temperature to the morphology and electrochemical activity of the films. The presence of anatase TiO2 and MWCNTs has been confirmed by X-ray diffraction (XRD), field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy, while conductivity and electrochemical properties of the nanocomposite MWCNΤs-TiO2 films were examined via cyclic voltammetry (CV) and spectroelectrochemistry. After successful protein immobilization (Cyt-c), the electrochemical and spectroelectrochemical behavior of these hybrid electrodes (Cyt-c/MWCNTs-TiO2) was examined in detail and particularly the effect of MWCNTs on the interfacial electron transfer between the film electrode and the adsorbed protein molecules.Schematic illustration of MWCNTs-TiO2 nanocomposite films used for Cyt-c immobilization. [ABSTRACT FROM AUTHOR]

The present work is to evaluate the neurotoxicity induced by pyrethroid insecticide 'Deltamethrin' at 0.32 mg/kg/day in two main regions of the Wistar rat brain (hippocampus and striatum) and the protective effects of Quercetin at 10 mg/kg/day on this toxicity after 90 days of exposure. The assay of brain parameters showed that Deltamethrin caused a significant increase of mitochondrial metabolite level (proteins, lipids, and carbohydrates) and enzyme activity (glutathione S-transferase and superoxide dismutase); a decreased amount of mitochondrial glutathione level and catalase and glutathione peroxidase activities; and an increase of malondialdehyde (MDA) acid levels of the two regions. Furthermore, mitochondrial functional testing in the brains of treated rats exhibited a significant increase in permeability followed by a mitochondrial swelling. Instead, a statistically significant decrease in mitochondrial respiration (O consumption) was recorded in the striatum and hippocampus. Our study showed that the pesticide caused a significant increase of the cytochrome c amount correlated with activation of neuronal apoptosis mechanisms by the significant increase of caspase-3 of hippocampus and striatum. In particular, the results of behavioral tests (open field, classic maze tests of sucrose, and Morris water maze) have significant changes, namely bad behavior of the treated rats, affecting the level of anxiety, learning, and memory, and general motor activity has mainly been shown in treated rats. In addition, the histological cuts clearly confirm cerebral necrosis in the hippocampus and the striatum caused by the pesticide. They allow us to consider the necrotic areas, black spots, reduction, and denaturation of these brain regions in the treated rats. On the other hand, we have studied the protective effects against the neurotoxicity of Deltamethrin (DLM). In this context, after the gavage of Quercetin at the dose of 10 mg/kg/day, we have noticed an improvement in the entire parameters: mitochondrial enzyme, metabolic, histological, and behavioral parameters. This confirmed the improvement of preventive and curative effect of Quercetin against free radicals induced by the DLM. [ABSTRACT FROM AUTHOR]

Objective: To investigate the positive effects of curcumin pie treatment to liver injury and the mechanism during heatstroke in dry-heat environment. Methods: 50 Sprague-Dawley rats were randomly divided into five groups (n=10): Control. HS. 50-cur. 100-cur and 200-cur. The Control and HS group were given a gavage of normal saline, while 50-cur. 100-cur. 200-cur group were given a gavage of curcumin with the concentration 50 mg/kg. 100 ing/kg, 200 mg/kg once a day for 7 consecutive days. At 8th day. all except Control group were transferred to the cabin (The Simulated Climate Cabin for Special Environment of Northwest of China. Ununqi of China) with the conditions of 41 ± 0.5 °C temperature. 10± 1 % relative humidity. At the time of 150th minute since the experiment began, the rats were in heatstroke, then be detected core temperature and anesthetized for collecting blood and liver samples. And same done to Control group. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) in the blood, malondialdehyde (MDA). superoxide dismutase (SOD), catalase (CAT) and cytochrome-c (Cyt-c) in the liver were detected, ultrastmctural changes of liver were observed by electron microscope. Results: (1) Tc of all rats in the cabin had risen to above 42 °C and conformed to the standard of heatstroke. Compared with HS group. Tc of curcumin pretreatment groups decreased (P<0.01). There were no significant differences among curcumin pretreatment groups (P>0.05). (2) Compared with Control group. ALT and AST of HS group increased (P<0.01). Compared with HS group, curcumin pretreatment decreased the levels of ALT and AST (P<0.05). The differences among curcumin pretreatment groups were significant (P<0.05). (3) Compared with Control group. MDA increased and SOD and CAT decreased in HS group (P<0.01). Compared with HS group. MDA decreased and SOD and CAT increased in curcumin pretreatment groups (P<0.01). The differences among curcumin pretreatment groups were significant (P<0.05). (4) Under the electron microscope,mitochondria swelled and proliferated with structure damaged and part of nucleus broken in HS group, while in 50-cur group, part of mitochondria swelled, but none of structure damaged, and in 100-cur and 200-cur group, only mitochondria proliferated obviously. (5) Compared with Control group (0.24± 0.02). Cyt-C expressed high in HS group (1.29± 0.19) (PO.Ol). Curcumin pretreatment decreased the expression of Cyt-c (the levels of Cyt-c in 50-cur. 100-cur. 200-cur were 0.75± 0.08. 0.64± 0.08. 0.48± 0.06) in the liver (P<0.05). The differences among curcumin pretreatment groups were significant (P<0.05). Conclusions: The liver injured during heatstroke in dry-heat environment and curcumin pretreatment could alleviate the liver injury through protecting mitochondria and inhibiting oxidative stress,and the protective effect was dose-dependent. [ABSTRACT FROM AUTHOR]

