Covalent Chemical Modification Research Articles

Covalent vs. Non covalent chemical modification of multiwalled carbon nanotubes based-nanofluids: Stability and thermal conductivity steadiness over temperature

Thermal conductivity enhancement coupled to nanoparticle dispersion stability over time are the two main conflicting requirements to overcome to develop carbon nanomaterial-based nanofluids. Both covalent and non-covalent approaches, having their own advantages and disadvantages, are commonly used for carbon nanotube based nanofluids development. However, they are rarely compared regarding their impact on stability and thermal conductivity performances for the same kind of nanofluid. In this work, multiwalled carbon nanotubes have been chemically modified by both covalent and non-covalent functionalization. For the covalent functionalization, a simple oxidation method is applied. It consists of refluxing the powdered MWCNTs in H2SO4/HNO3 at 5 M concentration. For the non-covalent approach, both an anionic (sodium dodecyl sulfate) and a non-ionic (Pluronic P123, comprising poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) in an alternating linear fashion, PEO-PPO-PEO) surfactants were employed at a concentration of 1 wt%. Nanofluids were prepared with multiwalled carbon nanotubes at a fixed concentration of 0.2 wt% for both chemical modification approaches. The oxidation reaction occurs without significant structure damaging as shown by transmission electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. UV–visible spectrophotometry analysis shows a remarkable stability with temperature in the 293.15–303.15 K range over one-week and for a longer period of 49 days for all the three types of nanofluids. Their thermal conductivity has been investigated in the same temperature range. For all the three nanofluid series prepared with a fixed concentration of carbon nanomaterials, a thermal conductivity enhancement compared to that of the base fluid has been observed. The thermal conductivity evolution with temperature is different for each type of nanofluid. The covalently oxidized multiwalled carbon nanotube based nanofluids showed the better thermal conductivity enhancement steadiness (around 7 %) with temperature. The discussion on the surfactant-based nanofluid behavior proposes the possible involved mechanisms as temperature increases for each surfactant.

Synthesis and characterization of cellulose derivatives for enhanced antimicrobial activity

AbstractThe need for biodegradable materials has driven the exploration of natural and biobased polymers, with cellulose emerging as a promising option due to its abundance and environmental sustainability. However, cellulose lacks inherent antimicrobial activity, prompting researchers to explore various strategies. In this work, the permanent covalent chemical modification of microcrystalline cellulose with 1-methylimidazole is reported to provide cellulose with antimicrobial properties. The modifications were confirmed using solid-state 13C-NMR and Fourier Transform Infrared (FT-IR) spectroscopies. The analysis from 13C-NMR and elemental analysis also confirmed the successful of the chemical modification occurred at C6 after chlorination with an estimated degree of substitution of 17%. The surface charge density of this cellulosic derivative was determined using ζ potential measurements, which confirmed its cationization. The crystalline and thermal properties of the resulting cellulose derivative were evaluated using X-ray diffraction and thermogravimetric analysis (TGA), respectively. Finally, the antimicrobial activity of the obtained cationic cellulose was assessed against both Gram-positive (Staphylococcus epidermidis) and Gram-negative (Pseudomona aeruginosa) bacteria, giving a reduction of > 99.99% and 99.6%, respectively.

Methylation status of potential genes in breast cancer patients and correlate them with gene expression

Breast cancer is one of the world’s most prevalent cancer among women. Globally approximately 2.3 million women are diagnosed with breast cancer in 2020. It arises due to epigenetic modification which outlooks aberrant methylation as its major cause. The methylation of DNA involves a covalent chemical modification which is one of the major drawback, promoting development of breast cancer. In addition, evidence and investigation suggest that these methylations at promoter region of CpG islands leads to silencing of gene associated with tumor suppressor which is involved in crucial gene expression leading to establishment of breast cancer. Among variety of hyper methylated gene involved in different function linked with breast cancer, the hyper methylation of BRCA1 gene is identified as the targeted gene in precipitating disorder. A lot of susceptible genes are identified and based on their penetrance ability are being classified In order to analyze DNA methylation status Bisulfite sequencing is employed which is based on the mechanism of converting every un-methylated cytosine into uracil. With the advancement in gene sequencing the concept of whole genome bisulfite sequencing is also on the way to determine the DNA methylation occurring at single base. Another robust and sensitive approach to determine the methylated status is RT-PCR, a diagnostic tool applied in order to detect the expression of single gene

Bind&Bite: covalently stabilized heterodimeric coiled-coil peptides for the site-selective, cysteine-free chemical modification of proteins.

