Biological Applications Research Articles

Triazole-linked Nucleic Acids: Synthesis, Therapeutics and Synthetic Biology Applications.

This article covers the triazole-linked nucleic acids where the triazole linkage (TL) replaces the natural phosphate backbone. The replacement is done at either a few selected linkages or all the phosphate linkages. Two triazole linkages, the four-atom TL1 and the six-atom TL2, have been discussed in detail. These triazole-modified oligonucleotides have found a wide range of applications, from therapeutics to synthetic biology. For example, the triazole-linked oligonucleotides have been used in the antisense oligonucleotide (ASO), small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as therapeutic agents. Due to the ease of the synthesis and a wide range of biocompatibility, the triazole linkage TL2 has been used to assemble a functional 300-mer DNA from alkyne- and azide-functionalized 100-mer oligonucleotides as well as an epigenetically modified variant of a 335 base-pair gene from ten short oligonucleotides. These outcomes highlight the potential of triazole-linked nucleic acids and open the doors for other TL designs and artificial backbones to fully exploit the vast potential of artificial nucleic acids in therapeutics, synthetic biology and biotechnology.

Control of artificial membrane fusion in physiological ionic solutions beyond the limits of electroformation

Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.

Design and Application of an in Situ Traceable NO Donor for Promoting the Healing of Wound Infections.

Therapies for wound infections require medications with antibacterial and wound-healing functions. However, it remains a challenge to produce a single drug that can perform dual functions. Nitric oxide (NO), with its antibacterial and wound-healing activities, is an ideal solution to address this challenge. However, many controlled-release strategies for NO rely on external probes for tracing the release in-situ, making it difficult to precisely assess the location and magnitude. To address this issue, this study describes a novel NO donor, DHU-NO1, capable of efficiently releasing NO under mild conditions (450nm illumination). Simultaneously, DHU-NO1 generates the fluorophore Azure B, which enables direct, non-consumptive tracing of the NO release by monitoring the fluorescence and absorption changes in Azure B. Given that NO can be conveniently traced, the amount of released NO can be controlled during biological applications, thereby allowing both functions of NO to be performed. When applied to the affected area, DHU-NO1, illuminated by both a simple LED light source and natural light, achieves significant antibacterial effects against wound infections and promotes wound healing in mice. This study offers a novel and effective approach for treating wound infections. This article is protected by copyright. All rights reserved.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

Patterning of COC Polymers by Middle‐Energy Ion Beams for Selective Cell Adhesion in Microfluidic Devices

AbstractMicrofluidic devices play a crucial role in advanced cell biology applications, including cell separations, cultivations, migration and interaction studies, diagnostic devices, and organ‐on‐chips. One of the frequent purposes of such devices is the ability to selectively address the attachment of cells at defined locations on the surface. This study explores the application of middle‐energy carbon, oxygen, and nitrogen ions to locally modify the surface of cyclic olefin copolymer (COC) thermoplastic material, allowing selective cell growth on patterned polymer surfaces. The investigation considers ion element type, ion beam energy, and ion irradiation fluence, analyzing their influence on the modification effect. Characterization of the modified surfaces involves various surface‐analytical methods such as contact angle, energy dispersive spectroscopy (SEM‐EDX), atomic force microscopy (AFM), x‐ray photoelectron spectroscopy (XPS), rutherford backscattering spectrometry (RBS), and elastic recoil detection analysis (ERDA). The study extends to practical aspects, with a representative cancer cell line, MCF‐7, grown on the patterned surface to evaluate the degree of selective attachment. Additionally, the stability of the irradiated patterns is tested under elevated temperatures beyond the glass transition temperature (Tg), demonstrating the compatibility of the approach with hot embossing technology. The findings underscore the potential of ion beam treatment for COC in cell‐biology‐related applications, offering insights into surface modification techniques for enhanced functionality in microfluidic devices.

The varying viscosity impact in an inclined peristaltic channel with diffusion‐thermo and thermo‐diffusion

AbstractA mathematical model is constructed to investigate the behavior of peristaltic flow of Jeffrey fluid in an inclined tapered asymmetric porous channel. The fluid viscosity is taken as space dependent variable quantity. Heat absorption, Soret and Dufour effects are also retained in the current scrutiny. These preferences have broad applications in engineering, biology and industry. We began our investigation by taking into account the two‐dimensional inclined asymmetric porous channel. In the context of mathematical modeling, the appropriate dimensional nonlinear equations for momentum, heat and mass transport are simplified into dimensionless equations by applying the essential estimation of long wavelength and low Reynolds number. The solution of the governing equations is executed numerically. A graphical depiction of many crucial physical characteristics on velocity, temperature, concentration, heat transfer rate, Nusselt number and Streamlines have been reported in ending section. Temperature profile exhibits an escalation with the augmentation of Brinkman number and Dufour number . For the growing values of Prandtl number , an increment in temperature profile is observed whilst a reverse tendency is captured for concentration profile. It is noted that concentration profile falls down owing to the enhancement in Soret number and Schmidt number . An oscillatory outlook is noticed for heat transfer rate and Nusselt number. The novelty of this proposed model in the research domain specifically depends on considerations of the combined study of the Variable viscosity, Darcy resistance, Viscous dissipation, Mixed convention, Heat absorption, Soret and Dufour effects in peristaltic flow of non‐ Newtonian Jeffrey fluid in an inclined Asymmetric tapered channel under the influence of convective boundary conditions.

