Comparison of PPAR Ligands as Modulators of Resolution of Inflammation,via Their Influence on Cytokines and Oxylipins Release in Astrocytes

Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARβ ({"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARβ ligands possessed the strongest effect. The PPARβ agonist, {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARβ agonist {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516. The PPARβ ligands, {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARβ ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.     

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca²⁺ concentration. However, the mutations differently affected the Ca²⁺ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca²⁺-dependent interaction of myosin heads with F-actin.     

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague–Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection—in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.     

Static fatigue of Hi-Nicalon™-S fiber at elevated temperature in air,steam, and silicic acid-saturated steam

A facility for testing SiC fiber tows in static fatigue and creep at elevated temperatures in air and steam was developed. Static fatigue of Hi-Nicalon™-S fibers was investigated at 800°C-1100°C at applied stresses between 115 and 1250 MPa in air, in Si(OH)₄(g)-saturated steam, and in unsaturated steam. Fibers tested in Si(OH)₄(g)-saturated steam and in air had silica scales throughout the test sections, but those tested in unsaturated steam did not develop scales near the steam injection point. Fiber lifetimes in static fatigue were shortest in unsaturated steam, intermediate in Si(OH)₄(g)-saturated steam, and longest in air. Failure strains did not exceed 0.3%. Steady-state strain rates and static fatigue lifetimes are modelled empirically by the Monkman-Grant relationship. Failure mechanisms are discussed.     

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.     

Stereodivergent Biocatalytic Formal Reduction of α-Angelica Lactone to (R)- and (S)-γ-Valerolactone in a One-Pot Cascade

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.     

Stochastic model of reaction rate oscillations during CO oxidation over zeolite-supported catalysts

The particle size effect on the oscillatory behaviour during CO oxidation over zeolite-supported Pd catalysts is simulated with the help of a deterministic point model and a stochastic mesoscopic model. The point model is developed on the basis of Sales, Turner and Maple (STM) model, which is modified to consider the effects of the oxidation of the Pd bulk upon the catalyst activity. It is demonstrated that the deterministic point model can simulate the main properties of regular reaction rate oscillations. The stochastic model is based on the developed point model and simulates the reaction by a Markovian chain of elementary transitions, which correspond to changes in numbers of atoms and molecules of reagent species on the surface of Pd particle due to elementary steps of reaction. The stochastic model explains the role of statistical fluctuations and correlations in the reaction dynamics on the surface of an nm-sized catalyst particle.     

Optimization of supercritical CO2 extraction of dried Helichrysum italicum flowers by response surface methodology: GC-MS profiles of the extracts and essential oil

Supercritical (SC) CO₂ extraction of dried Immortelle flowers was performed at different process parameters. The optimal extraction conditions related to the yield were determined by response surface methodology (yield 4.09% at 20 MPa and 52°C). The extracts were analyzed by GC-MS and tremetone derivatives dominated: bitalin A, 12-acetoxytremetone, gnaphaliol, 1-[2-(2-methyl-2,3-dihydroxypropyl)-2,3-dihydro-1-benzofuran-5-yl]ethanone, isobutyl bitalin A, and 1-[2-(acetylprop-1-en-2-yl)-3-hydroxy-2,3-dihydro-1-benzofuran-5-yl]ethanone. Striking differences were found among the essential oil and SC-CO₂ extracts composition. The major oil compounds were γ-curcumene, α-pinene, β-selinene, α-selinene, and limonene. Mono- and sesquiterpene were found among minor constituents of the extracts. Neryl acetate was present in the extracts and essential oil.     

Bioactivity of Phenanthrenes from Juncus acutus on Selenastrum capricornutum

Twenty-five 9,10-dihydrophenanthrenes, four phenanthrenes, a dihydrodibenzoxepin, and a pyrene, isolated from the wetland plant Juncus acutus, were tested to detect their effects on the green alga Selenastrum capricornutum. Nine of the compounds were isolated and identified for the first time. Most of the compounds caused inhibition of algal growth. The 9,10-dihydrophenanthrenes 1, 5, 21, and 22 were the most active.     

Geranylgeraniol and Neurological Impairment: Involvement of Apoptosis and Mitochondrial Morphology

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.