Ensuring site-selectivity in covalent chemical modification of proteins is one of the major challenges in chemical biology and related biomedical disciplines. Most current strategies either utilize the selectivity of proteases, or are based on reactions involving the thiol groups of cysteine residues. We have modified a pair of heterodimeric coiled-coil peptides to enable the selective covalent stabilization of the dimer without using enzymes or cysteine moieties. Fusion of one peptide to the protein of interest, in combination with linking the desired chemical modification to the complementary peptide, facilitates stable, regio-selective attachment of the chemical moiety to the protein, through the formation of the covalently stabilized coiled-coil. This ligation method, which is based on the formation of isoeptide and squaramide bonds, respectively, between the coiled-coil peptides, was successfully used to selectively modify the HIV-1 envelope glycoprotein. Covalent stabilization of the coiled-coil also facilitated truncation of the peptides by one heptad sequence. Furthermore, selective addressing of individual positions of the peptides enabled the generation of mutually selective coiled-coils. The established method, termed Bind&Bite, can be expected to be beneficial for a range of biotechnological and biomedical applications, in which chemical moieties need to be stably attached to proteins in a site-selective fashion.

Pseudo-Isolated α-Helix Platform for the Recognition of Deep and Narrow Targets.

Although interest in stabilized α-helical peptides as next-generation therapeutics for modulating biomolecular interfaces is increasing, peptides have limited functionality and stability due to their small size. In comparison, α-helical ligands based on proteins can make steric clash with targets due to their large size. Here, we report the design of a monomeric pseudo-isolated α-helix (mPIH) system in which proteins behave as if they are peptides. The designed proteins contain α-helix ligands that do not require any covalent chemical modification, do not have frayed ends, and importantly can make sterically favorable interactions similar to isolated peptides. An optimal mPIH showed a more than 100-fold increase in target selectivity, which might be related to the advantages in conformational selection due to the absence of frayed ends. The α-helical ligand in the mPIH displayed high thermal stability well above human body temperature and showed reversible and rapid folding/unfolding transitions. Thus, mPIH can become a promising protein-based platform for developing stabilized α-helix pharmaceuticals.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Photoreactivity and Enhanced Mechanical Properties and Water Stability in Polysaccharide-Based Films Using Vanadium Ion Coordination

Different strategies have been employed to improve the physicochemical and mechanical properties of soft polysaccharide-based materials. Some of these strategies include covalent cross-linking or chemical modification and blending of polymers. In an alternative approach, we used vanadium ion coordination to create dynamic covalent bonds and obtain films with enhanced properties. We prepared bio-based films using pectin and chitosan, two naturally occurring polysaccharides, with glycerol added as a plasticizer to increase the flexibility and elasticity of the material. We incorporated the vanadium ion into the films via soaking in V(V) solutions. Films were characterized through FT-IR to examine the different interactions inside the films. Scanning electron microscopy showed laminated sheets separated by ∼0.16 μm in the V(V)-containing films as compared to ∼0.75 μm in the films without V(V). Rheological studies showed that V(V)-coordinated films possessed better mechanical properties (higher storage modulus) and water stability than films without vanadium. Upon irradiation with blue light, diffuse reflectance spectroscopy and color analysis showed the changes in the color of the film with irradiation from yellow to blue, consistent with the photochemical reduction of V(V) to V(IV). A small change in modulus was observed during light irradiation without significant structural changes observed within the film in scanning electron microscopy. These results show that it is possible to create robust and water-stable polysaccharide-based materials through coordination of the functional groups of polysaccharides with metal ions.