Optical Force Sensor Based on Plasmon Modulated Upconversion Luminescence

AbstractA novel force sensor exploiting the interaction between plasmonic nanostructures and upconversion nanoparticles (UCNPs) is reported. The nanosensor is composed of a gold nanodisk and UCNPs separated by a flexible polymer layer. The gold nanodisk is designed to exhibit a plasmon resonance that selectively enhances one of the emission bands of the UCNPs while leaving the other ones largely unaffected. As the nanosensor is compressed or stretched by an external force, the polymer layer thickness changes, modulating the plasmon‐UCNP coupling. The resulting changes in the luminescence intensity provide the basis for sensing. Furthermore, the nanosensor employs ratiometric sensing, which makes it highly robust against any environmental variations. The nanosensors exhibit two orders of magnitude higher responsivity than previously reported UCNP‐based force sensors. They can be prepared as an on‐chip sensor array or in a colloidal solution, making them suitable for a variety of applications in biology and robotics.

Molecular differentiation analysis of ten putative species of Fannia (Diptera: Fanniidae) collected in carrion-baited traps from Colombia.

The genus Fannia is the most representative of the Fannidae family of true flies with worldwide distribution. Some species are attracted to decomposing materials and live vertebrate animals, which makes them important in forensics, medical and veterinary fields. However, identifying Fannia species can be difficult due to the high similarity in the external morphology of females and limited descriptions and morphological keys. Herein, molecular markers could provide a complementary tool for species identification. However, molecular identification has still limited application since databases contain few data for neotropical species of Fannia. This study assessed the potential of two molecular markers, the COI-3' region and internal transcribed spacer 2 (ITS2), to differentiate 10 putative species of the genus Fannia from Colombia using distance-based and tree-based approaches. The partial ITS2 and/or COI-3' regions allowed molecular diagnosis of six species, while pairs of species Fannia colazorrensis + F. dodgei and F. laclara + F. aburrae are conflicting. Although these results might suggest that conflicting pair species are conspecific, consistent morphological differences between males do not support this hypothesis. The lack of differentiation at the nuclear and mitochondrial molecular markers for the conflicting species may be due to incomplete evolutionary lineage separation, hybridization, or introgression events. In addition, sexual selection on male morphological traits before species-specific differences in molecular markers emerge may partially explain the results. Our study provides a valuable dataset to identify and confirm some Fannia species molecularly. Further, they could be used to associate females and immature stages with their conspecifics as a baseline to deep into their biology, ecology, distribution and potential applications in forensic and medico-veterinary entomology.

Resveratrol: A Review on the Biological Activity and Applications

Resveratrol: A Review on the Biological Activity and Applications

Based ATP-gating mechanism for detection of alkaline phosphatase in single-glass micropipettes functionalized by three-dimensional DNA network.

The construction of gating system in artificial channels is a cutting-edge research direction in understanding biological process and application sensing. Here, by mimicking the gating system, we report a device that easily synthesized single-glass micropipettes functionalized by three-dimensional (3D) DNA network, which triggers the gating mechanism for the detection of biomolecules. Based on this strategy, the gating mechanism shows that single-glass micropipette assembled 3D DNA network is in the "OFF" state, and after collapsing in the presence of ATP, they are in the "ON" state, at which point they exhibit asymmetric response times. In the "ON" process of the gating mechanism, the ascorbic acid phosphate (AAP) can be encapsulated by a 3D DNA network and released in the presence of adenosine triphosphate (ATP), which initiates a catalyzed cascade reaction under the influence of alkaline phosphatase (ALP). Ultimately, the detection of ALP can be responded to form the fluorescence signal generated by terephthalic acid that has captured hydroxyl radicals, which has a detection range of 0-250mU/mL and a limit of detection of 50mU/mL. This work provides a brand-new way and application direction for research of gating mechanism.

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data.

