Ekspresija i pročišćavanje glavnoga proteina (OmpH) bakterije Pasteurella multocida P52 proizvedenoga u bakteriji Escherichia coli.

Porin H (OmpH) is the major outer membrane protein in the envelope of Pasteurella multocida. The gene ompH, encoding major outer membrane protein was amplified by PCR excluding the region coding for signal peptide and cloned in the pQE32 prokaryotic expression vector. The recombinant OmpH was expressed as a fusion protein with 6-His tag at N-terminal in E. coli M15 cells transformed with recombinant plasmid pQE32-ompH. The expressed protein was purified from E. coli and characterized by SDS-PAGE and western blot analysis. The fusion recombinant protein eluted had a molecular mass of about 37 kDa. The expressed recombinant protein was confirmed with western blot analysis using RGS-His antibody and anti-P. multocida serum raised against whole cell lysate.Porin H (OmpH) je glavni protein stanične stijenke bakterije Pasteurella multocida. Gen ompH, koji kodira njegovu tvorbu, isključujući područje za tvorbu signalnog peptida, bio je umnožen lančanom reakcijom polimerazom i kloniran u prokariotskom vektoru pQE32. Rekombinantni OmpH bio je izražen kao fuzijski protein sa 6-His tag na N-kraju u stanicama E. coli M15 transformiranima rekombinantnim plazmidom pQE32- ompH. Proizveden protein bio je pročišćen iz E. coli i identificiran SDS-PAGE-om i western blotom. Izdvojeni fuzijski rekombinantni protein imao je molekularnu masu oko 37 kDa. Identitet proizvedenog rekombinantnog proteina bio je povrđen western blot analizom uporabom protutijela za RGS-His i antiseruma za lizat cjelovite stanice P. multocida

AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia

Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5′ AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2′,7′-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40phox, gp91phox, AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD+/NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40phox and gp91phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD+. Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD+/SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia

Inhibition of neuroinflammation by thymoquinone requires activation of Nrf2/ARE signalling

Thymoquinone is an antioxidant phytochemical that has been shown to inhibit neuroinflammation. However, little is known about the potential roles of intracellular antioxidant signalling pathways in its anti-inflammatory activity. The objective of this study was to elucidate the roles played by activation of the Nrf2/ARE antioxidant mechanisms in the anti-inflammatory activity of this compound. Thymoquinone inhibited lipopolysaccharide (LPS)-induced neuroinflammation through interference with NF-B signalling in BV2 microglia. Thymoquinone also activated Nrf2/ARE signalling by increasing nuclear localisation, DNA binding and transcriptional activity of Nrf2, as well as increasing protein levels of HO-1 and NQO1. Suppression of Nrf2 activity through siRNA or with the use of trigonelline resulted in the loss of anti-inflammatory activity by thymoquinone. Taken together, our studies show that thymoquinone inhibits NF-kappaB-dependent neuroinflammation in BV2 microglia, by targeting antioxidant pathway involving activation of both Nrf2/ARE. We propose that activation of Nrf2/ARE signalling pathway by thymoquinone probably results in inhibition of NF-kappaB-mediated neuroinflammation

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.Peer reviewe

Investigation of optical properties of InP, AIN and Sapphire for applications in non-contact semiconductor process monitoring

The objective of this thesis was to develop a reliable multi-wavelength pyrometer for simultaneous measurement of the wafer temperature and its optical properties in the wavelength range of 1 to 20 microns and temperature range of 30 to 1500° C. The spectral emissometer has been utilized for measurement of the temperature dependent optical properties of InP, AlN and Sapphire. The experimental results presented in this thesis showed that the measurement of high temperature optical properties could be performed reliably with a novel approach using the spectral ernissometer. The temperature determination capability of the emissometer was tested and verified using a standard thermocouple embedded in a silicon wafer. The temperature measurement accuracy, with the emissometer, was found to be within +/- 10° C of the thermocouple temperature for a temperature range of 30 to 300° C. A particularly interesting results were the observed sharp peak in the emissivity of Fe doped InP at 14 microns, the deconvoluted values of the refractive indices from the measured optical properties of Fe doped InP are within +/- 10 % of the limited refractive index data available in the literature, sapphire exhibiting emissivity value of ~1 at 8 microns and the refractive indices of sapphire approach high values in the wavelength range of 12 to 16 microns resulting from its high reflectance. Spectral emissometry has been established as a reliable technique for simultaneously measurement of temperature and optical properties of semiconductors