Molecular Engineering of Aptamer Self-Assemblies Increases in Vivo Stability and Targeted Recognition.

Functionally modified aptamer conjugates are promising tools for targeted imaging or treatment of various diseases. However, broad applications of aptamer molecules are limited by their in vivo instability. To overcome this challenge, current strategies mostly rely on covalent chemical modification of aptamers, a complicated process that requires case-by-case sequence design, multiple-step synthesis, and purification. Herein, we report a covalent modification-free strategy to enhance the in vivo stability of aptamers. This strategy simply utilizes one-step molecular engineering of aptamers with gold nanoclusters (GNCs) to form GNCs@aptamer self-assemblies. Using Sgc8 as a representative aptamer, the resulting GNCs@Sgc8 assemblies enhance cancer-cell-specific binding and sequential internalization by a receptor-mediated endocytosis pathway. Importantly, the GNCs@aptamer self-assemblies resist nuclease degradation for as long as 48 h, compared to the degradation of aptamer alone at 3 h. In parallel, the tumor-targeted recognition and retention of GNCs@aptamer self-assemblies are dramatically enhanced, indicated by a 9-fold signal increase inside the tumor compared to the aptamer alone. This strategy is to avoid complicated chemical modification of aptamers and can be extended to all aptamers. Our work provides a simple, effective, and universal strategy for enhancing the in vivo stability of any aptamer or its conjugates, thus expanding their imaging and therapeutic applications.

Covalent chemical modification of myofibrillar proteins to improve their gelation properties: A systematic review.

To improve the quality of meat products is a constant focus for both the meat industry and scientists. As major components in meat protein, the gelation properties of myofibrillar proteins (MPs) predominantly determine the sensory quality and product yield of the final product. Naturally or artificially occurring covalent modifications are known to largely affect MP functionality by changing the protein structure and forming aggregates, leading to both favorable and unfavorable outcomes. The review aims to summarize the mechanisms associated with several covalent modifications and the recent developments in enhancing MP gelation properties. Various extrinsic and intrinsic parameters controlling oxidation, phenolic-protein interactions, enzyme catalysis, glycation, and isoelectric solubilization/precipitation, and their effects on the characteristics of heat-induced MP gels are discussed. This article provides an improved understanding of the covalent modifications that occur mainly in the MP system and how they can be utilized to promote its gelation properties. Covalent modifications exhibited dose-dependent and dual-role manners for MP gelation properties. Mild oxidation, enzyme catalysis, and isoelectric solubilization/precipitation treatment would be beneficial to form more aligned and cross-linked three-dimensional networks for MP gels because of moderate protein aggregation. However, an excessive aggregate impedes the MP gelation behavior, leading to reduced gelation quality. Glycation effectively increased hydrophilicity of MPs and phenolic conjugation provides MPs with novel bioactivity. A proper utilization of such a process or even a rational combination of them allowed us to enhance the gelation properties of MP with assorted appreciated functionalities and further improve the quality of meat products.

The impact of epitranscriptomic marks on post-transcriptional regulation in plants.

Ribonucleotides within the various RNA molecules in eukaryotes are marked with more than 160 distinct covalent chemical modifications. These modifications include those that occur internally in messenger RNA (mRNA) molecules such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), as well as those that occur at the ends of the modified RNAs like the non-canonical 5' end nicotinamide adenine dinucleotide (NAD+) cap modification of specific mRNAs. Recent findings have revealed that covalent RNA modifications can impact the secondary structure, translatability, functionality, stability and degradation of the RNA molecules in which they are included. Many of these covalent RNA additions have also been found to be dynamically added and removed through writer and eraser complexes, respectively, providing a new layer of epitranscriptome-mediated post-transcriptional regulation that regulates RNA quality and quantity in eukaryotic transcriptomes. Thus, it is not surprising that the regulation of RNA fate mediated by these epitranscriptomic marks has been demonstrated to have widespread effects on plant development and the responses of these organisms to abiotic and biotic stresses. In this review, we highlight recent progress focused on the study of the dynamic nature of these epitranscriptome marks and their roles in post-transcriptional regulation during plant development and response to environmental cues, with an emphasis on the mRNA modifications of non-canonical 5' end NAD+ capping, m6A and several other internal RNA modifications.