The Nilgai or Blue Bull (Boselaphus tragocamelus) is the largest Asian antelope which is endemic to Nepal, Pakistan and Indian regions, where these animals are found in packs. Due to rampant hunting, deforestation and habitat degradation, this vulnerable specie is near threatened in many parts of the world. There is need of conservation efforts both at genomic and geographic level. The present study was designed for genomic characterization of Boselaphus tragocamelus in Pakistan by using phylogenetic analysis. Samples from Boselaphus tragocamelus were collected from different parks, zoos and natural habitats. DNA was extracted by standard inorganic extraction method. Primer3 software was used for primer designing targeting Mitochondrial specie identification markers such as d-loop, cytochrome-b and cytochrome-c. After amplification, PCR product was sequenced. Bioinformatics tools were applied for identification of polymorphic loci. Allelic frequency of each locus was calculated. Multidimensional scaling plot illustrated low level of generic diversity among individuals. Phylogenetic analysis revealed conserved neighbouring pattern among different individuals as they shared common ancestry. This is the first report on genomic characterization of Nilgai from Pakistan. The information of selected species of deer is prerequisite for designing effective strategy in future effective practices for conservation. However further genomic investigations should be carried out at a larger scale. [ABSTRACT FROM AUTHOR]

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many “on-line„ systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ΔpilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ΔS. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ.

This study aimed to develop a robust approach for the early diagnosis and treatment of tumors. Short circular DNA nanotechnology synthesized a stiff and compact DNA nanotubes (DNA-NTs) framework. TW-37, a small molecular drug, was loaded into DNA-NTs for BH3-mimetic therapy to elevate the intracellular cytochrome-c levels in 2D/3D hypopharyngeal tumor (FaDu) cell clusters. After anti-EGFR functionalization, the DNA-NTs were tethered with a cytochrome-c binding aptamer, which can be applied to evaluate the elevated intracellular cytochrome-c levels via in situ hybridization (FISH) analysis and fluorescence resonance energy transfer (FRET). The results showed that DNA-NTs were enriched within the tumor cells via anti-EGFR targeting with a pH-responsive controlled release of TW-37. In this way, it initiated the triple inhibition of "BH3, Bcl-2, Bcl-xL, and Mcl-1". The triple inhibition of these proteins caused Bax/Bak oligomerization, leading to the perforation of the mitochondrial membrane. This led to the elevation of intracellular cytochrome-c levels, which reacted with the cytochrome-c binding aptamer to produce FRET signals. In this way, we successfully targeted 2D/3D clusters of FaDu tumor cells and achieved the tumor-specific and pH-triggered release of TW-37, causing tumor cell apoptosis. This pilot study suggests that anti-EGFR functionalized, TW-37 loaded, and cytochrome-c binding aptamer tethered DNA-NTs might be the hallmark for early tumor diagnosis and therapy. • We developed a FRET-based, stimuli-responsive, and aptamer-tethered DNA framework. • DNA nanotubes (DNA-NTs) were evaluated for FISH-switch-on bioimaging of pre-apoptosis. • Anti-EGFR@DNA-NTs hindered the proliferation of hypopharyngeal tumor (FaDu) cells. • DNA-NTs carrying TW-37 caused triple inhibition of "BH3, Bcl-2/Bcl-xl, and Mcl-1". • BH3-mimetic therapy restored cell apoptosis and sensed elevated cytochrome c levels. [ABSTRACT FROM AUTHOR]