Sample-wise deconvolution methods estimate cell-type proportions and gene expressions in bulk tissue samples, yet their performance and biological applications remain unexplored, particularly in human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk tissue RNA sequencing (RNA-seq), single-cell/nuclei (sc/sn) RNA-seq, and immunohistochemistry. A total of 1,130,767 nuclei per cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expressions. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk tissue or single-cell eQTLs did alone. Differential gene expressions associated with Alzheimer's disease, schizophrenia, and brain development were also examined using the deconvoluted data. Our findings, which were replicated in bulk tissue and single-cell data, provided insights into the biological applications of deconvoluted data in multiple brain disorders.

In vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis

Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Protein Adsorption on Solid Surfaces: Data Mining, Database, Molecular Surface-Derived Properties, and Semiempirical Relationships.

Protein adsorption on solid surfaces is a process relevant to biological, medical, industrial, and environmental applications. Despite this wide interest and advancement in measurement techniques, the complexity of protein adsorption has frustrated its accurate prediction. To address this challenge, here, data regarding protein adsorption reported in the last four decades was collected, checked for completeness and correctness, organized, and archived in an upgraded, freely accessible Biomolecular Adsorption Database, which is equivalent to a large-scale, ad hoc, crowd-sourced multifactorial experiment. The shape and physicochemical properties of the proteins present in the database were quantified on their molecular surfaces using an in-house program (ProMS) operating as an add-on to the PyMol software. Machine learning-based analysis indicated that protein adsorption on hydrophobic and hydrophilic surfaces is modulated by different sets of operational, structural, and molecular surface-based physicochemical parameters. Separately, the adsorption data regarding four "benchmark" proteins, i.e., lysozyme, albumin, IgG, and fibrinogen, was processed by piecewise linear regression with the protein monolayer acting as breakpoint, using the linearization of the Langmuir isotherm formalism, resulting in semiempirical relationships predicting protein adsorption. These relationships, derived separately for hydrophilic and hydrophobic surfaces, described well the protein concentration on the surface as a function of the protein concentration in solution, adsorbing surface contact angle, ionic strength, pH, and temperature of the carrying fluid, and the difference between pH and the isoelectric point of the protein. When applying the semiempirical relationships derived for benchmark proteins to two other "test" proteins with known PDB structure, i.e., β-lactoglobulin and α-lactalbumin, the errors of this extrapolation were found to be in a linear relationship with the dissimilarity between the benchmark and the test proteins. The work presented here can be used for the estimation of operational parameters modulating protein adsorption for various applications such as diagnostic devices, pharmaceuticals, biomaterials, or the food industry.

Coarse-grained force field for ZIF-8: A study on adsorption, diffusion, and structural properties.

Metal-organic frameworks (MOFs) are revolutionizing a spectrum of industries, from groundbreaking gas storage solutions to transformative biological system applications. The intricate architecture of these materials necessitates the use of advanced computational techniques for a comprehensive understanding of their molecular structure and prediction of their physical properties. Coarse-grained (CG) simulations shine a spotlight on the often-neglected influences of defects, pressure effects, and spatial disorders on the performance of MOFs. These simulations are not just beneficial but indispensable for high-demand applications, such as mixed matrix membranes and intricate biological system interfaces. In this work, we propose an optimized CG force field tailored for ZIF-8. Our work provides a deep dive into sorption isotherms and diffusion coefficients of small molecules. We demonstrate the structural dynamics of ZIF-8, particularly how it responds to pressurization, which affects its crystal structure and leads to local changes in aperture size and area. Emphasizing the game-changing potential of CG simulations, we explore the characteristics of amorphization in ZIF-8. Through computational exploration, we aim to bridge the knowledge gap, enhancing the potential applications of nanoporous materials for various applications.

Chitinases: expanding the boundaries of knowledge beyond routinized chitin degradation.

Chitinases, enzymes that degrade chitin, have long been studied for their role in various biological processes. They play crucial roles in the moulting process of invertebrates, the digestion of chitinous food, and defense against chitin-bearing pathogens. Additionally, chitinases are involved in physiological functions in crustaceans, such as chitinous food digestion, moulting, and stress response. Moreover, chitinases are universally distributed in organisms from viruses to mammals and have diverse functions including tissue degradation and remodeling, nutrition uptake, pathogen invasion, and immune response regulation. The discovery of these diverse functions expands our understanding of the biological significance and potential applications of chitinases. However, recent research has shown that chitinases possess several other functions beyond just chitin degradation. Their potential as biopesticides, therapeutic agents, and tools for bioremediation underscores their significance in addressing global challenges. More importantly, we noted that they may be applied as bioweapons if ethical regulations regarding production, engineering and application are overlooked.

Development and Characterization of Hemoglobin Microbubbles for Acoustic Blood Oxygen Level Dependent Imaging.

