Single-stranded siRNAs activate RNAi in animals.

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.

The TMEM189 gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens.

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri.

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99-92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4-C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12-C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.

Cytochrome c is an oxidative stress-activated plasmalogenase that cleaves plasmenylcholine and plasmenylethanolamine at the sn-1 vinyl ether linkage.

Plasmalogens are phospholipids critical for cell function and signaling that contain a vinyl ether linkage at the sn-1 position and are highly enriched in arachidonic acid (AA) at the sn-2 position. However, the enzyme(s) responsible for the cleavage of the vinyl ether linkage in plasmalogens has remained elusive. Herein, we report that cytochrome c, in the presence of either cardiolipin (CL), O2 and H2O2, or oxidized CL and O2, catalyzes the oxidation of the plasmalogen vinyl ether linkage, promoting its hydrolytic cleavage and resultant production of 2-AA-lysolipids and highly reactive α-hydroxy fatty aldehydes. Using stable isotope labeling in synergy with strategic chemical derivatizations and high-mass-accuracy MS, we deduced the chemical mechanism underlying this long sought-after reaction. Specifically, labeling with either 18O2 or H218O, but not with H218O2, resulted in M + 2 isotopologues of the α-hydroxyaldehyde, whereas reactions with both 18O2 and H218O identified the M + 4 isotopologue. Furthermore, incorporation of 18O from 18O2 was predominantly located at the α-carbon. In contrast, reactions with H218O yielded 18O linked to the aldehyde carbon. Importantly, no significant labeling of 2-AA-lysolipids with 18O2, H218O, or H218O2 was present. Intriguingly, phosphatidylinositol phosphates (PIP2 and PIP3) effectively substituted for cardiolipin. Moreover, cytochrome c released from myocardial mitochondria subjected to oxidative stress cleaved plasmenylcholine in membrane bilayers, and this was blocked with a specific mAb against cytochrome c Collectively, these results identify the first plasmalogenase in biology, reveal the production of previously unanticipated signaling lipids by cytochrome c, and present new perspectives on cellular signaling during oxidative stress.

Cytochrome c takes on plasmalogen catabolism.

Plasmalogens-phospholipids containing a characteristic vinyl ether group-are precursors of lipids important for cellular signaling such as arachidonic acid. Plasmalogen catabolism involves cleavage of the vinyl ether bond, but the identity of the corresponding enzyme that cleaves the sn-1 vinyl ether bond was unknown. New research shows that cytochrome c, with some help from another lipid, catalyzes the oxidative cleavage of this bond. This discovery, and the subsequent mechanistic dissection, provides exciting new directions for lipid signaling research.

Anton Chekhov and Robert Koch Cheek to Cheek: A Proteomic Study.

Five different letters and post cards as well as the shirt worn by Anton Chekhov on his death bed, stored in the State Literary-Memorial Museum-Reserve A. P. Chekhov Melikhovo (nearby Moscow), have been analyzed by applying EVA (an ethyl vinyl acetate foil studded with crushed strong anion and cation exchangers and with C8 resins) diskettes to these surfaces. Three different eluates (under acidic and basic conditions and with acetonitrile) were analyzed by high resolution mass spectrometry. The environmental microbiota present on samples and the Mycobacterium tuberculosis strain were described by a meta-proteomics approach. Eight identified M. tuberculosis proteins confirmed the presence of the bacterium and the cause of Chekhov's death, in addition to several sequenced peptides belonging to other bacterial species. The human plasma proteins and human keratins, detected on a tiny blood spot on the shirt, demonstrated the power of the combined approach.

A novel assay for the introduction of the vinyl ether double bond into plasmalogens using pyrene-labeled substrates.

Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1-O-pyrenedecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and the lyso-substrate, 1-O-pyrenedecyl-sn-glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1-O-pyrenedecyl-sn-glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.

2-(2-Nitrovinyl) furan exacerbates oxidative stress response of Escherichia coli to bacteriostatic and bactericidal antibiotics.

The influence of 2-(2-nitrovinyl) furan on the activities of selected bacteriostatic and bactericidal antibiotics was investigated. Minimum inhibitory concentration and fractional inhibitory concentration index were determined to evaluate the interaction between 2-(2-nitrovinyl) furan and the antibiotics. 2-(2-nitrovinyl) furan exhibited additive interactions with chloramphenicol, erythromycin, lincomycin and gemifloxacin. However, synergistic interaction was observed with amoxicillin, ampicillin and ciprofloxacin. Superoxide anion content of Escherichia coli exposed to antibiotics with/without 2-(2-nitrovinyl) furan increased significantly (p < .05). Furthermore, reduced glutathione decreased significantly with a corresponding increase in glutathione disulphide. In addition, malondialdehyde, a product of lipid peroxidation, increased significantly in E. coli exposed to antibiotics and 2-(2-nitrovinyl) furan. It can be deduced from this study that 2-(2-nitrovinyl) furan enhanced bacteriostatic and bactericidal antibiotics-mediated bacterial death possibly by potentiating reactive oxygen species generation and oxidative stress.

