Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

A simple and fast method for metabolomic analysis by gas liquid chromatography-mass spectrometry.

INTRODUCTION: The metabolomic profile is an essential tool for understanding the physiological processes of biological samples and their changes. In addition, it makes it possible to find new substances with industrial applications or use as drugs. As GC-MS is a very common tool for obtaining the metabolomic profile, a simple and fast method for sample preparation is required. OBJECTIVES: The aim of this research was to develop a direct derivatization method for GC-MS to simplify the sample preparation process and apply it to a wide range of samples for non-targeted metabolomic analysis purposes. METHODS: One pot combined esterification of carboxylic acids with methanol and silylation of the hydroxyl groups was achieved using a molar excess of chlorotrimethylsilane with respect to methanol in the presence of pyridine. RESULTS: The metabolome profile obtained from different samples, such as bilberry and cherry cuticles, olive leaves, P. aeruginosa and E. coli bacteria, A. niger fungi and human sebum from the ceruminous gland, shows that the procedure allows the identification of a wide variety of metabolites. Aliphatic fatty acids, hydroxyfatty acids, phenolic and other aromatic compounds, fatty alcohols, fatty aldehydes dimethylacetals, hydrocarbons, terpenoids, sterols and carbohydrates were identified at different MSI levels using their mass spectra. CONCLUSION: The metabolomic profile of different biological samples can be easily obtained by GC-MS using an efficient simultaneous esterification-silylation reaction. The derivatization method can be carried out in a short time in the same injection vial with a small amount of reagents.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

Preparation and Uses of Chlorinated Glycerol Derivatives.

Crude glycerol (C3H8O3) is a major by-product of biodiesel production from vegetable oils and animal fats. The increased biodiesel production in the last two decades has forced glycerol production up and prices down. However, crude glycerol from biodiesel production is not of adequate purity for industrial uses, including food, cosmetics and pharmaceuticals. The purification process of crude glycerol to reach the quality standards required by industry is expensive and dificult. Novel uses for crude glycerol can reduce the price of biodiesel and make it an economical alternative to diesel. Moreover, novel uses may improve environmental impact, since crude glycerol disposal is expensive and dificult. Glycerol is a versatile molecule with many potential applications in fermentation processes and synthetic chemistry. It serves as a glucose substitute in microbial growth media and as a precursor in the synthesis of a number of commercial intermediates or fine chemicals. Chlorinated derivatives of glycerol are an important class of such chemicals. The main focus of this review is the conversion of glycerol to chlorinated derivatives, such as epichlorohydrin and chlorohydrins, and their further use in the synthesis of additional downstream products. Downstream products include non-cyclic compounds with allyl, nitrile, azide and other functional groups, as well as oxazolidinones and triazoles, which are cyclic compounds derived from ephichlorohydrin and chlorohydrins. The polymers and ionic liquids, which use glycerol as an initial building block, are highlighted, as well.

Synthesis and Thermophysical Characterization of Fatty Amides for Thermal Energy Storage.

Nine monoamides were synthesized from carboxylic acids (C8-C18) and crude glycerol. The final monoamides were the result of a rearrangement of the acyl chain during the final hydrogenation process. The purity of the final compounds was determined by spectroscopic and mass spectrometry (MS) techniques. The thermophysical properties of solid monoamides were investigated to determine their capability to act as phase change materials (PCM) in thermal energy storage. Thermophysical properties were determined with a differential scanning calorimeter (DSC). The melting temperatures of the analyzed material ranged from 62.2 °C to 116.4 °C. The analyzed enthalpy of these monoamides ranged from 25.8 kJ/kg to 149.7 kJ/kg. Enthalpy values are analyzed considering the carbon chain and the formation of hydrogen bonds.

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the corresponding small subunit gene APS2b in rice leaves.

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

Patterns of CRISPR/Cas9 activity in plants, animals and microbes.

The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.

From symmetric glycerol derivatives to dissymmetric chlorohydrins.

The anticipated worldwide increase in biodiesel production will result in an accumulation of glycerol for which there are insufficient conventional uses. The surplus of this by-product has increased rapidly during the last decade, prompting a search for new glycerol applications. We describe here the synthesis of dissymmetric chlorohydrin esters from symmetric 1,3-dichloro-2-propyl esters obtained from glycerol. We studied the influence of two solvents: 1,4-dioxane and 1-butanol and two bases: sodium carbonate and 1-butylimidazole, on the synthesis of dissymmetric chlorohydrin esters. In addition, we studied the influence of other bases (potassium and lithium carbonates) in the reaction using 1,4-dioxane as the solvent. The highest yield was obtained using 1,4-dioxane and sodium carbonate.

