Metathesis as a versatile tool in oleochemistry

Olefin metathesis, awarded with the Nobel Prize in Chemistry 2005 for Chauvin, Grubbs and Schrock, has emerged as a powerful tool for organic as well as polymer chemistry. In oleochemistry, this reaction is well known and has been applied for many decades. Examples include the functionalization of the double bonds of different oleochemicals or the (direct) polymerization of plant oils via metathesis. More recent developments, particularly the development of better and more robust catalysts, allow for highly efficient cross‐metathesis reactions opening new possibilities for the direct introduction of chemical functionalities. Within this contribution, the development of metathesis in oleochemistry will be discussed, covering self‐metathesis as well as more recent developments in the field of cross‐metathesis that lead to desired platform chemicals.     

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased β-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.     

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.     

The influence of transition metals – Fe,Co, Cu on transformation of organic matters from Domanic rocks in hydrothermal catalytic system

Character of conversion of organic matter from Domanic rocks of Pervomaiskoye field (Tatarstan) of Semiluki horizon of upper Devonian deposits in the hydrothermal-catalytic system at temperature of 300?°C in carbon dioxide medium was studied with the application of complex of oil-soluble precursors of catalysts containing Fe, Co, and Cu. In presence of catalysts complex, content of organic extract increases, in which content of hydrocarbon fractions, saturated and aromatic hydrocarbons, increases 1.5 times, while resins content decreases by two times. As result of kerogen destruction in products of experiments, the content of asphaltenes and carbonaceous substances such as carbenes and carboides increase.     

An ontology-based approach for formalisation and semantic organisation of conformance requirements in construction

This paper presents an ontology-based method for the formalisation and application of construction conformance requirements for effective code checking. This research continues our work on the development of a generic model automating the conformance checking of construction projects against building codes. We start from the analysis of the related research on the formalisation and organisation of building codes that allows us to formulate our approach for semantic annotation and scheduling of conformance requirements for conformance checking task. Our approach comprises 5 main steps: formalisation, semantic annotation, classification, context-based scheduling and semantic search of conformance requirements. They are implemented as corresponding key components of the C3R (Conformance Checking in Construction — Reasoning) prototype that we have developed to model the conformance checking process in construction. Finally, we discuss the ongoing work and perspectives of our research: the validation of our model by construction practices and the enrichment of our approach by usage-based knowledge.     

Starch‐capped sulphur nanoparticles synthesised from bulk powder sulphur and their anti‐phytopathogenic activity against Clavibacter sepedonicus

Water‐soluble, stable nanoparticles of elemental sulphur with a size of 9‐52 nm have been synthesised using the stabilising potential of starch. Sulphide anions were used as sulphur precursors that were generated earlier from the bulk powder sulphur in the base‐reduction system NaOH‐N₂H₄·H₂O followed by their oxidation with molecular oxygen to element sulphur atoms. Using a set of modern spectral and microscopic methods (XRD, optical spectroscopy, DLS, TEM), the phase state, elemental composition of the nanocomposites and their nanomorphological characteristics have been investigated. It was found that nanocomposites are formed as sulphur particles with the shape which is nearly spherical dispersed in the polysaccharide starch matrix with a pronounced tendency to cluster into ring formations. Water solubility and stability of the obtained nanoparticles is ensured by sorption of starch macromolecules on the surface of sulphur nanoparticles, with the thickness of the stabilising shell in a range of 10‐171 nm. In vitro experiments were carried out to study the anti‐microbial activity of the obtained sulphur nanocomposite (1.6% S) using the propidium iodide fluorescent dye staining method and the diffusion method. It showed that the water solution of the starch‐capped sulphur nanoparticles at the concentration of 6.25 µg/ml had a pronounced anti‐phytopathogenic activity against the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus.     

Visualizing biochemical activities in living cells through chemistry

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.     