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol.

Divinyl-132,173-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The 1H NMR spectra in CDCl3 showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C132 and C173 positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.

Contrasting growth properties of Nocardioides JS614 on threedifferent vinyl halides.

Ethene (ETH)-grown inocula of Nocardioides JS614 grow on vinyl chloride (VC), vinyl fluoride (VF), or vinyl bromide (VB) as the sole carbon and energy source, with faster growth rates and higher cell yields on VC and VF than on VB. However, whereas acetate-grown inocula of JS614 grow on VC and VF after a lag period, growth on VB did not occur unless supplemental ethene oxide (EtO) was present in the medium. Despite inferior growth on VB, the maximum rate of VB consumption by ETH-grown cells was ~ 50% greater than the rates of VC and VF consumption, but Br- release during VB consumption was non-stoichiometric with VB consumption (~ 66%) compared to 100% release of Cl- and F- during VC and VF consumption. Evidence was obtained for VB turnover-dependent toxicity of cell metabolism in JS614 with both acetate-dependent respiration and growth being significantly reduced by VB turnover, but no VC or VF turnover-dependent toxicity of growth was detected. Reduced growth rate and cell yield of JS614 on VB probably resulted from a combination of inefficient metabolic processing of the highly unstable VB epoxide (t0.5 = 45 s), accompanied by growth inhibitory effects of VB metabolites on acetate-dependent metabolism. The exact role(s) of EtO in promoting growth of alkene repressed JS614 on VB remains unresolved, with evidence of EtO inducing epoxide consuming activity prior to an increase in alkene oxidizing activity and supplementing reductant supply when VB is the growth substrate.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3.

Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.

Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells.

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether.

Long-chain bases (LCBs) are the precursors to ceramide and sphingolipids in eukaryotic cells. They are formed by the action of serine palmitoyl-CoA transferase (SPT), a complex of integral membrane proteins located in the endoplasmic reticulum. SPT activity is negatively regulated by Orm proteins to prevent the toxic overaccumulation of LCBs. Here we show that overaccumulation of LCBs in yeast results in their conversion to a hitherto undescribed LCB derivative, an LCB vinyl ether. The LCB vinyl ether is predominantly formed from phytosphingosine (PHS) as revealed by conversion of odd chain length tracers C17-dihydrosphingosine and C17-PHS into the corresponding LCB vinyl ether derivative. PHS vinyl ether formation depends on ongoing acetyl-CoA synthesis, and its levels are elevated when the LCB degradative pathway is blocked by deletion of the major LCB kinase, LCB4, or the LCB phosphate lyase, DPL1. PHS vinyl ether formation thus appears to constitute a shunt for the LCB phosphate- and lyase-dependent degradation of LCBs. Consistent with a role of PHS vinyl ether formation in LCB detoxification, the lipid is efficiently exported from the cells.

Divinyl chlorophyll a in the marine eukaryotic protist Alexandrium ostenfeldii (Dinophyceae).

Here it is reported the first detection of DV-chl a together with the usual chl a in the marine dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Growth response and photosynthetic parameters were examined at two irradiances (80 and 240 µmol photons m(-2) s(-1)) and temperatures (15 °C and 19 °C) in a divinylic strain (AOTV-OS20) versus a monovinylic one (AOTV-OS16), using in vivo chl a fluorescence kinetics of PSII to characterize photosynthetic parameters by pulse amplitude modulated fluorescence, (14)C assimilation rates and toxin analyses. The divinylic isolate exhibited slower growth and stronger sensitivity to high irradiance than normal chl a strain. DV-chl a : chl a ratios decreased along time (from 11.3 to < 0.5 after 10 months) and to restore them sub-cloning and selection of strains with highest DV-chl a content was required. A mutation and/or epigenetic changes in the expression of divinyl reductase gene/s in A. ostenfeldii may explain this altered pigment composition. Despite quite severe limitations (reduced fitness and gradual loss of DV-chl a content), the DV-chl a-containing line in A. ostenfeldii could provide a model organism in photosynthetic studies related with chl biosynthesis and evolution.

Indomethacin-Kollidon VA64 Extrudates: A Mechanistic Study of pH-Dependent Controlled Release.

