End Modification Research Articles

Reducing end modification of nanocellulose as a novel approach for high-performance sustainable composites

The development of nanocellulose sustainable materials is considered as one of the most promising alternatives to address plastic pollution issues, as global plastic wastes may increase to 11 billion tonnes by 2025. However, how to achieve the homogeneous dispersion of nanocelluloses (CNCs) and strong interfacial interactions with matrix materials, while well maintaining its percolation networks, is a challenge in this field. As opposed to the conventional surface chemical modification strategy, the reducing end modification of CNCs as a novel approach provides an opportunity to achieve this objective, which also opens a new door for the design of stimuli-responsive CNC sustainable composites, such as vitrimer materials and stimuli-responsive Pickering emulsions.

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Synthesis of New Type Polymers by Quasi-Living Atom Transfer Radical Polymerization.

Thanks to the polymer revolution of the 20th century, plastics are now part of our everyday lives. We use plastics as naturally as if they had always been an integral part of our lives. However, in the recent past, we were still predominantly using wood, metal, and glass objects, which were replaced by plastic products at an explosive rate. In many cases, this replacement has resulted in products with better physical, chemical, or biological properties. The changeover was too rapid, and the consequences were not recognized in time. This is evidenced by the huge scale of plastic pollution worldwide today. It is therefore in the interests of the future of both humans and animals that we must pay particular attention to the direct and indirect environmental impact of plastics introduced in animal husbandry. Starting from the tetrafunctional initiator produced as the first step of my work, poly(n-butyll acrylate) star polymers of different molecular weights were synthesized by atom transfer radical polymerization, using the so-called “core first” method. The bromine chain end of the produced star polymers was replaced by an azide group using a substitution reaction. Propalgyl telechelic PEGs were synthesized as a result of lattice end modification of poly(ethylene glycol) with different molecular weights. The azidated star polymers were connected with propalgyl telechelic PEGs using Huisgen’s “click” chemical process, and as a result of the “click” connection, amphiphilic polymer networks with several different structures were obtained.

Noncanonical metabolite RNA caps: Classification, quantification, (de)capping, and function.

The 5' cap of eukaryotic mRNA is a hallmark for cellular functions from mRNA stability to translation. However, the discovery of novel 5'-terminal RNA caps derived from cellular metabolites has challenged this long-standing singularity in both eukaryotes and prokaryotes. Reminiscent of the 7-methylguanosine (m7G) cap structure, these noncanonical caps originate from abundant coenzymes such as NAD, FAD, or CoA and from metabolites like dinucleoside polyphosphates (NpnN). As of now, the significance of noncanonical RNA caps is elusive: they differ for individual transcripts, occur in distinct types of RNA, and change in response to environmental stimuli. A thorough comparison of their prevalence, quantity, and characteristics is indispensable to define the distinct classes of metabolite-capped RNAs. This is achieved by a structured analysis of all present studies covering functional, quantitative, and sequencing data which help to uncover their biological impact. The biosynthetic strategies of noncanonical RNA capping and the elaborate decapping machinery reveal the regulation and turnover of metabolite-capped RNAs. With noncanonical capping being a universal and ancient phenomenon, organisms have developed diverging strategies to adapt metabolite-derived caps to their metabolic needs, but ultimately to establish noncanonical RNA caps as another intriguing layer of RNA regulation. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5′-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5′-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

How fluorescent labels affect the kinetics of the toehold-mediated DNA strand displacement reaction

End modification of the toehold domain or near to it using fluorophore dyes or quenchers can significantly modulate the kinetics of the toehold-mediated strand displacement reaction (TMSDR) by almost two orders of magnitude.

The influence of chain‐end functionalization and stereocomplexation on the degradation stability under alkaline condition

AbstractHydrolytic degradation of polylactides (PLAs) bearing cinnamic acid derivatives was conducted and their thermal degradation temperature at 10% weight loss (T10) and melting point temperature (Tm) were measured, to confirm that a PLA stereocomplex could be modified for use in a wide range of application. The improved T10 values of PLA films by chain end modification were maintained after the alkaline degradation. The increase in Tm by stereocomplex formation was also maintained after degradation with a slight change in the melting point of the attached homopolymer. The present results indicate the suitability of chain end modification for the additional functionality of PLA stereocomplex films.