Combined immunodeficiency and hypoglycemia associated with mutations in hypoxia upregulated 1

Non peer reviewe

Ketogenic diet attenuates hepatopathy in mouse model of respiratory chain complex III deficiency caused by a Bcs1l mutation

Mitochondrial disorders are among the most prevalent inborn errors of metabolism but largely lack treatments and have poor outcomes. High-fat, low-carbohydrate ketogenic diets (KDs) have shown beneficial effects in mouse models of mitochondrial myopathies, with induction of mitochondrial biogenesis as the suggested main mechanism. We fed KD to mice with respiratory chain complex III (CIII) deficiency and progressive hepatopathy due to mutated BCS1L, a CIII assembly factor. The mutant mice became persistently ketotic and tolerated the KD for up to 11 weeks. Liver disease progression was attenuated by KD as shown by delayed fibrosis, reduced cell death, inhibition of hepatic progenitor cell response and stellate cell activation, and normalization of liver enzyme activities. Despite no clear signs of increased mitochondrial biogenesis in the liver, CIII assembly and activity were improved and mitochondrial morphology in hepatocytes normalized. Induction of hepatic glutathione transferase genes and elevated total glutathione level were normalized by KD. Histological findings and transcriptome changes indicated modulation of liver macrophage populations by the mutation and the diet. These results reveal a striking beneficial hepatic response to KD in mice with mitochondrial hepatopathy and warrant further investigations of dietary modification in the management of these conditions in patients.Peer reviewe

Antimalarial drug artemether inhibits neuroinflammation in BV2 microglia through Nrf2-dependent mechanisms

Artemether, a lipid-soluble derivative of artemisinin has been reported to possess anti-inflammatory properties. In this study, we have investigated the molecular mechanisms involved in the inhibition of neuroinflammation by the drug. The effects of artemether on neuroinflammation-mediated HT22 neuronal toxicity were also investigated in a BV2 microglia/HT22 neuron co-culture. To investigate effects on neuroinflammation, we used LPS-stimulated BV2 microglia treated with artemether (5-40µM) for 24 hours. ELISAs and western blotting were used to detect pro inflammatory cytokines, nitric oxide, PGE2, iNOS, COX-2 and mPGES-1. BACE-1 activity and Aβ levels were measured with ELISA kits. Protein levels of targets in NF-kappaB and p38 MAPK signalling, as well as HO-1, NQO1 and Nrf2 were also measured with western blot. NF-kappaB binding to the DNA was investigated using EMSA. MTT, DNA fragmentation and ROS assays in BV2-HT22 neuronal co-culture were used to evaluate the effects of artemether on neuroinflammation-induced neuronal death. The role of Nrf2 in the anti-inflammatory activity of artemether was investigated in BV2 cells transfected with Nrf2 siRNA. Artemether significantly suppressed pro-inflammatory mediators (NO/iNOS, PGE2/COX-2/mPGES-1, TNFα, and IL-6), Aβ and BACE-1 in BV2 cells following LPS stimulation. These effects of artemether were shown to be mediated through inhibition of NF-kappaB and p38MAPK signalling. Artemether produced increased levels of HO-1, NQO1 and GSH in BV2 microglia. The drug activated Nrf2 activity by increasing nuclear translocation of Nrf2 and its binding to antioxidant response elements in BV2 cells. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of both anti-inflammatory and neuroprotective activities of artemether. We conclude that artemether induces Nrf2 expression and suggest that Nrf2 mediates the anti-inflammatory effect of artemether in BV2 microglia. Our results suggest that this drug has a therapeutic potential in neurodegenerative disorders

Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology

Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOX(Rosa26) mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOX(Rosa26) mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.Peer reviewe