Oxygen levels in tissues and organs are crucial for their normal functioning, and approaches to monitor them non-invasively have wide biological and clinical applications. In this study, we developed a method of acoustically detecting oxygenation using contrast-enhanced ultrasound (CEUS) imaging. Our approach involved the use of specially designed hemoglobin-based microbubbles (HbMBs) that reversibly bind to oxygen and alter the state-dependent acoustic response. We confirmed that the bioactivity of hemoglobin remained intact after the microbubble shell was formed, and we did not observe any significant loss of heme. We conducted passive cavitation detection (PCD) experiments to confirm whether the acoustic properties of HbMBs vary based on the level of oxygen present. The experiments involved driving the HbMBs with a 1.1 MHz focused ultrasound transducer. Through the PCD data collected, we observed significant differences in the subharmonic and harmonic responses of the HbMBs when exposed to an oxygen-rich environment versus an oxygen-depleted one. We used a programmable ultrasound system to capture high-frame rate B mode videos of HbMBs in both oxy and deoxy conditions at the same time in a two-chambered flow phantom and observed that the mean pixel intensity of deoxygenated HbMB was greater than in the oxygenated state using B-mode imaging. Finally, we demonstrated that HbMBs can circulate in vivo and are detectable by a clinical ultrasound scanner. To summarize, our results indicate that CEUS imaging with HbMB has the potential to detect changes in tissue oxygenation and could be a valuable tool for clinical purposes in monitoring regional blood oxygen levels.

Detection of tetanus toxoid with iron magnetic nanobioprobe

Diagnosis of diseases with low facilities, speed, accuracy and sensitivity is an important matter in treatment. Bioprobes based on iron oxide nanoparticles are a good candidate for early detection of deadly and infectious diseases such as tetanus due to their high reactivity, biocompatibility, low production cost and sample separation under a magnetic field. In this study, silane groups were coated on surface of iron oxide nanoparticles using tetraethoxysilane (TEOS) hydrolysis. Also, NH2 groups were generated on the surface of silanized nanoparticles using 3-aminopropyl triethoxy silane (APTES). Antibody was immobilized on the surface of silanized nanoparticles using TCT trichlorothriazine as activator. Silanization and stabilized antibody were investigated by using of FT-IR, EDX, VSM, SRB technique. UV/vis spectroscopy, fluorescence, agglutination test and ELISA were used for biosensor performance and specificity. The results of FT-IR spectroscopy showed that Si–O–Si and Si-O-Fe bonds and TCT chlorine and amine groups of tetanus anti-toxoid antibodies were formed on the surface of iron oxide nanoparticles. The presence of Si, N and C elements in EDX analysis confirms the silanization of iron oxide nanoparticles. VSM results showed that the amount of magnetic nanoparticles after conjugation is sufficient for biological applications. Antibody stabilization on nanoparticles increased the adsorption intensity in the uv/vis spectrometer. The fluorescence intensity of nano bioprobe increased in the presence of 10 ng ml−1. Nanobio probes were observed as agglomerates in the presence of tetanus toxoid antigen. The presence of tetanus antigen caused the formation of antigen-nanobioprobe antigen complex. Identification of this complex by HRP-bound antibody confirmed the specificity of nanobioprobe. Tetanus magnetic nanobioprobe with a diagnostic limit of 10 ng ml−1 of tetanus antigen in a short time can be a good tool in LOC devices and microfluidic chips.

Michael Addition Reaction between Dehydroalanines and Phosphites Enabled the Introduction of Phosphonates into Oligopeptides.

A method for introducing a range of phosphonates into oligopeptides through a Michael addition reaction between dehydroalanine and phosphite is presented. The method offers a mild, cheap, and straightforward approach to peptide phosphorylation that has potential applications in chemical biology and medicinal chemistry. Moreover, the introduction of a phosphonate group into short antibacterial peptides is described to demonstrate its utility, leading to the discovery of phosphonated antibacterial peptides with potent broad-spectrum antibacterial activity.

Na in diamond: high spin defects revealed by the ADAQ high-throughput computational database

Color centers in diamond are at the forefront of the second quantum revolution. A handful of defects are in use, and finding ones with all the desired properties for quantum applications is arduous. By using high-throughput calculations, we screen 21,607 defects in diamond and collect the results in the ADAQ database. Upon exploring this database, we find not only the known defects but also several unexplored defects. Specifically, defects containing sodium stand out as particularly relevant because of their high spins and predicted improved optical properties compared to the NV center. Hence, we studied these in detail, employing high-accuracy theoretical calculations. The single sodium substitutional (NaC) has various charge states with spin ranging from 0.5 to 1.5, ZPL in the near-infrared, and a high Debye-Waller factor, making it ideal for biological quantum applications. The sodium vacancy (NaV) has a ZPL in the visible region and a potential rare spin-2 ground state. Our results show sodium implantation yields many interesting spin defects that are valuable additions to the arsenal of point defects in diamond studied for quantum applications.