Monitoring of bio-catalytic events by using nano-probes is of immense interest due to unique optical properties of metal nanoparticles. In the present study, tunneling of enzyme activity was achieved using redox cofactors namely oxidized cytochrome-c (Cyt-c) and Co-enzyme-Q (Co-Q) immobilized on Quantum dots (QDs) which acted as a bio-probe for NAD + dependent dehydrogenase catalyzed reaction. We studied how electron transfer from substrate to non-native electron acceptors can differentially modify photoluminescence properties of CdTe QDs. Two probes were designed, QD-Ox-Cyt-c and QD-Ox-Co-Q, which were found to quench the fluorescence of QDs. However, formaldehyde dehydrogenase (FDH) catalyzed reduction of Cyt-c and Co-Q on the surface of QDs lead to fluorescence turn-on of CdTe QDs. This phenomenon was successfully used for the detection of HCHO in the range of 0.01–100,000 ng/mL (LOD of 0.01 ng/mL) using both QD-Ox-Cyt-c (R 2 =0.93) and QD-Ox-Co-Q (R 2 =0.96). Further probe performance and stability in samples like milk, wine and fruit juice matrix were studied and we could detect HCHO in range of 0.001−100,000 ng/mL (LOD of 0.001 ng/mL) with good stability and sensitivity of probe in real samples (R 2 =0.97). Appreciable recovery and detection sensitivity in the presence of metal ions suggests that the developed nano-probes can be used successfully for monitoring dehydrogenase based bio-catalytic events even in the absence of NAD + . Proposed method is advantageous over classical methods as clean up/ derivatization of samples is not required for formaldehyde detection. [ABSTRACT FROM AUTHOR]

Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20–40 μM evodiamine treatment for 24–48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survival of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer. [ABSTRACT FROM AUTHOR]

Microemulsions (µEs), comprising water as polar component, pluronic (normal, L35 and reverse, 10R5) as surfactant and a hydrophobic ionic liquid (HIL) as non-polar component have been prepared and characterized. Owing to higher surface activity, pluronics have promoted the formation of µEs without the use of co-surfactant. Thus prepared µEs have been utilized as nano-reactors for the oxidation of guaiacol in the presence of Cytochrome-c (Cyt-c) at 15, 20, and 25 °C. A 3.2- and 1.3-fold increase in the rate of formation of product of enzymatic catalysis in direct µE (HIL-in-water) with reverse pluronic (10R5) is observed at 15 and 20 °C as compared to that in buffer. However, negligible enzymatic activity is observed in the direct µE formed by normal pluronic (L35). The catalytic activity of Cyt-c decreases in reverse µEs (water-in-HIL) as compared to direct µEs irrespective of the nature of pluronic used. The contrasting nature of nano-interfaces formed by pluronics in µEs and the extent of hydration of these nano-interfaces controlled by temperature exerts varying influence on the catalytic activity of Cyt-c. It is expected that the present work would result in providing a versatile platform for the creation of new IL and pluronic-based µEs for bio-catalytic applications, which have never been reported. [Display omitted] • Ionic liquid and pluronic-based microemulsions (µEs) are prepared. • Molecular structure of pluronic directed the nature of formed nano-interfaces. • Nano-interfaces influenced the catalytic activity of Cytochrome-c positively. • Cytochrome-c exhibits 3.2-fold higher activity in direct-µEs than in buffer. [ABSTRACT FROM AUTHOR]