N-Heterocyclic Linkages Are Produced from Condensation of Amidines onto Graphitic Carbon

Covalent chemical modification is a powerful strategy for endowing low-cost graphitic carbon substrates with designer functionality. However, the surface chemistry of carbon is complex, making it d...

Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite.For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs.Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.

Studies on covalent functionalization of single layer black phosphorus from GW calculations based on the many body perturbation theory

The major challenge of black phosphorus (BP) is its fast-oxidative degradation in air. Organic covalent chemical modification of BP flakes could suppress the chemical degradation. Here we focus on the effects of covalent chemical modification on the electronic structures of single layer BP. We employed the state-of-the-art first principles GW method, which is based on the many body green functions, to study the covalent modification of single layer BP by forming P–C bonds. Our results show that the functional group chemisorbed at perfect lattice site results in deep in-gap state, which may deteriorate its performance, while the P–C bonds forming at single vacancies are electrically inactive. In addition, our results show that functional groups at low concentration would be preferably chemisorbed at single vacancies. At high concentration, functional groups would lead to the breaking of P–P bonds, and hence, oxygen impurity will easily insert in the interstitial position. Finally, we propose that functional groups at low concentration would protect BP from deterioration, and at high concentrations, functional groups would accelerate the deterioration of BP performance.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

Epigenetic Control of Mitochondrial Function in the Vasculature.

The molecular signatures of epigenetic regulation and chromatin architecture are emerging as pivotal regulators of mitochondrial function. Recent studies unveiled a complex intersection among environmental factors, epigenetic signals, and mitochondrial metabolism, ultimately leading to alterations of vascular phenotype and increased cardiovascular risk. Changing environmental conditions over the lifetime induce covalent and post-translational chemical modification of the chromatin template which sensitize the genome to establish new transcriptional programs and, hence, diverse functional states. On the other hand, metabolic alterations occurring in mitochondria affect the availability of substrates for chromatin-modifying enzymes, thus leading to maladaptive epigenetic signatures altering chromatin accessibility and gene transcription. Indeed, several components of the epigenetic machinery require intermediates of cellular metabolism (ATP, AcCoA, NADH, α-ketoglutarate) for enzymatic function. In the present review, we describe the emerging role of epigenetic modifications as fine tuners of gene transcription in mitochondrial dysfunction and vascular disease. Specifically, the following aspects are described in detail: (i) mitochondria and vascular function, (ii) mitochondrial ROS, (iii) epigenetic regulation of mitochondrial function; (iv) the role of mitochondrial metabolites as key effectors for chromatin-modifying enzymes; (v) epigenetic therapies. Understanding epigenetic routes may pave the way for new approaches to develop personalized therapies to prevent mitochondrial insufficiency and its complications.

Charge transfer and band gap opening of a ferrocene/graphene heterostructure

The objective of this paper was to study the electrochemical and electronic behavior of pristine graphene used as a catalyst for redox reactions and the charge transport mechanisms that occur in the interface between graphene and covalently attached ferrocene. The covalent grafting and chemical modification of epitaxial graphene by a ferrocene redox marker was performed through electrochemical oxidation of ethylene diamine that provided a low coverage density and a short distance between the ferrocene and graphene. The electrochemical activity, as well as, the electronic and structural properties of the nanomaterials were characterized by Cyclic Voltammetry, Electrochemical Impedance Spectroscopy, X-ray Photoelectron Spectroscopy, RAMAN spectroscopies and Angle-Resolved-Photoelectron Spectroscopy. We show that the proposed modification improved the electronic properties of graphene where p-doping was observed and a band gap opening of about 60 meV was demonstrated. The electrochemical activity and electron transfer ability was inproved. A heterogeneous electron transfer rate of KS = 7 s−1, was obtained thanks to a low density of immobilzed ferrocene and the small spacer provided by ethylene diamine giving a fast tunneling electron transfer. We believe promising perspectives for the development of self-organized graphene electrodes with various electrochemical properties by tailoring the attached redox functional groups.

