Response surface methodology as a tool to optimize the extraction of bioactive compounds from plant sources.

Response surface methodology (RSM) is a widely used mathematical and statistical technique for modeling and optimizing the process for the extraction of bioactive compounds. This review explains the optimization approach through the use of experimental design and empirical models for response prediction and the utilization of the desirability function for multiple response optimization. This paper also reviews recent studies on the application of RSM to optimize bioactive compound extraction processes such as conventional solvent extraction, microwave-assisted extraction, supercritical fluid extraction, and ultrasound-assisted extraction. Finally, the challenges associated with the use of RSM and the efforts made to improve RSM in the extraction process are also highlighted. Overall, this review informs many aspects of RSM that are occasionally ignored or insufficiently discussed with regard to the optimization of bioactive compound extraction processes, and it summarizes significant applications where RSM proved suitable. © 2022 Society of Chemical Industry.

DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position.

The carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct modification of the 5-position by electrophiles or by further derivatization of the 5-methylpyrimidines. Here, we review the structural diversity of these modified bases, the variety of cofactors that serve as carbon donors, and the common principles shared by enzymatic mechanisms generating them.

Implementation of good manufacturing practices (GMP) to improve the quality of smoked fish (Scombercolias).

For several years, fish smoking has been the widely adopted processing method among artisanal fish smokers located along the coastal zones in many parts of West Africa including Ghana. However, several issues pertaining to biochemical and microbiological contaminants still remain, mainly because of the suboptimal, unhygienic fish handling during the processing. To help curtail the problem, we developed and implemented a simple good manufacturing practice (GMP) system for experimentation at two local fish smoking facilities (Facility A, FA; Facility B, FB) to assess the effectiveness for improving the quality of smoked fish. The implementation of GMP did not affect the physical properties of the smoked fish but improved the peroxide value, total volatile base nitrogen, polyaromantic hydrocarbons and histamine levels. The total aerobic counts decreased from 3.96 ± 0.12 cfu/g to 1.52 ± 0.28 cfu/g (FA) or from 4.10 ± 0.2 cfu/g to 1.85 ± 0.85 cfu/g, (FB). The coliforms and Escherichia coli decreased respectively from 1.69 ± 0.12 cfu/g and 1.15 ± 0.21 cfu/g (FA) and from 1.74 ± 0.37 cfu/g and 1.24 ± 0.37 cfu/g, (FB) to below detection (no observed colony) after introducing the single use of potable water, use of smoking oven and fish core temperature of 108.1 ± 7.5 °C and 82.5 ± 3.9 °C, respectively for 2 h, wearing of safety apparels, drying and cooling of smoked fish under nets, and the use of waste disposal bins. The results show that sensitization and training of fish smokers in GMP may be relevant for improving the microbial and overall quality of smoked fish.

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA.

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7.

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge.

A propargyl containing guanosine phosphoramidite was synthesized and incorporated into siRNA, enabling click-ligation with an azido fluorophore onto the nucleobase sugar edge. Duplex stability was not affected by labeling at this new site, which allowed deconvolution of the effects of label, structure and attachment site on RNAi activity.

Single-molecule FRET reveals a cooperative effect of two methyl group modifications in the folding of human mitochondrial tRNA(Lys).

Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA(Lys) is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m(2)G10 alone does not facilitate the folding of tRNA(Lys), but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m(1)A9 exceeds the contribution of m(1)A alone. This constitutes an unprecedented cooperative effect of two nucleotide modifications in the context of a naturally occurring RNA, which may be of general importance for tRNA structure and help understanding several recently described decay pathways for hypomodified tRNAs.