Innovation and new technology in ureteral stents

Ureteral stent insertion is a procedure performed extensively by all urologists. Nevertheless, stent-related symptoms and stent encrustation are still common complications pushing the innovation and development of novel ureteral stents. Developments are focussing on three significant aspects: material, design, and removal technique. Various materials including silicone, polymers, and metals are frequently utilized, with or without an additional coating. The use of biodegradable materials is looking promising but these is a lack of proven clinical trials in association with this in humans. The new designs focus on the reduction of stent-related symptoms through the modification of the bladder end. The new stent removal techniques with extraction strings or novel magnetic end may exclude subsequent cystoscopic procedures. Finally, utilization of a ureteral stent tracker application helps in reminding both physicians and patients to remove the stent at the appropriate time.

Dextran-Coated Latex Nanoparticles via Photo-RAFT Mediated Polymerization Induced Self-Assembly.

Polysaccharide coated nanoparticles represent a promising class of environmentally friendly latex to replace those stabilized by small toxic molecular surfactants. We report here an in situ formulation of free-surfactant core/shell nanoparticles latex consisting of dextran-based diblock amphiphilic copolymers. The synthesis of copolymers and the immediate latex formulation were performed directly in water using a photo-initiated reversible addition fragmentation chain transfer-mediated polymerization induced self-assembly strategy. A hydrophilic macromolecular chain transfer-bearing photosensitive thiocarbonylthio group (eDexCTA) was first prepared by a modification of the reducing chain end of dextran in two steps: (i) reductive amination by ethylenediamine in the presence of sodium cyanoborohydride, (ii) then introduction of CTA by amidation reaction. Latex nanoparticles were then formulated in situ by chain-extending eDexCTA using 2-hydroxypropyl methacrylate (HPMA) under 365 nm irradiation, leading to amphiphilic dextran-b-poly(2-hydroxypropyl methacrylate) diblock copolymers (DHX). Solid concentration (SC) and the average degree of polymerization -- of PHPMA block (X) were varied to investigate their impact on the size and the morphology of latex nanoparticles termed here SCDHX. Light scattering and transmission electron microscopy analysis revealed that SCDHX form exclusively spherical nano-objects. However, the size of nano-objects, ranging from 20 nm to 240 nm, increases according to PHPMA block length.

Post-transcriptional regulation in spermatogenesis: all RNA pathways lead to healthy sperm.

Multiple RNA pathways are required to produce functional sperm. Here, we review RNA post-transcriptional regulation during spermatogenesis with particular emphasis on the role of 3' end modifications. From early studies in the 1970s, it became clear that spermiogenesis transcripts could be stored for days only to be translated at advanced stages of spermatid differentiation. The transition between the translationally repressed and active states was observed to correlate with the shortening of the transcripts' poly(A) tail, establishing a link between RNA 3' end metabolism and male germ cell differentiation. Since then, numerous RNA metabolic pathways have been implicated not only in the progression through spermatogenesis, but also in the maintenance of genomic integrity. Recent studies have characterized the elusive 3' biogenesis of Piwi-interacting RNAs (piRNAs), identified a critical role for messenger RNA (mRNA) 3' uridylation in meiotic progression, established the mechanisms that destabilize transcripts with long 3' untranslated regions (3'UTRs) in post-mitotic cells, and defined the physiological relevance of RNA exonucleases and deadenylases in male germ cells. In this review, we discuss RNA processing in the male germline in the light of the most recent findings. A brief recollection of different RNA-processing events will aid future studies exploring post-transcriptional regulation in spermatogenesis.

Syntheses of benzhydryl 2-propanoyl-functionalized trithiocarbonates and its use as chain transfer agents in the RAFT polymerization of styrene