Because of its weakly acidic nature (pKa of 4.5), indomethacin presents an aqueous solubility that significantly increases when changing from acidic to neutral/alkaline pH (1.5 µg/mL at pH 1.2 and 105.2 µg/mL at pH 7.4). We have therefore investigated the impact of the dissolution medium pH on the dissolution performance of indomethacin:Kollidon VA64 extrudates. The impact of the drug loading on the dissolution properties of these systems was also examined (5%, 15%, 30%, 50%, 70%, and 90% drug loading). Time-resolved Raman spectroscopy along with in-line UV-vis spectrophotometry was employed to directly relate changes in dissolution behavior to physicochemical changes that occur to the extrudate during the test. The dissolution tests were performed in pH 2 HCl (to mimic the stomach conditions), and this was then switched during the experiment to pH 6.8 phosphate buffer (to simulate the poststomach conditions). The rotating disc dissolution rate test was also used to simultaneously measure the dissolution rate of both the drug and the polymer. We found that in pH 2 HCl buffer, for the 15% or higher drug-loaded extrudates, Kollidon VA64 preferentially dissolves from the exterior of the compact leaving an amorphous drug-rich hydrophobic shell, which, similarly to an enteric coating, inhibits the drug release. The in situ formation of an enteric coating has been previously hypothesized, and this has been the first time that is directly observed in a pH-variable dissolution test. The dissolution medium switch to pH 6.8 phosphate buffer, due to the large increase of the aqueous solubility of indomethacin at this pH, leads to rapid dissolution of the material forming the coating and therefore total drug release. In contrast, the 5% extrudate is fully hydrated and quickly dissolves at low pH pointing to a dissolution performance dependent on highly water-soluble Kollidon VA64.

Synergistic effect of phosphorothioate, 5'-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.

Chemical modifications are essential to improve metabolic stability and pharmacokinetic properties of siRNA to enable their systemic delivery. We investigated the effect of combing the phosphorothioate (PS) modification with metabolically stable phosphate analog (E)-5'-vinylphosphonate and GalNAc cluster conjugation on the activity of fully 2'-modified siRNA in cell culture and mice. Our data suggest that integrating multiple chemical approaches in one siRNA molecule improved potency 5-10 fold and provide a roadmap for developing more efficient siRNA drugs.

Chemoenzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol.

We report the synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) by a three-step method. Vinyl oleate was first synthesized by transvinylation between vinyl acetate and oleic acid. This was further reacted with glycerol at 35 °C for 8 h using 10% (w/v) Novozym 435 to synthesize 1,3-diolein. The 1,3-diolein content in the crude reaction mixture was 90.8% and was obtained at 82.3% (w/w) yield with 98.6% purity after purification. Finally, OPO was chemically synthesized by reacting purified 1,3-diolein with palmitic acid. 94.8% OPO was produced in the crude reaction mixture and the regiopurity of OPO after purification was 98.7% at 90.5% yield based on positional distribution analysis. This is an innovative approach for the synthesis of 1,3-diolein in a solvent-free system as an alternative to previously presented studies that applied solvent system.

Purification and immobilization of the recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as potential biocatalyst for the production of chlorophyllide and phytol.

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.

Evolution of a new chlorophyll metabolic pathway driven by the dynamic changes in enzyme promiscuous activity.

Organisms generate an enormous number of metabolites; however, the mechanisms by which a new metabolic pathway is acquired are unknown. To elucidate the importance of promiscuous enzyme activity for pathway evolution, the catalytic and substrate specificities of Chl biosynthetic enzymes were examined. In green plants, Chl a and Chl b are interconverted by the Chl cycle: Chl a is hydroxylated to 7-hydroxymethyl chlorophyll a followed by the conversion to Chl b, and both reactions are catalyzed by chlorophyllide a oxygenase. Chl b is reduced to 7-hydroxymethyl chlorophyll a by Chl b reductase and then converted to Chl a by 7-hydroxymethyl chlorophyll a reductase (HCAR). A phylogenetic analysis indicated that HCAR evolved from cyanobacterial 3,8-divinyl chlorophyllide reductase (DVR), which is responsible for the reduction of an 8-vinyl group in the Chl biosynthetic pathway. In addition to vinyl reductase activity, cyanobacterial DVR also has Chl b reductase and HCAR activities; consequently, three of the four reactions of the Chl cycle already existed in cyanobacteria, the progenitor of the chloroplast. During the evolution of cyanobacterial DVR to HCAR, the HCAR activity, a promiscuous reaction of cyanobacterial DVR, became the primary reaction. Moreover, the primary reaction (vinyl reductase activity) and some disadvantageous reactions were lost, but the neutral promiscuous reaction (NADH dehydrogenase) was retained in both DVR and HCAR. We also show that a portion of the Chl c biosynthetic pathway already existed in cyanobacteria. We discuss the importance of dynamic changes in promiscuous activity and of the latent pathways for metabolic evolution.

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay.

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.