DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for split GFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at ≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator split GFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated split GFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

Preventive effect of fermented whey protein mediated by Lactobacillus gasseri IM13 via the PI3K/AKT/FOXO pathway in muscle atrophy.

This study investigated the preventive effects of whey protein fermented with Lactobacillus gasseri IM13 (F-WP) against dexamethasone (DEX)-induced muscle atrophy. C2C12 muscle cells were treated with F-WP followed by DEX treatment. Dexamethasone treatment inhibited myotube formation and the expression of myogenic regulatory factors; however, pretreatment with F-WP attenuated DEX-induced damage. The F-WP significantly activated the phosphorylation of the IGF-1/PI3K/AKT pathway and improved muscle homeostasis suppressed by DEX. Moreover, F-WP alleviated the phosphorylation of mTOR, S6K1, and 4E-BP1 and enhanced muscle protein synthesis. Muscle-specific ubiquitin ligases and autophagy lysosomes, which were activated by the dephosphorylation of FOXO3a by DEX treatment, were significantly attenuated by F-WP pretreatment of myotubes. For peptidomic analysis, F-WP was fractionated using preparative HPLC (prep-HPLC), and the AA sequences of 11 peptides were identified using MALDI-TOF/MS/MS. In conclusion, fermentation of whey protein by the specific probiotic strain IM13 produced bioactive peptides with high antioxidant and anti-sarcopenic-sarcopenic effects, which markedly enhanced myogenesis and muscle protein synthesis while diminishing muscle protein degradation compared with intact whey protein.

Biomimetic Diatom Biosilica and Its Potential for Biomedical Applications and Prospects: A Review.

Diatom biosilica is an important natural source of porous silica, with three-dimensional ordered and nanopatterned structures referred to as frustules. The unique features of diatom frustules, such as their high specific surface area, thermal stability, biocompatibility, and adaptable surface chemistry, render diatoms valuable materials for high value-added applications. These attributes make diatoms an exceptional cost-effective raw material for industrial use. The functionalization of diatom biosilica surface improves its biophysical properties and increases the potential applications. This review focuses on the potential uses of diatom biosilica including traditional approaches and recent progress in biomedical applications. Not only well-studied drug delivery systems but also promising uses on bone regeneration and wound healing are covered. Furthermore, considerable aspects and possible future directions for the use of diatom biosilica materials are proposed to develop biomedical applications and merit further exploration.

Biomineral-Based Composite Materials in Regenerative Medicine.

Regenerative medicine aims to address substantial defects by amplifying the body's natural regenerative abilities and preserving the health of tissues and organs. To achieve these goals, materials that can provide the spatial and biological support for cell proliferation and differentiation, as well as the micro-environment essential for the intended tissue, are needed. Scaffolds such as polymers and metallic materials provide three-dimensional structures for cells to attach to and grow in defects. These materials have limitations in terms of mechanical properties or biocompatibility. In contrast, biominerals are formed by living organisms through biomineralization, which also includes minerals created by replicating this process. Incorporating biominerals into conventional materials allows for enhanced strength, durability, and biocompatibility. Specifically, biominerals can improve the bond between the implant and tissue by mimicking the micro-environment. This enhances cell differentiation and tissue regeneration. Furthermore, biomineral composites have wound healing and antimicrobial properties, which can aid in wound repair. Additionally, biominerals can be engineered as drug carriers, which can efficiently deliver drugs to their intended targets, minimizing side effects and increasing therapeutic efficacy. This article examines the role of biominerals and their composite materials in regenerative medicine applications and discusses their properties, synthesis methods, and potential uses.

Nanoengineered Silica-Based Biomaterials for Regenerative Medicine.

The paradigm of regenerative medicine is undergoing a transformative shift with the emergence of nanoengineered silica-based biomaterials. Their unique confluence of biocompatibility, precisely tunable porosity, and the ability to modulate cellular behavior at the molecular level makes them highly desirable for diverse tissue repair and regeneration applications. Advancements in nanoengineered silica synthesis and functionalization techniques have yielded a new generation of versatile biomaterials with tailored functionalities for targeted drug delivery, biomimetic scaffolds, and integration with stem cell therapy. These functionalities hold the potential to optimize therapeutic efficacy, promote enhanced regeneration, and modulate stem cell behavior for improved regenerative outcomes. Furthermore, the unique properties of silica facilitate non-invasive diagnostics and treatment monitoring through advanced biomedical imaging techniques, enabling a more holistic approach to regenerative medicine. This review comprehensively examines the utilization of nanoengineered silica biomaterials for diverse applications in regenerative medicine. By critically appraising the fabrication and design strategies that govern engineered silica biomaterials, this review underscores their groundbreaking potential to bridge the gap between the vision of regenerative medicine and clinical reality.

