Efficient and safe small RNA delivery to macrophage using peptide-based nanocomplex.

As one of the gene therapies, RNA interference (RNAi) effectively suppresses only specific genes, targeting various diseases in which they are involved. For the successful process of RNAi, efficient and safe delivery of small RNAs, including small interfering RNA and short hairpin RNA, is essential. Herein, an S-R11 fusion peptide, SPACE peptide conjugated with poly-arginine, was introduced to deliver small RNAs into immune cells that are difficult to transfect. This S-R11 peptide stably formed a spontaneous self-assembling nanocomplex through electrostatic attraction and hydrogen bonding with small RNAs. The nanocomplex showed about 5.3-fold better permeation efficiency than the conventional Lipofectamine™ 2000 for RAW 264.7 macrophage cells. Moreover, it induced about 66.2% silencing effect of the target gene in the cells activated with polyinosinic:polycytidylic acid (poly (I:C)). In addition, the cell viability of fusion peptide was ensured even in a concentration range exceeding the concentration used in the nanocomplex. Based on these results, it is expected that the nanocomplex in this study can be used as a new gene delivery system that can overcome the challenge of gene therapies to immune cells.

Novel fusion peptide-mediated siRNA delivery using self-assembled nanocomplex.

BACKGROUND: Gene silencing using siRNA can be a new potent strategy to treat many incurable diseases at the genetic level, including cancer and viral infections. Treatments using siRNA essentially requires an efficient and safe method of delivering siRNA into cells while maintaining its stability. Thus, we designed novel synergistic fusion peptides, i.e., SPACE and oligoarginine. RESULTS: Among the novel fusion peptides and siRNAs, nanocomplexes have enhanced cellular uptake and gene silencing effect in vitro and improved retention and gene silencing effects of siRNAs in vivo. Oligoarginine could attract siRNAs electrostatically to form stable and self-assembled nanocomplexes, and the SPACE peptide could interact with the cellular membrane via hydrogen bonding. Therefore, nanocomplexes using fusion peptides showed improved and evident cellular uptake and gene silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) via the lipid raft-mediated endocytosis pathway, especially to the HDFn cells of the skin, and all of the fusion peptides were biocompatible. Also, intratumorally injected nanocomplexes had increased retention time of siRNAs at the site of the tumor. Finally, nanocomplexes demonstrated significant in vivo gene silencing effect without overt tissue damage and immune cell infiltration. CONCLUSIONS: The new nanocomplex strategy could become a safe and efficient platform for the delivery of siRNAs into cells and tissues to treat various target diseases through gene silencing.

SARS-CoV-2 Variants: Mutations and Effective Changes.

One of the primary threats to the goal of controlling and eventually defeating SARS-CoV-2 is that of mutation. Recognizing this, a great amount of effort and dedicated study is being given to the matter. Due to the novel coronavirus's general prevalence and rate of mutation, this is an extremely dynamic area with constant new developments. Therefore, understanding the virus's pathogenesis and how mutations affect it is crucial. This review attempts to aid in understanding the currently most important strains and what primary changes they entail in connection to more specific mutations, and how they each affect infectivity, antigen resistance, and other properties. In an attempt to maintain relevance to the time at which this paper will be published, priority has been given to variants classified by the WHO and the CDC as of Sep. 23, 2021, as "Variants of Concern". Of particular interest in B.1.1.7, B.1.351, B.1.617.2, P.1 are the mutations affecting the Spike protein and Receptor Binding Domain, as they directly affect infectivity and susceptibility to neutralization. Certain mutations (D614G, E484K, N501Y, K417N, L452R and P681R) have appeared across several different strains, often accompanied by others that may be complementary working together to confer increased infectivity, fitness, or resistance to neutralization. We anticipate that the understanding of such COVID-19 mutations will, in the near future, prove important for diagnosis, treatment development, and vaccine development.

Colorimetric biosensor using dual-amplification of enzyme-free reaction through universal hybridization chain reaction system.