Whether seizures might determine the activation of cell death pathways and what could be the relevance of seizure-induced cell death in epilepsy are still highly debated issues. We recently developed an experimental model of acquired focal cortical dysplasia (the MAM-pilocarpine or MP rat) in which the occurrence of status epilepticus – SE – and subsequent seizures induced progressive cellular/molecular abnormalities and neocortical/hippocampal atrophy. Here, we exploited the same model to verify when, where, and how cell death occurred in neurons and glia during epilepsy course. We analyzed Fluoro Jade (FJ) staining and the activation of c-Jun- and caspase-3-dependent pathways during epilepsy, from few hours post-SE up to six months of spontaneous recurrent seizures. FJ staining revealed that cell injury in MP rats was not temporally restricted to SE, but extended throughout the different epileptic stages. The region-specific pattern of FJ staining changed during epilepsy, and FJ + neurons became more prominent in the dorsal and ventral hippocampal CA at chronic epilepsy stages. Phospho-c-Jun- and caspase-3-dependent pathways were selectively activated respectively in neurons and glia, at early but even more conspicuously at late chronic stages. Phospho-c-Jun activation was associated with increased cytochrome-c staining, particularly at chronic stages, and the staining pattern of cytochrome-c was suggestive of its release from the mitochondria. Taken together, these data support the content that at least in the MP rat model the recurrence of seizures can also sustain cell death mechanisms, thus continuously contributing to the pathologic process triggered by the occurrence of SE. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying stress-induced depression have not been fully outlined. Hence, the current study aimed at testing the link between behavioral changes in chronic mild stress (CMS) model and changes in hippocampal energy metabolism and the role of paroxetine (PAROX) in ameliorating these changes. Male Wistar rats were divided into three groups: vehicle control, CMS-exposed rats, and CMS-exposed rats receiving PAROX (10 mg/kg/day intraperitoneally). Sucrose preference, open-field, and forced swimming tests were carried out. Corticosterone (CORT) was measured in serum, while adenosine triphosphate and its metabolites, cytosolic cytochrome-c (Cyt-c), caspase-3 (Casp-3), as well as nitric oxide metabolites (NOx) were measured in hippocampal tissue homogenates. CMS-exposed rats showed a decrease in sucrose preference as well as body weight compared to control, which was reversed by PAROX. The latter further ameliorated the CMS-induced elevation of CORT in serum (91.71±1.77 ng/mL vs 124.5±4.44 ng/mL, P<0.001) as well as the changes in adenosine triphosphate/adenosine diphosphate (3.76±0.02 nmol/mg protein vs 1.07±0.01 nmol/mg protein, P<0.001). Furthermore, PAROX reduced the expression of Cyt-c and Casp-3, as well as restoring NOx levels. This study highlights the role of PAROX in reversing depressive behavior associated with stress-induced apoptosis and changes in hippocampal energy metabolism in the CMS model of depression. [ABSTRACT FROM AUTHOR]

Objective : In the present study we analyzed neuroprotective and antiapoptotic effect of the difumarate salt S-15176, as an anti-ischemic, an antioxidant and a stabilizer of mitochondrial membrane in secondary damage following spinal cord injury (SCI) in a rat model. Methods : Three groups were performed with 30 Wistar rats; control (1), trauma (2), and a trauma+S-15176 (10 mg/kg i.p., dimethyl sulfoxide) treatment (3). SCI was performed at the thoracic level using the weight-drop technique. Spinal cord tissues were collected following intracardiac perfusion in 3rd and 7th days of posttrauma. Hematoxylin and eosin staining for histopatology, terminal deoxynucleotidyl transferase dUTP nick end labeling assay for apoptotic cells and immunohistochemistry for proapoptotic cytochrome-c, Bax and caspase 9 were performed to all groups. Functional recovery test were applied to each group in 3rd and 7th days following SCI. Results : In trauma group, edematous regions, diffuse hemorrhage, necrosis, leukocyte infiltration and severe degeneration in motor neurons were observed prominently in gray matter. The number of apoptotic cells was significantly higher (p<0.05) than control group. In the S-15176-treated groups, apoptotic cell number in 3rd and 7th days (p<0.001), also cytochrome-c (p<0.001), Bax (p<0.001) and caspase 9 immunoreactive cells (p<0.001) were significantly decreased in number compared to trauma groups. Hemorrhage and edema in the focal areas were also noticed in gray matter of treatment groups. Results of the locomotor test were significantly increased in treatment group (p<0.05) when compared to trauma groups. Conclusion : We suggest that difumarate salt S-15176 prevents mitochondrial pathways of apoptosis and protects spinal cord from secondary injury and helps to preserve motor function following SCI in rats. [ABSTRACT FROM AUTHOR]

Abstract: The study of noncovalent interactions between pharmaceutical molecules and proteins is essential for understanding molecular mechanisms of protein function, and provides foundations for de novo therapeutic agent design. Electrospray ionization mass spectrometry (ESI-MS) has nowadays become a popular tool for analyzing the noncovalent protein complexes, however it usually has difficulty in determining the interaction sites and binding mechanisms. In this work, a new structure-differential binding (SDB) method, combined with ESI-MS and molecular docking (MD) techniques (SDB–ESIMS–MD), was developed and applied to a study of the binding interactions in noncovalent protein–small drug molecule complexes for the characterization of binding sites and binding modes. Using this developed method, protein complexes of flavonoid and flavonoid glycoside ligands and cytochrome-c (Cyt-c) were studied in detail. ESI-MS was used to determine the relative binding affinities and dissociation constants of flavonoid–Cyt-c complexes, and to measure the changes in the stability of the protein complexes with the structural modifications of the ligands for identifying effective binding functional groups. Molecular docking simulations complemented ESI-MS experiments by providing the protein–ligand interaction profile of each complex and displaying the binding mode for each interaction. This SDB–ESIMS–MD method can be applied to a broad range of protein–drug interactions and used to guide further research in the study of structure–binding relationship between drug molecules and targeted biomacromolecules. [Copyright &y& Elsevier]