Biominerals and Bioinspired Materials in Biosensing: Recent Advancements and Applications.

Inspired by nature's remarkable ability to form intricate minerals, researchers have unlocked transformative strategies for creating next-generation biosensors with exceptional sensitivity, selectivity, and biocompatibility. By mimicking how organisms orchestrate mineral growth, biomimetic and bioinspired materials are significantly impacting biosensor design. Engineered bioinspired materials offer distinct advantages over their natural counterparts, boasting superior tunability, precise controllability, and the ability to integrate specific functionalities for enhanced sensing capabilities. This remarkable versatility enables the construction of various biosensing platforms, including optical sensors, electrochemical sensors, magnetic biosensors, and nucleic acid detection platforms, for diverse applications. Additionally, bioinspired materials facilitate the development of smartphone-assisted biosensing platforms, offering user-friendly and portable diagnostic tools for point-of-care applications. This review comprehensively explores the utilization of naturally occurring and engineered biominerals and materials for diverse biosensing applications. We highlight the fabrication and design strategies that tailor their functionalities to address specific biosensing needs. This in-depth exploration underscores the transformative potential of biominerals and materials in revolutionizing biosensing, paving the way for advancements in healthcare, environmental monitoring, and other critical fields.

Elucidating TH301's influence on the torsion angle of CRY1 W399 using replica exchange with solute tempering (REST) molecular dynamics (MD) simulations.

Cryptochrome 1 (CRY1) is a protein involved in the circadian clock and associated with various diseases. Targeting CRY1 for drug development requires the discovery of competitive inhibitors that target its FAD binding site through ubiquitination. During the development of compounds to regulate CRY1, an intriguing compound called TH301 was identified. Despite binding to CRY1, TH301 does not induce the expected reaction and is considered an inactive compound. However, it has been observed that TH301 affects the torsion angle of CRY1's W399 residue, which plays a crucial role in the regulation of ubiquitination by influencing the movement of the lid loop. In our research, we aimed to understand how TH301 induces the torsion angle of CRY1's W399 to shift to an "out-form" by performing REST-based MD simulations. The cyclopentane of TH301 tends to align parallel with W292, creating a repulsive force when W399 is in the "in-form", leading to a flip. In the "out-form", W399's side chain interacts with TH301's chlorobenzene through a π-π interaction, stabilizing this pose. This analysis helps identify compounds binding to CRY1 and filter out inactive ones. We found that assessing the interaction energy between TH301 and W399 is crucial to evaluate whether W399 flips or not. These findings contribute to the development of drugs targeting CRY1 and enhance our understanding of its regulatory mechanisms.

Self-Entrapment of Antimicrobial Peptides in Silica Particles for Stable and Effective Antimicrobial Peptide Delivery System.

Antimicrobial peptides (AMPs) have emerged as a promising solution to tackle bacterial infections and combat antibiotic resistance. However, their vulnerability to protease degradation and toxicity towards mammalian cells has hindered their clinical application. To overcome these challenges, our study aims to develop a method to enhance the stability and safety of AMPs applicable to effective drug-device combination products. The KR12 antimicrobial peptide was chosen, and in order to further enhance its delivery and efficacy the human immunodeficiency virus TAT protein-derived cell-penetrating peptide (CPP) was fused to form CPP-KR12. A new product, CPP-KR12@Si, was developed by forming silica particles with self-entrapped CPP-KR12 peptide using biomimetic silica precipitability because of its cationic nature. Peptide delivery from CPP-KR12@Si to bacteria and cells was observed at a slightly delivered rate, with improved stability against trypsin treatment and a reduction in cytotoxicity compared to CPP-KR12. Finally, the antimicrobial potential of the CPP-KR12@Si/bone graft substitute (BGS) combination product was demonstrated. CPP-KR12 is coated in the form of submicron-sized particles on the surface of the BGS. Self-entrapped AMP in silica nanoparticles is a safe and effective AMP delivery method that will be useful for developing a drug-device combination product for tissue regeneration.