On-site genetic detection needs to develop a sensitive and straightforward biosensor without special equipment, which can detect various genetic biomarkers. Hybridization chain reaction (HCR) amplifying signal isothermally could be considered as a good candidate for on-site detection. Here, we developed a novel genetic biosensor on the basis of enzyme-free dual-amplification of universal hybridization chain reaction (uHCR) and hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme. The uHCR is the strategy which enables simple design for multiple target detection by the introduction of target-specific trigger hairpin without changing the whole system according to a target change. Also, HRP-mimicking DNAzyme could produce a sensitive and quantitative colorimetric signal with increased stability with a limit of detection (LOD) of 5.67 nM. The universality of the uHCR biosensor was proven by the detection of four different targets (miR-21, miR-125b, KRAS-Q61K, and BRAF-V600E) for cancer diagnosis. The uHCR biosensor showed specificity that could discriminate single-nucleotide polymorphism. Moreover, the uHCR biosensor could detect targets in the diluted serum sample. Overall, the uHCR biosensor demonstrated the potential for field testing with a simple redesign without complicated steps or special equipment using a universal hairpin system and enzyme-free amplification. This strategy could enable stable and sensitive detection of a variety of targets. Therefore, it could be applied to urgent detection of various pathogens, remote diagnosis, and self-screening of diseases.

Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

Biomimetic repeat protein derived from Xenopus tropicalis for fibrous scaffold fabrication.

Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiber-based sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein-based material. The codon-optimized recombinant frog repeat protein, referred to as 'xetro', was produced at a high rate in a bacterial system, and an acid extraction-based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet-spun xetro microfibers demonstrated about 2- and 1.5-fold higher tensile strength compared with synthetic polymer polylactic acid and cross-linked collagen, respectively. In addition, the wet-spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber-based material for biomedical engineering.

Mussel-Inspired Protein Nanoparticles Containing Iron(III)-DOPA Complexes for pH-Responsive Drug Delivery.

A novel bioinspired strategy for protein nanoparticle (NP) synthesis to achieve pH-responsive drug release exploits the pH-dependent changes in the coordination stoichiometry of iron(III)-3,4-dihydroxyphenylalanine (DOPA) complexes, which play a major cross-linking role in mussel byssal threads. Doxorubicin-loaded polymeric NPs that are based on Fe(III)-DOPA complexation were thus synthesized with a DOPA-modified recombinant mussel adhesive protein through a co-electrospraying process. The release of doxorubicin was found to be predominantly governed by a change in the structure of the Fe(III)-DOPA complexes induced by an acidic pH value. It was also demonstrated that the fabricated NPs exhibited effective cytotoxicity towards cancer cells through efficient cellular uptake and cytosolic release. Therefore, it is anticipated that Fe(III)-DOPA complexation can be successfully utilized as a new design principle for pH-responsive NPs for diverse controlled drug-delivery applications.

Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

In vivo residue-specific dopa-incorporated engineered mussel bioglue with enhanced adhesion and water resistance.

Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNA(Tyr) by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.

Aptamer-modified tetrahedral DNA nanostructure-immobilized liposome for specific gene delivery and potential cancer theragnostic.

Targeted delivery of therapeutic agents to cancer cells is crucial for effective cancer treatment without adverse effects. In this study, we developed a novel delivery carrier, Aptamer-modified tetrahedral DNA nanostructure (TDN) immobilized Liposome (ApTL), for specific delivery to nucleolin-overexpressing cancer cells. We demonstrated that targeted ApTL was highly effective in delivering plasmid and mRNA to nucleolin-overexpressing cancer cells compared to non-targeted ApTL with a non-specific aptamer. ApTL, which is highly negative and nano-sized, specifically delivered nucleic acids to MDA-MB-231 and HeLa cancer cells, primarily via lipid-raft-mediated endocytosis. Furthermore, the co-delivery of mRNA and doxorubicin resulted in increased apoptosis and reduced cancer cell viability. Interestingly, co-delivery of mRNA and Dox did not show a significant difference in EGFP expression at 24 h but dramatically increased EGFP expression at 48 h, making ApTL/mEGFP/Dox a promising candidate for detecting live cancer cells after targeted cancer drug treatment. Our results suggest that ApTL can be a promising tool for the targeted delivery of therapeutic agents to nucleolin-overexpressing cancer cells, providing a new strategy for cancer theragnostic.

Electrosprayable Levan-Coated Nanoclusters and Ultrasound-Responsive Drug Delivery for Cancer Therapy.