Peanut Allergy: Characteristics and Approaches for Mitigation.

Peanut allergy has garnered significant attention because of the high sensitization rate, increase in allergy, and severity of the reaction. Sufficiently reliable therapies and efficient mitigating techniques to combat peanut allergy are still lacking. Current management relies on avoiding peanuts and nuts and seeds with homologous proteins, although adverse events mostly occur with accidental ingestion. There is a need for hypoallergenic peanut products to protect sensitized individuals and perhaps serve as immunotherapeutic products. Alongside traditional practices of thermal and chemical treatment, novel processing approaches such as high-pressure processing, pulsed ultraviolet light, high-intensity ultrasound, irradiation, and pulsed electric field have been performed toward reducing the immunoreactivity of peanut. Covalent and noncovalent chemical modifications to proteins also have the tendency to alter peanut allergenicity. Enzymatic hydrolysis seems to be the most advantageous technique in diminishing the allergenic potential of peanut. Furthermore, the combined processing approach (hurdle technologies) such as enzymatic hydrolysis followed by, or in conjunction with, roasting, high pressure and heat, ultrasound with enzymatic treatment, or germination have shown a significant reduction of peanut immunoreactivity and may emerge as useful techniques in reducing the allergenicity of peanut and other foods. This study represents our current knowledge about the alterations in allergenic properties of peanut via different processing mechanisms as well as evaluating its future potential, geographical based data on increasing sensitization, clinical relevance, eliciting dose, and current management of peanut allergy. Furthermore, the molecular characteristics and clinical relevance of peanut allergens have been discussed.

Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

We report herein the synthesis and DNA/RNA binding properties of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids. By virtue of a new bivalent side chain on bPNA+, similar DNA affinity and hybrid thermostability can be obtained with half the molecular footprint of previously reported bPNA. Lysine derivatives bearing two melamine bases (K2M) can be prepared on multigram scale by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine side chain that affords both peptide solubility and selective base-triple formation with 4 T/U bases; the Fmoc-K2M derivative can be used directly in solid phase peptide synthesis, rendering bPNA+ conveniently accessible. A compact bPNA+binding site of two U6 domains can be genetically encoded to replace existing 6 bp stem elements at virtually any location within an RNA transcript. We thus replaced internal 6 bp RNA stems that supported loop regions with 6 base-triple hybrid stems using fluorophore-labeled bPNA+. As the loop regions engaged in RNA tertiary interactions, the labeled hybrid stems provided a fluorescent readout; bPNA+ enabled this readout without covalent chemical modification or introduction of new structural elements. This strategy was demonstrated to be effective for reporting on widely observed RNA tertiary interactions such as intermolecular RNA-RNA kissing loop dimerization, RNA-protein binding, and intramolecular RNA tetraloop-tetraloop receptor binding, illustrating the potential general utility of this method. The modest 6 bp stem binding footprint of bPNA+ makes the hybrid stem replacement method practical for noncovalent installation of synthetic probes of RNA interactions. We anticipate that bPNA+ structural probes will be useful for the study of tertiary interactions in long noncoding RNAs.

HAMR: High-Throughput Annotation of Modified Ribonucleotides.

Ribonucleotides can be decorated with over 100 types of covalent chemical modifications. These modifications change the structure, function, and catalytic activity of RNAs, forming a layer of posttranscriptional regulation termed the epitranscriptome. Recent advances in high-throughput mapping have demonstrated these modifications are abundant and mark nearly all classes of RNAs, including messenger RNAs. Here, we outline one such technique called high-throughput annotation of modified ribonucleotides (HAMR). HAMR exploits the tendency of certain modified ribonucleotides to interfere with base pairing, leading to errors in complementary DNA synthesis during RNA sequencing library preparation. In total, we present a computational protocol for in silico identification of modifications with HAMR, which can be retroactively applied to a variety of RNA sequencing techniques.

Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis.

Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.