Abstract The preparation of three new benzhydryl 2-propanoyl-functionalized trithiocarbonate RAFT agents, benzhydryl 2-(ethylsulfanylthiocarbonylsulfanyl)propanoate, benzhydryl 2-(butylsulfanylthiocarbonylsulfanyl)propanoate, benzhydryl 2-(dodecylsulfanylthiocarbonylsulfanyl)propanoate is described. The RAFT polymerization of styrene, mediated by the appropriate benzhydryl 2-(alkylsulfanylthiocarbonylsulfanyl)propanoate as the chain transfer agent, with AIBN as initiator at 70 0C, proceeds via reversible-deactivation radical polymerization processes to produce the corresponding α-benzhydryl 2-propanoyl polystyrene derivative, with the benzhydryl ester group introduced at the α-terminus of the polymer chain. Polymerization kinetics measurements show that each RAFT polymerization reaction follows first order rate kinetics with respect to percentage monomer consumption and the number average molecular weights of the functionalized polymers increase linearly with the percentage monomer conversion to produce functionalized polymers with narrow molecular weight distributions. The percentage monomer conversion with time for each polymerization reaction was monitored by 1H NMR spectroscopy by evaluating the consumption of styrene with respect to polymerization time. Post-RAFT polymerization chain end modification of α-benzhydryl 2-propanoyl-functionalized polystyrene by the acid catalyzed ester hydrolysis reactions with concentrated sulfuric acid afforded the corresponding α-carboxyl functionalized polystyrene derivative, with the retention of the thiocarbonylthio moiety at the ω-terminus of the polymer chain. The benzhydryl 2-propanoyl-functionalized trithiocarbonate chain transfer agents and the different functionalized polystyrene derivatives were characterized by 1H NMR, 13C NMR and FTIR spectroscopy, size exclusion chromatography, thin layer chromatography and mass spectrometry measurements.

Tuning Photodynamic Properties of BODIPY Dyes, Porphyrins' Little Sisters.

The photodynamic properties of a series of non-halogenated, dibrominated and diiodinated BODIPYs with a phthalimido or amino end modification on the phenoxypentyl and phenoxyoctyl linker in the meso position were investigated. Halogen substitution substantially increased the singlet oxygen production based on the heavy atom effect. This increase was accompanied by a higher photodynamic activity against skin melanoma cancer cells SK-MEL-28, with the best compound reaching an EC50 = 0.052 ± 0.01 µM upon light activation. The dark toxicity (toxicity without light activation) of all studied dyes was not detected up to the solubility limit in cell culture medium (10 µM). All studied BODIPY derivatives were predominantly found in adiposomes (lipid droplets) with further lower signals colocalized in either endolysosomal vesicles or the endoplasmic reticulum. A detailed investigation of cell death indicated that the compounds act primarily through the induction of apoptosis. In conclusion, halogenation in the 2,6 position of BODIPY dyes is crucial for the efficient photodynamic activity of these photosensitizers.

Division of labor in epitranscriptomics: What have we learnt from the structures of eukaryotic and viral multimeric RNA methyltransferases?

The translation of an mRNA template into the corresponding protein is a highly complex and regulated choreography performed by ribosomes, tRNAs, and translation factors. Most RNAs involved in this process are decorated by multiple chemical modifications (known as epitranscriptomic marks) contributing to the efficiency, the fidelity, and the regulation of the mRNA translation process. Many of these epitranscriptomic marks are written by holoenzymes made of a catalytic subunit associated with an activating subunit. These holoenzymes play critical roles in cell development. Indeed, several mutations being identified in the genes encoding for those proteins are linked to human pathologies such as cancers and intellectual disorders for instance. This review describes the structural and functional properties of RNA methyltransferase holoenzymes, which when mutated often result in brain development pathologies. It illustrates how structurally different activating subunits contribute to the catalytic activity of these holoenzymes through common mechanistic trends that most likely apply to other classes of holoenzymes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Processing > Capping and 5' End Modifications.

A Timely Review of Cross-Kingdom Regulation of Plant-Derived MicroRNAs.

MicroRNAs (miRNAs) belong to a class of non-coding RNAs that suppress gene expression by complementary oligonucleotide binding to the sites in target messenger RNAs. Numerous studies have demonstrated that miRNAs play crucial role in virtually all cellular processes of both plants and animals, such as cell growth, cell division, differentiation, proliferation and apoptosis. The study of rice MIR168a has demonstrated for the first time that exogenous plant MIR168a influences cholesterol transport in mice by inhibiting low-density lipoprotein receptor adapter protein 1 expression. Inspired by this finding, the cross-kingdom regulation of plant-derived miRNAs has drawn a lot of attention because of its capability to provide novel therapeutic agents in the treatment of miRNA deregulation-related diseases. Notably, unlike mRNA, some plant miRNAs are robust because of their 3′ end modification, high G, C content, and the protection by microvesicles, miRNAs protein cofactors or plant ingredients. The stability of these small molecules guarantees the reliability of plant miRNAs in clinical application. Although the function of endogenous miRNAs has been widely investigated, the cross-kingdom regulation of plant-derived miRNAs is still in its infancy. Herein, this review summarizes the current knowledge regarding the anti-virus, anti-tumor, anti-inflammatory, anti-apoptosis, immune modulation, and intestinal function regulation effects of plant-derived miRNAs in mammals. It is expected that exploring the versatile role of plant-derived miRNAs may lay the foundation for further study and application of these newly recognized, non-toxic, and inexpensive plant active ingredients.