In this study, we synthesized levan shell hydrophobic silica nanoclusters encapsulating doxorubicin (L-HSi-Dox) and evaluated their potential as ultrasound-responsive drug delivery systems for cancer treatment. L-HSi-Dox nanoclusters were successfully fabricated by integrating a hydrophobic silica nanoparticle-doxorubicin complex as the core and an amphiphilic levan carbohydrate polymer as the shell by using an electrospray technique. Characterization analyses confirmed the stability, size, and composition of the nanoclusters. In particular, the nanoclusters exhibited a controlled release of Dox under aqueous conditions, demonstrating their potential as efficient drug carriers. The levanic groups of the nanoclusters enhanced the targeted delivery of Dox to specific cancer cells. Furthermore, the synergism between the nanoclusters and ultrasound effectively reduced cell viability and induced cell death, particularly in the GLUT5-overexpressing MDA-MB-231 cells. In a tumor xenograft mouse model, treatment with the nanoclusters and ultrasound significantly reduced the tumor volume and weight without affecting the body weight. Collectively, these results highlight the potential of the L-HSi-Dox nanoclusters and ultrasound as promising drug delivery systems with an enhanced therapeutic efficacy for biomedical applications.

Multiple suppressing small interfering RNA for cancer treatment-Application to triple-negative breast cancer.

Certain cancers, such as triple-negative breast cancer (TNBC), pose a challenging prognosis due to the absence of identifiable hormone-related receptors and effective targeted therapies. Consequently, novel therapeutics are required for these cancers, offering minimal side effects and reduced drug resistance. Unexpectedly, siRNA-7, initially employed as a control, exhibited significant efficacy in inhibiting cell viability in MDA-MB-231 cells. Through a genome-wide search of seed sequences, the targets of siRNA-7 were identified as cancer-related genes, namely PRKCE, RBPJ, ZNF737, and CDC7 in MDA-MB-231 cells. The mRNA repression analysis confirmed the simultaneous suppression by siRNA-7. Combinatorial administration of single-targeting siRNAs demonstrated a comparable reduction in viability to that achieved by siRNA-7. Importantly, siRNA-7 selectively inhibited cell viability in MDA-MB-231 cells, while normal HDF-n cells remained unaffected. Furthermore, in a xenograft mouse model, siRNA-7 exhibited a remarkable 76% reduction in tumor volume without any loss in body weight. These findings position siRNA-7 as a promising candidate for a novel, safe, specific, and potent TNBC cancer therapeutic. Moreover, the strategy of multiple suppressing small interfering RNA holds potential for the treatment of various diseases associated with gene overexpression.

Actively cross-linking hemostatic sealant enables rapid hemostasis and wound closure.

A rapid hemostatic sealant can save a patient's life from shock and death due to severe trauma or excessive bleeding from the wound site during surgery. However, an ideal hemostatic sealant needs to meet the standards of safety, efficacy, usability, cost, and approvability and overcome new challenges. Here, we devised a combinatorial hemostatic sealant of PEG succinimidyl glutarate-based cross-linking branched polymers (CBPs) and the active hemostatic peptide (AHP). After ex vivo optimization, the best hemostatic combination was called an active cross-linking hemostatic sealant (ACHS). Interestingly, ACHS formed cross-links with serum proteins, blood cells, and tissue and interconnected coating on blood cells, which might induce hemostasis and tissue adhesion based on SEM images. Moreover, ACHS showed the highest coagulation efficacy, formation, and agglomeration of thrombi within 12 s, and in vitro biocompatibility. Mouse model experiments represented rapid hemostasis within 1 min, wound closure of the liver incision, and less bleeding than the commercialized sealant with tissue biocompatibility. ACHS has the advantages of rapid hemostasis, mild sealant, and easy supply by chemical synthesis without inhibition by anticoagulants, which might minimize bacterial infection by immediate wound closure. Therefore, ACHS could become a new-type hemostatic sealant to match surgical needs for internal bleeding.

Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples.

Synergistic gene delivery by self-assembled nanocomplexes using fusion peptide and calcium phosphate.

Gene therapy can be a promising therapeutic approach to cure the fundamental causes of incurable genetic diseases. Because virus carriers are costly and can cause inflammation and immunogenicity, efficient non-viral carriers need to be developed for broader gene therapy applications. Therefore, we designed novel synergistic nanocomplexes for efficient transfection incorporated by the fusion of nuclear localization signal and cell-penetrating peptides with calcium phosphate. Fusion peptides were able to package large plasmid DNAs into nanocomplexes spontaneously and efficiently. After optimization, S-R/CaP or S-S/CaP nanocomplexes significantly improved specific luciferase expression up to 2-fold compared to Lipofectamine® 2000. In addition, the large Cas9-encoding plasmids were transfected into HEK293T cells more efficiently than Lipofectamine® 2000. Furthermore, subcutaneously injected cells to mice maintained more stable protein expression until 10 days than Lipofectamine® 2000. Moreover, the biocompatibility was revealed by observing negligible cytotoxicity, histological difference, and inflammatory cytokine release. Consequently, the new chimeric strategy will be an efficient and safe gene carrier into cells and tissues to treat various genetic diseases through gene therapy.