Abstract: In the present study, we examined the molecular mechanism by which Piperine (bioactive compound of Piper nigrum) inhibits neuronal cell apoptosis. We further investigated the anti-inflammatory effect of Piperine on 6-OHDA induced Parkinson''s disease. Consistent with its antioxidant properties, Piperine (10mg/kg bwt) reduced 6-OHDA-induced lipid peroxidation and stimulated glutathione levels in striatum of rats. Furthermore, Piperine treatment diminished cytochrome-c release from mitochondria and reduced caspase-3 and caspase-9 activation induced by 6-OHDA. Treatment with Piperine markedly inhibited poly(ADP-ribose) polymerase activation, pro-apoptotic Bax levels and elevation of Bcl-2 levels. Piperine reduces contralateral rotations induced by apomorphine. Further narrow beam test and rotarod also showed improvement in motor coordination and balance behavior in rats treated with Piperine. In addition Piperine depletes inflammatory markers, TNF-α and IL-1β in 6-OHDA-induced Parkinson''s rats. We propose that, in addition to its antioxidant properties Piperine exerts a protective effect via anti-apoptotic and anti-inflammatory mechanism on 6-OHDA induced Parkinson''s disease. [Copyright &y& Elsevier]

Objectives To investigate the effect of tibolone and raloxifene on the serum apoptotic markers soluble Fas (sFas), soluble Fas ligand (sFasL) and cytochrome-c (cyt-c) in postmenopausal women. Methods A total of 89 healthy postmenopausal women, attending the University Menopause Clinic, were randomly allocated to tibolone ( n =30), raloxifene ( n =29) or no treatment ( n =30). Serum apoptotic markers sFas, sFasL and cyt-c were measured at baseline and at 6 months. Results Serum sFasL decreased significantly in women receiving tibolone (baseline: 53.8±28.3 pg/ml, 6 months: 40.45±19.2 pg/ml, p =0.001), whilst sFas levels did not significantly change in this group. Serum sFas or sFasL did not change either in the raloxifene group or in the control group. Serum cyt-c concentrations were under the detection limit of the assay in all women assessed. Conclusions Tibolone use resulted in a significant decrease in serum sFasL, but not in serum sFas. Raloxifene had no effect on either sFas or sFasL. These results may indicate that tibolone use is associated with a decrease in receptor-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9MPa, and 1MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1kHz pulse repetition frequency, and 1min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology. [Copyright &y& Elsevier]

Abstract: Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H2O2) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H2O2 showed increases in miR-1 expression, mitochondria dysfunction, cytochrome-c release and apoptosis, Addition of IGF-1 into the iPS cell cultures reduced the H2O2 cytotoxicity. Prediction algorithms showed that 3′-untranslated regions of IGF-1 gene as a target of miR-1. Moreover, miR-1 mimic, but not miR-1 mimic negative control, diminished the protective effect of IGF-1 on H2O2-induced mitochondrial dysfunction, cytochrome-c release and apoptosis in iPS cells. In conclusion, IGF-1 inhibits H2O2-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. IGF-1′s effect is, at least partially, regulated by miR-1 in iPS cells. [Copyright &y& Elsevier]