Biophysical Characterization and Computational Modeling of Histone mRNA Degradation Intermediates

Histone mRNA is the only known metazoan mRNA that is not polyadenylated at its 3′ end; instead, it ends in a highly conserved stem-loop formed by Watson-Crick base pairing. In histone mRNA degradation, the stem-loop is trimmed by 3′ human exonuclease (3′hExo) and uridylated by terminal uridylyl transferase 7. This process creates uridylated intermediates—some of which are more common in the cell than expected. It is unknown how this trimming and uridylation affects the binding interactions between the stem-loop and its protein binding partners, the stem-loop binding protein (SLBP) and 3′hExo. In this work, we characterized the binding interactions of a wild-type stem-loop, a uridylated intermediate, and a truncated stem-loop using a combination of experimental and computational biophysical techniques. The uridylated intermediate represents one of the most prominent degradation intermediates identified in the cell, and the truncated stem-loop represents the stem-loop post-trimming and pre-uridylation. Through native electrophoretic mobility shift assays, we show that SLBP and 3′hExo are able to bind both the uridylated intermediate and the truncated stem-loop despite 3′ end modification. We also performed molecular dynamics simulations (1 µs) to evaluate structural changes induced by stem-loop modification and to understand how these changes impact SLBP and 3′hExo binding. RNA-protein systems were generated by applying AMBER force fields to modified versions of the histone mRNA ternary complex crystal structures (PDB 4QOZ and 4L8R). This work shows that modification of the stem-loop through trimming and uridylation does not interfere with the tertiary structure of the ternary complex.

Evolutionary Glycomics: A Comprehensive Study of Vertebrate Host Serum/Plasma Glycome Using Orthogonal Glycomics Techniques

Eukaryotic Vertebrate host glycosylation machinery has been subject to immense evolutionary pressure. Besides, being crucial for host sustenance, glyco-epitopes are also key-regulators of inter-species pathogen transmission. Significant evolutionary events such as loss of N-glycolyl neuraminic acid (Neu5Gc) or alpha Galactose (α-Gal) epitopes in species such as humans have been relevant for species development and have significantly impacted the ongoing arms race between pathogen and their hosts. Comprehensive knowledge on Vertebrate species-specific glycosylation is still scattered, thereby leaving a substantial gap in our understanding of their glycome evolution. Serum/plasma N- and O-glycomes of 35 different Vertebrate species (mammals, marsupials, birds, reptiles and fish) were analysed by two orthogonal glycomics methods, Porous Graphitised Carbon nano- Liquid Chromatography coupled to Electrospray Ionisation Tandem Mass Spectrometry (PGC nano-LC ESI-MS/MS) and Matrix Associated Laser Desorption Ionisation Time-of-flight Mass Spectrometry (MALDI TOF MS). Sialic acid diversity was additionally captured using High Performance Liquid Chromatography (HPLC) analyses of 1,2 Diamino Methylenedioxybenzene dichloride (DMB)- labelled sialic acids. Vertebrate serum/plasma glycome predominantly contained complex di-, mono sialylated glycans. Core fucosylation of these N-glycans was strongly species dependent, as were the non-reducing end modifications. Most species showed a preference for incorporation of either N-acetyl neuraminic acid (NeuAc) or NeuGc, with exceptions such as rats that use both. Anseriformes (Goose, Duck) N-glycans carried GalNAc-GlcNAc (LacDiNAc) epitopes that, however, were absent in Galliformes (Chicken, Turkey). Reptilians (e.g. Green sea turtles) had high levels of α-Gal epitopes, and Saltwater Crocodile uniquely showed high amounts of oligomannose N-glycans in serum. This largest, vertebrate species-specific serum/plasma glycan and MS/MS spectral data is currently in the process of being made available open access through our collaboration with glycan focussed database such as Glyconnect and UniCarbDB.

Biological implications of decapping: beyond bulk mRNA decay.