Novel A-Ring Chalcone Derivatives of Oleanolic and Ursolic Amides with Anti-Proliferative Effect Mediated through ROS-Triggered Apoptosis

A series of A-ring modified oleanolic and ursolic acid derivatives including C28 amides (3-oxo-C2-nicotinoylidene/furfurylidene, 3β-hydroxy-C2-nicotinoylidene, 3β-nicotinoyloxy-, 2-cyano-3,4-seco-4(23)-ene, indolo-, lactame and azepane) were synthesized and screened for their cytotoxic activity against the NCI-60 cancer cell line panel. The results of the first assay of thirty-two tested compounds showed that eleven derivatives exhibited cytotoxicity against cancer cells, and six of them were selected for complete dose–response studies. A systematic study of local SARs has been carried out by comparative analysis of potency distributions and similarity relationships among the synthesized compounds using network-like similarity graphs. Among the oleanane type triterpenoids, C2-[4-pyridinylidene]-oleanonic C28-morpholinyl amide exhibited sub-micromolar potencies against 15 different tumor cell lines and revealed particular selectivity for non-small cell lung cancer (HOP-92) with a GI₅₀ value of 0.0347 μM. On the other hand, superior results were observed for C2-[3-pyridinylidene]-ursonic N-methyl-piperazinyl amide 29, which exhibited a broad-spectrum inhibition activity with GI₅₀ < 1 μM against 33 tumor cell lines and <2 μM against all 60 cell lines. This compound has been further evaluated for cell cycle analysis to decipher the mechanism of action. The data indicate that compound 29 could exhibit both cytostatic and cytotoxic activity, depending on the cell line evaluated. The cytostatic activity appears to be determined by induction of the cell cycle arrest at the S (MCF-7, SH-SY5Y cells) or G₀/G₁ phases (A549 cells), whereas cytotoxicity of the compound against normal cells is nonspecific and arises from apoptosis without significant alterations in cell cycle distribution (HEK293 cells). Our results suggest that the antiproliferative effect of compound 29 is mediated through ROS-triggered apoptosis that involves mitochondrial membrane potential depolarization and caspase activation.     

Novel spray-dried PHA microparticles for antitumor drug release

The production of poly-3-hydroxybutyrate (P3HB) and poly-3-hydroxybutyrate/polyethylene glycol (PEG)-based microparticles, loaded with antitumor drugs paclitaxel (PTX) and 5-Fluorouracil (5-FU) by spray-drying technique, was investigated. The average diameter of microparticles was found to be 3.4?±?0.5?µm and zeta potential was about ?44?mV. The addition of surfactant PEG did not show any effect on the morphological characteristics of the particles. But the chemical structure of drug influenced on the properties. Microparticles had heterogeneous pores on the surface when the hydrophobic PTX was encapsulated. It was established that the addition of surfactant positively influenced on the properties of particles and led to the loading of 5-FU directly into the matrix. This is confirmed by the results of electron microscopy and dynamics of drug release in vitro. As a whole, the release profiles of PTX and 5-FU from composite P3HB/PEG microparticles were less than from P3HB microparticles. The results of the morphological evaluation of Hela cells demonstrated that the use of cytostatic drugs loaded in P3HB microparticles induces morphological changes associated with apoptosis (chromatin condensation, core fragmentation, margination of nucleus). Thus, the obtained results can serve as the basis for the development of new antitumor drugs of prolonged action, intended for various modes of administration.     

New Investigations with Lupane Type A-Ring Azepane Triterpenoids for Antimycobacterial Drug Candidate Design

Twenty lupane type A-ring azepano-triterpenoids were synthesized from betulin and its related derivatives and their antitubercular activity against Mycobacterium tuberculosis, mono-resistant MTB strains, and nontuberculous strains Mycobacterium abscessus and Mycobacterium avium were investigated in the framework of AToMIc (Anti-mycobacterial Target or Mechanism Identification Contract) realized by the Division of Microbiology and Infectious Diseases, NIAID, National Institute of Health. Of all the tested triterpenoids, 17 compounds showed antitubercular activity and 6 compounds were highly active on the H37Rv wild strain (with MIC 0.5 µM for compound 7), out of which 4 derivatives also emerged as highly active compounds on the three mono-resistant MTB strains. Molecular docking corroborated with a machine learning drug-drug similarity algorithm revealed that azepano-triterpenoids have a rifampicin-like antitubercular activity, with compound 7 scoring the highest as a potential M. tuberculosis RNAP potential inhibitor. FIC testing demonstrated an additive effect of compound 7 when combined with rifampin, isoniazid and ethambutol. Most compounds were highly active against M. avium with compound 14 recording the same MIC value as the control rifampicin (0.0625 µM). The antitubercular ex vivo effectiveness of the tested compounds on THP-1 infected macrophages is correlated with their increased cell permeability. The tested triterpenoids also exhibit low cytotoxicity and do not induce antibacterial resistance in MTB strains.