Ziram as a dithiocarbamate fungicide is widely used throughout the world in agriculture. We previously found that ziram significantly inhibited cytotoxic T lymphocyte activity in a dose-dependent manner. To explore the mechanism of this inhibition, we investigated ziram-induced apoptosis in human T lymphocytes. Jurkat T cells were treated with ziram at 0.031-1 μM for 2-24 h. Freshly isolated primary human T cells were treated with ziram at 0.0625-1 μM for 15 and 24 h. Apoptosis was determined by FITC-Annexin V/PI staining and the TUNEL assay. To explore the mechanism of apoptosis, intracellular levels of active caspases 3, 3/7, 8, and 9 and pan-caspase and mitochondrial cytochrome-c release were determined by flow cytometry. Disruption to mitochondrial transmembrane potential was determined with a MitoLight Apoptosis Detection Kit. We found that ziram induced apoptosis in a time- and dose-dependent manner in both Jurkat cells and primary human T cells. The primary human T cells were more sensitive to ziram than the Jurkat cell line. Ziram induced increases in active caspases 3, 3/7, 8, and 9 and pan-caspase in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, partially but significantly inhibited the apoptosis. Moreover, a general caspase inhibitor, Z-VAD-FMK, significantly and almost completely blocked the apoptosis. Ziram also disrupted mitochondrial transmembrane potential and caused mitochondrial cytochrome-c release. These findings indicate that ziram can induce apoptosis in human T cells, and the apoptosis is mediated by both the caspase-cascade and the mitochondria/cytochrome-c pathways. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS) is known to be associated with pulmonary cytokine production resulting in infiltration of neutrophils and pulmonary injury. The study presented here is focused on the changes in cell death regulating proteins during lung injury and studied the relationship between the LPS challenged ARDS and the subsequent role of surfactant therapy against ARDS. Apoptotic features, such as induction of DNA fragmentation ladders in lung tissue, expression of cytoplasmic proteins (Bcl-2, Bax), mitochondrial proteins (cytochrome c, Apaf-1) and activation of caspases were detected following LPS exposure. Instillation of the surfactant derived from the natural source as well as synthetic surfactant prevents LPS induced apoptosis. These results suggest that the mechanism of induction of apoptosis by LPS depends on mitochondrial dysfunction and the anti-apoptotic effects of surfactant treatment conferred protection in the rat model during lung injury mediated via suppression of mitochondrial intrinsic pathway. [ABSTRACT FROM AUTHOR]

Unilamellar liposomes, prepared from synthetic lipid mixture of DMPC and DMPG either by sonication or extrusion, were used to entrap water soluble and water insoluble molecules to investigate the efficacy of encapsulation by different liposome preparation methods. In the case of entrapment of hydrophilic protein cytochrome-C, the solutions were subjected to a series of ultrafiltration steps to eliminate any free protein outside the vesicles. It was observed that the protein could be encapsulated by the vesicles only if cholesterol was present in the bilayer. The release of cytochrome-C was observed spectrophotometrically upon vesicle-breakdown. The amount of protein encapsulated depended on the method of preparation and was found to be 10 times greater in extruded liposomes compared to those produced by sonication. Hydrophobic Vitamin E, on the other hand, could be encapsulated in the liposome bilayer, independently of the presence of cholesterol and the method of preparation. These fundamental results can be used to develop more efficient drug encapsulations and to have better understanding about their release. [ABSTRACT FROM AUTHOR]

Abstract: Avermectins are a series 16-membered macrocyclic lactone derivatives derived from the actinomycete Streptomyces avermectinius, which has been applied worldwide in veterinary, human medicine and agriculture. Abamectin is a mixture of avermectins containing ⩾80% avermectin B1a and ⩽20% avermectin B1b. In this paper, the apoptotic induction effect of abamectin on Spodoptera frugiperda (Sf9) cells was detected by using various techniques including cell proliferation assay, flow cytometry, morphology analysis with inverted phase contrast microscope, scanning electron microscope, transmission electron microscopy, enzyme activity assay and Western blot. The results revealed that within the concentrations of 5–15μg/mL, abamectin inhibited the growth of the Sf9 cells and induced apoptotic cell death in a time- and dose-dependent manner. The proliferation inhibition rates of Sf9 cells were 6.45±0.47%, 11.51±0.20% and 12.97±0.46% after 12h of treatment with avermectin at the concentrations of 5, 10 and 15μg/mL, which increased to 30.74±0.57%, 36.14±0.25% and 39.27±0.63% after 72h of the same treatments, respectively. The maximum apoptosis rate of each concentration was 24.13±0.43%, 24.69±0.38% and 27.285±0.84%, respectively. Treated cells showed typical apoptosis morphological changes including cell swelling, chromatin condensation, apoptotic bodies, swollen mitochondria and high content of phagosome. Both mitochondrial events including mitochondrial membrane potential (Δψm) loss as well as cytochrome-c release into cytosol and significant activation of caspase-3 occurred during apoptosis. These data confirmed the apoptosis inducing effects of abamectin on S. frugiperda (Sf9) cell line, and provide preliminary studies on its mechanism. [Copyright &y& Elsevier]