Novel enzymatic single-nucleotide modification of DNA oligomer: prevention of incessant incorporation of nucleotidyl transferase by ribonucleotide-borate complex.

Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.

Stabilized and Immobilized Carbonic Anhydrase on Electrospun Nanofibers for Enzymatic CO2 Conversion and Utilization in Expedited Microalgal Growth.

Carbonic anhydrases convert CO2 to bicarbonate at a high turnover rate up to 106 s-1, but their actual applications in CO2 conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO2 to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO2 reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO2 conversion and utilization technologies.

Fusion tags to enhance heterologous protein expression.

Escherichia coli is the most widely used heterologous protein expression system. However, this system remains a challenge due to the low solubility of proteins, insufficient yield, and inclusion body formation. Numerous approaches have sought to address these issues. The use of a fusion tag is one of the most powerful strategies for obtaining large amounts of heterologous protein in E. coli expression system. Here, recent advances in fusion tags that increase the expression of proteins are reviewed. In addition, proposed concepts for designing peptide tags to increase protein expression are discussed.

The NT11, a novel fusion tag for enhancing protein expression in Escherichia coli.

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.

Improvement in the Reproducibility of a Paper-based Analytical Device (PAD) Using Stable Covalent Binding between Proteins and Cellulose Paper.

Paper-based analytical devices (PADs) have been widely used in many fields because they are affordable and portable. For reproducible quantitative analysis, it is crucial to strongly immobilize proteins on PADs. Conventional techniques for immobilizing proteins on PADs are based on physical adsorption, but proteins can be easily removed by weak physical forces. Therefore, it is difficult to ensure the reproducibility of the analytical results of PADs using physical adsorption. To overcome this limitation, in this study, we showed a method of covalent binding of proteins to cellulose paper. This method consists of three steps, which include periodate oxidation of paper, the formation of a Schiff base, and reductive amination. We identified aldehyde and imine groups formed on paper using FT-IR analysis. This covalent bonding approach enhanced the binding force and binding capacity of proteins. We confirmed the activity of an immobilized antibody through a sandwich immunoassay. We expect that this immobilization method will contribute to the commercialization of PADs with high reproducibility and sensitivity.

Expression and characterization of a codon-optimized alkaline-stable carbonic anhydrase from Aliivibrio salmonicida for CO2 sequestration applications.

The CO2 mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO2, the accumulation of which in the atmosphere is the main cause of global warming. Here, we characterize a highly stable form of the cloned CA from the Gram-negative marine bacterium Aliivibrio salmonicida, named ASCA that can promote CO2 absorption in an alkaline solvent required for efficient carbon capture. We designed a mature form of ASCA (mASCA) using a codon optimization of ASCA gene and removal of ASCA signal peptide. mASCA was highly expressed (255 mg/L) with a molecular weight of approximately 26 kDa. The mASCA enzyme exhibited stable esterase activity within a temperature range of 10-60 °C and a pH range of 6-11. mASCA activity remained stable for 48 h at pH 10. We also investigated its inhibition profiles using inorganic anions, such as acetazolamide, sulfanilamide, iodide, nitrate, and azide. We also demonstrate that mASCA is capable of catalyzing the conversion of CO2 to CaCO3 (calcite form) in the presence of Ca2+. It should be noted that mASCA enzyme exhibits high production yield and sufficient stabilities against relatively high temperature and alkaline pH, which are required conditions for the development of more efficient enzymatic CCS systems.

A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon Candidatus Nitrosopumilus koreensis.

OBJECTIVES: To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine at elevated pH and high temperature. RESULTS: A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis. This protein contains key tyrosinase domains, such as copper-binding domains and an O2-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l(-1) and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio. CONCLUSIONS: The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

OBJECTIVES: To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized. RESULTS: A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l(-1) with a purity of >95 %. It efficiently formed a complex with Ca(2+). Ca(2+)-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20-30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein. CONCLUSIONS: Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

Translocation and stability of replicative DNA helicases upon encountering DNA-protein cross-links.

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.

A multiplex single nucleotide polymorphism genotyping method using ligase-based mismatch discrimination and CE-SSCP.

Accuracy, simplicity, and cost-effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase-based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray-based detection, the design process and multiplex assay procedure are complicated because of the DNA size-dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition-optimized ligation and high-resolution CE-based SSCP. With this high-resolution CE-SSCP system, we are able to use similar-sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single-base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.

Solution structure and stability of the DNA undecamer duplexes containing oxanine mismatch.

Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.