It is well established that mRNA steady-state levels do not directly correlate with transcription rate. This is attributed to the multiple post-transcriptional mechanisms, which control both mRNA turnover and translation within eukaryotic cells. One such mechanism is the removal of the 5' end cap structure of RNAs (decapping). This 5' cap plays a fundamental role in cellular functions related to mRNA processing, transport, translation, quality control, and decay, while its chemical modifications influence the fate of cytoplasmic mRNAs. Decapping is a highly controlled process, performed by multiple decapping enzymes, and regulated by complex cellular networks. In this review, we provide an updated synopsis of 5' end modifications and functions, and give an overview of mRNA decapping enzymes, presenting their enzymatic properties. Focusing on DCP2 decapping enzyme, a major component on the 5'-3' mRNA decay pathway, we describe cis-elements and trans-acting factors that affect its activity, substrate specificity, and cellular localization. Finally, we discuss current knowledge on the biological functions of mRNA decapping and decay factors, highlighting the major questions that remain to be addressed.

Traceless Staudinger Ligation for Bioconjugation of RNA.

Staudinger ligation is an attractive bioorthogonal reaction for use in studying biomolecules due to its capacity to form a native amide bond between a tag and a biomolecule. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. The procedure involves (i) synthesis of phosphine-containing reactive groups, affinity purification tags, or photoactivatable benzophenone probe, (ii) synthesis of 2'-azido dinucleotides and 24-nt RNA, and (iii) traceless Staudinger ligation experiments. Each phosphine was characterized by 1 H, 13 C, and 31 P NMR and high-resolution spectrometry and the functionalized nucleotides were characterized by LC/MS. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphines Basic Protocol 2: Synthesis of dinucleotides 4 and 5 Basic Protocol 3: Synthesis of modified RNA 6 Basic Protocol 4: Traceless Staudinger reactions on a dinucleotide Basic Protocol 5: Traceless Staudinger reaction on RNA.

Facile detection of RNA phospho-methylation in cells and tissues.

RNAs from various cells and tissues are modified in nearly 200 chemically distinct ways. These modifications can be deposited either on the 5' or 3' ends, or internally on the nucleobases or sugar backbone. 5'-end modifications are crucial for protecting RNAs from untimely degradation/processing, regulating their cellular functions, or discriminating endogenous RNAs from pathogenic RNAs. 5'-end phospho-methylation is a remarkable RNA modification that is enzymatically deposited either on the γ-phosphate of nascent triphosphorylated RNAs by human BCDIN3/MePCE, or on the α-phosphate of processed monophosphorylated RNAs by human BCDIN3D. These 5'-phospho-methyltransferases are part of the BIN3 family of O-methyltransferases conserved from S. pombe to humans and play important cellular and biological roles, many of which await further elucidation. Here, we quickly recapitulate historical methods for the detection of 5'-end phospho-methyl modifications, and focus more specifically on a method that can be used to detect and quantify α-monophosphate methylation from as low as 10-100ng of total RNA from cells or tissues. This method is important for deciphering the roles of BCDIN3D and its homologs across species, as well as serves as starting point for the development of new methods for detection of 5'-end modifications.

Quantitative Characterization of the Amount and Length of (1,3)-β-d-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-d-glucan Synthase.

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely uncharacterized due to the absence of informative activity assays. Previously, a radioactive assay and reducing end modification have been used to characterize GS activity. The radioactive assay determines only the total amount of glucan formed through glucose incorporation and does not report the length of the polymers produced. The glucan length has been characterized by reducing end modification, but this method is unsuitable for mechanistic studies due to the very high detection limit of millimolar amounts and the labor intensiveness of the technique. Consequently, fundamental aspects of GS catalysis, such as the polymer length specificity, remain ambiguous. We have developed a size exclusion chromatography (SEC)-based method that allows detailed functional and mechanistic characterization of GS. The approach harnesses the pH-dependent solubility of (1,3)-β-d-glucan, where (1,3)-β-d-glucan forms water-soluble random coils under basic pH conditions, and can be analyzed by SEC using pulsed amperometric detection (PAD) and radioactivity counting (RC). This approach allows quantitative characterization of the total amount and length of glucan produced by GS with minimal workup and a d-glucose (Glc) detection limit of ~100 pmol. Consequently, this approach was successfully used for the kinetic characterization of GS, providing the first detailed mechanistic insight into GS catalysis. Due to its sensitivity, the assay is applicable to the characterization of GS from any fungi and can be adapted to study other polysaccharide synthases.