A rapid and reliable direct method for quantifying meat acylglycerides with monomode microwave irradiation.

A rapid methodology for direct analysis of meat acylglycerides is proposed. A transesterification is carried out in a microwave reactor consisting of a monomode oven using chlorotrimethylsilane (CTMS) and methanol. High-temperature gas chromatography was used to check the absence of underivatized acylglycerides. Whereas transesterification is complete after 30s at 90 degrees C in the microwave method, the reference method needs 2h to complete this process. Moreover, the CTMS-microwave method shows higher recoveries of individual saturated, monounsaturated and polyunsaturated fatty acids. No influence of microwave irradiation on the composition of the fatty acids was observed.

[BMIM][PF(6)] promotes the synthesis of halohydrin esters from diols using potassium halides.

Haloesterification of diverse diols with various carboxylic acids was achieved using potassium halides (KX) as the only halide source in ionic liquids. The best yield was obtained in [BMIM][PF(6)] when 1,2-octanediol, palmitic acid and KBr were used. This yield was 85% and the regioisomer with the bromine in primary position was present in a 75:25 ratio. The regioisomeric ratio could be improved using either KCl or some phenylcarboxylic acids. [BMIM][PF(6)] acts as both reaction media and catalyst of the reaction. To the best of our knowledge, this type of combined reaction using an ionic liquid is unprecedented. The other solvents tested did not lead either to the same yield or to the same regioisomeric ratio.

Solid-phase synthesis of a combinatorial library of dihydroceramide analogues and its activity in human alveolar epithelial cells.

Solid-phase synthesis of a small combinatorial library of dihydroceramide analogues as mixtures of erythro and threo diastereomers is described. Some dihydroceramide analogues cause growth arrest and apoptosis in a dose-dependent manner in human alveolar epithelial cells. This activity is likely due to the threo isomers, as evidenced by cellular studies with a pair of diastereomerically pure N-acyldihydrosphingosines. The apoptotic activity reported in this work provides information for the design of new compounds that may provide the basis for the generation of biochemical tools for the study of different pathologies where ceramide and/or dihydroceramide are involved.

Synthesis and use of stereospecifically deuterated analogues of palmitic Acid to investigate the stereochemical course of the delta11 desaturase of the processionary moth.

Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the immediate sex pheromone precursor. In this article, we describe the synthesis of different stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher the stereochemical course of the T. pityocampa Delta(11) desaturase. The synthesis of probes has been carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental evidence is presented that the Delta(11) desaturase of T. pityocampa transforms palmitic acid into (Z)-11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12.

Synthesis of fluorinated analogs of myristic acid as potential inhibitors of Egyptian armyworm (Spodoptera littoralis) delta11 desaturase.

To study the activity of the different desaturases present in the pheromone biosynthetic pathway of the Egyptian armyworm, Spodoptera littoralis, we prepared a series of mono- and gem-difluorinated analogs of myristic acid with halogen substitution at the C8-C11 positions of the aliphatic chain via specifically positioned dithiane precursors. Thus, transformation of dithianes by treatment with N-bromosuccinimide in the presence of H2O followed by reduction with LiAlH4 afforded the appropriate alcohols, which reacted with diethylaminosulfur trifluoride to give rise to the corresponding monofluoroderivative intermediates. Alternatively, the introduction of the gem-difluoro functionality was carried out by reaction of the appropriate dithiane intermediate with 1,3-dibromo-5,5-dimethylhydantoin in the presence of HF/pyridine. The activity of these fluorinated FA as substrates and inhibitors of the desaturases involved in the biosynthesis of the sex pheromonal blend of S. littoralis has been studied. In this case, 11-fluorotetradecanoic acid elicited a moderate inhibitory activity of delta11 desaturase.

Enzymatic desaturation of fatty acids: delta11 desaturase activity on cyclopropane acid probes.

The formation of methylenecyclopropanes by enzymatic desaturation of 11-cyclopropylundecanoic acid (1) and its disubstituted derivatives cis- and trans-3-5 has been investigated using the Delta(11) desaturase of Spodoptera littoralis as model enzyme. Gas chromatography coupled to mass spectrometry analyses of methanolyzed lipidic extracts from tissues incubated with each probe revealed that all the cyclopropyl fatty acids were transformed into the corresponding 11-cyclopropylidene acids, except for compound trans-5 (5b), which was not desaturated at C11. The formation of methylenecyclopropane 9 as the only reaction product from 1 indicates that a potential radical intermediate is too short-lived to allow rearrangement reactions. Information on the Delta(11) desaturase substrate binding domain is provided considering the cyclopropyl probes 3-5 as conformationally restricted analogues of the straight-chain substrates.