Pattern-mapped multiple detection of 11 pathogenic bacteria using a 16s rDNA-based oligonucleotide microarray.

Pathogen detection is an important issue in human health due to the threats posed by severe communicable diseases. In the present study, to achieve efficient and accurate multiple detection of 11 selected pathogenic bacteria, we constructed a 16S rDNA oligonucleotide microarray containing doubly specific capture probes. Many target pathogens were specifically detected by the microarray with the aid of traditional perfect match-based analysis using our previously proposed two-dimensional visualization plot tool. However, some target species or subtypes were difficult to discriminate by perfect match analysis due to nonspecific binding of conserved 16S rDNA-derived capture probes with high sequence similarity. We noticed that the patterns of specific spots for each strain were somewhat different in the two-dimensional gradation plot. Therefore, to discriminate subtle differences between phylogenetically related pathogens, a pattern-mapping statistical model was established using an artificial neural network algorithm trained by experimental repeats. The oligonucleotide microarray system harboring doubly specific capture probes combined with the pattern-mapping analysis tool resulted in successful detection of all target pathogens including even subtypes of two closely related species showing strong nonspecific binding. Collectively, the results indicate that our novel combined system of a 16S rDNA-based DNA microarray and a pattern-mapping statistical analysis tool is a simple and effective method for detecting multiple pathogens.

A functional carbohydrate chip platform for analysis of carbohydrate-protein interaction.

A carbohydrate chip based on glass or other transparent surfaces has been suggested as a potential tool for high-throughput analysis of carbohydrate-protein interactions. Here we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has di-amine groups at both ends. The modified carbohydrates were covalently attached to an amino-reactive NHS-activated glass surface by formation of stable amide bonds. This proposed method was applied for efficient construction of a carbohydrate microarray to analyze carbohydrate-protein interactions. The carbohydrate chip prepared using our method can be successfully used in diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions, and carbohydrate sensor chip or microarray development for diagnosis and screening.

Plasmonic Biosensor Controlled by DNAzyme for On-Site Genetic Detection of Pathogens.

On-site predetection of pathogens could significantly decrease of a disease outbreak or national loss in most of the countries. However, conventional detection techniques are limited in use for on-site detection due to the necessity of specialized skill or equipment. Therefore, it is necessary to develop a new technique that can predetect pathogens in the field without special skills or equipment. Here, a DNAzyme strategy to control a plasmonic biosensor for rapid and simple visual detection of Salmonella choleraesuis is adopted. Multicomponent DNAzyme formed by target addition can cleave the linker effectively at 50 °C. Linker cleavage induces dispersion of two DNA-immobilized gold nanoparticles and color change. Under optimized assay conditions, the target could be detected via visual discrimination sensitively and specifically. Moreover, the biosensor shows the possibility of practical use with contaminants and a 16S rRNA real target. As a result, the proposed plasmonic biosensor can visually detect S. choleraesuis without unstable enzymes, a specialized technique, or equipment. Therefore, these advantages could allow that this biosensor would be used for on-site predetection to lower the risk of transmission of infectious diseases.

Newly Identified HNP-F from Human Neutrophil Peptide-1 Promotes Hemostasis.

Active hemostatic agents can play a crucial role in saving patients' lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide-1 (HNP-1) has been previously reported with the hemostatic mechanism, which part of HNP-1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP-F) promoting hemostasis, originating from HNP-1, has been newly identified by the blood coagulation ability test. HNP-F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP-F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP-F's biosafety. It is hypothesized that the surface charge and structure of HNP-F could be favorable to interact with fibrinogen or thrombospondin-1. Collectively, because HNP-F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.

Quantitative oligonucleotide microarray data analysis with an artificial standard probe strategy.

Quantitative data analysis is an important element in several applications of DNA microarray, including mRNA expression profiling and estimation of infectious doses for pathogens. Here, we introduce an artificial standard probe strategy for quantitative pathogen detection using an oligonucleotide chip as a model system. The standard capture probe sequence was artificially designed to prevent non-specific hybridization with bacterial targets. Based on the fluorescence intensities of artificial standard spots, the raw fluorescence intensity data for specific spots could be corrected to generate linear correlations with target concentrations. Therefore, our novel artificial standard probe may be effectively applied for the correction of chip-to-chip variations and quantitative data analysis of a one-color labeled DNA microarray system.