Search

A significant heart attack known as a myocardial infarction (MI) occurs when the blood supply to the heart is suddenly interrupted, harming the heart muscles due to a lack of oxygen. The incidence of myocardial infarction is increasing worldwide. A relationship between COVID-19 and myocardial infarction due to the recent COVID-19 pandemic has also been revealed.We propose a biomarker and a method that can be used for the diagnosis of myocardial infarction, and an aptamer-based approach.For the diagnosis of myocardial infarction, an algorithm-based diagnosis method was developed using electrocardiogram data. A diagnosis method through biomarker detection was then developed.Myocardial infarction is a disease that is difficult to diagnose based on the aspect of a single factor. For this reason, it is necessary to use a combination of various methods to diagnose myocardial infarction quickly and accurately. In addition, new materials such as aptamers must be grafted and integrated into new ways.The incidence of myocardial infarction is increasing worldwide, and some studies are being conducted on the association between COVID-19 and myocardial infarction. The key to properly treating myocardial infarction is early detection, thus we aim to do this by offering both tools and techniques as well as the most recent diagnostic techniques.Myocardial infarction is diagnosed using an electrocardiogram and echocardiogram, which utilize cardiac signals. It is required to identify biomarkers of myocardial infarction and use biomarker-based ELISA, SPR, gold nanoparticle, and aptamer technologies in order to correctly diagnose myocardial infarction.

We isolated S-formylglutathione hydrolase (scSFH) from Sphingobium chungbukense strain KCTC2955 and expressed it in Escherichia coli. The extracted scSFH showed activity toward p-nitrophenyl butyrate (p-NB), and a protein–substrate interaction was confirmed through 3D molecular docking simulation. The DNA sequence of the cloned scSFH gene (900 bp) has an open reading frame encoding 300 amino acids with a predicted molecular mass of 32.82 kDa; the sequence contains Ser-His-Asp conserved in the active site and a GHSMGG motif. The cloned scSFH gene successfully overproduced scSFH protein under the T7 promoter of the expression vectors pET21a and pGEX4T-1 in E. coli BL21(DE3). We analyzed E. coli-expressed scSFH protein for esterase activity using p-NB as a substrate. Furthermore, we confirmed the interaction between the scSFH protein and p-NB substrate. The scSFH protein of the SFH family was overexpressed in E. coli BL21(DE3) and purified using affinity Ni–NTA agarose resin. The activity of purified scSFH against p-NB was 6.87 mg L−1 for Km and 131.16 mg L−1 protein min−1 for Vmax. Additionally, we confirmed that the serine residue of the GHSMGG motif in the obtained protein interacts with p-NB through molecular docking prediction by the molecular operating environment. We identified a novel scSFH gene from S. chungbukense and overexpressed it in E. coli. The recombined scSFH protein showed activity toward p-NB, and the interaction was confirmed through 3D molecular docking prediction. This research could be applied to identify new genes and confirm their functions.

Sphinganine is a key precursor for the synthesis of ceramide during sphingolipid biosynthesis in eukaryotes. In the sphinganine biosynthetic pathway, serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase (3KSR) catalyze the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine, and the conversation of 3-ketosphinganine to dehydrosphingosine (DHS), respectively. Phytosphingosine among cosmeceutical ingredients exists primarily on the surface of the skin and serves as a potent anti-microbial and anti-inflammatory agent. To overexpressing produce phytosphingosine in bacteria, we designed and synthesized the oligonucleotide of two major genes (3KSR and SPT) involved in the biosynthesis of sphingolipid for industrial applications. The genes encoding SPT from the Gram-negative bacterium Sphingobium chungbukense DJ77 and 3KSR from the eukaryotic Saccharomyces cerevisiae were expressed in Escherichia coli BL21 (DE3) to produce DHS efficiently. Codon optimization was utilized for 3KSR expression in E. coli to facilitate the expression of eukaryotic proteins in the bacterial host. The synthetic 3KSR (3sKSR) protein activity was improved approximately 1.8 times compared to the wild-type 3KSR. Additionally, we successfully overexpressed the spt gene obtained from S. chungbukense DJ77, which can be used in efficient production of the sphenoid bases, with codon-optimized 3sKSR. The enhanced accumulation of DHS by SPT-3sKSR suggests that the codon-optimized enzyme (3sKSR) is more efficient for heterogeneous expression in E. coli. Our results demonstrate that sphingolipid metabolism can be configured in engineered E. coli by the simultaneous overexpression of two key enzymes, SPT and 3sKSR, thereby producing sphingosine.

In the described experiments, we identify and clone a novel serine palmitoyltransferase (SPT) isolated from the gram-negative obligatory aerobic bacteria Sphingobium chungbukense strain KCTC2955. SPT catalyzes the first rate-limiting reaction in the de novo biosynthesis of sphingolipids, in S. chungbukense. The DNA sequence of the cloned SPT gene (1791 bp) has an open reading frame encoding 405 amino acids with a predicted molecular mass of 41 kDa; the sequence contains the consensus PLP-binding motif (GTFSKSXXXXGG) which is conserved among serine palmitoyltransferase. The ribosomal binding site GGGA lays 6 bp upstream of the ORF of 1218 bp, which is initiated by an ATG codon and terminated by TGA. The cloned SPT gene succeeded in overproducing the SPT protein under the T7 promoter of the expression vector pET 21a in Escherichia coli BL21(DE3), in which the product amounted to about 20–30% of the total protein isolated from the cell extract. Sphingobium SPT showed about a 68.4–89.3% homology with other enzymes in the serine palmitoyltransferase family, and amino acid residues proposed to be involved in catalysis were conserved. The activity of purified recombinant-SPT using Ni-NTA metal-affinity chromatography was measured via the radioactivity of the [3H]-3-ketodihydrosphingosone (KDS) formation using l-[3H]-serine. We have identified a novel SPT gene from S. chungbukense KCTC2955 and overexpressed the recombinant SPT in E. coli. This study is a first step in understanding the reaction mechanism of SPT in bacteria as well as the first focused on the genus Sphingobium.

Development of genetically modified crops has rapidly increased in last few years. The most widely grown GM crops express genes that confer herbicide tolerance and insect resistance. Detection system of GM crops is important for safety evaluation before its consumption. The purpose of this research is to detect GM crops, especially PAT, in food-samples. The bar gene (PAT protein, herbicide resistant) was cloned in pGEX-4T-1 and expressed by E. coli. The high-affinity PAT-specific single-stranded DNA (ssDNA) aptamers were obtained from a random DNA library. MOE docking study was performed to identify the potential binding region of the selected aptamers on PAT. Aptamer-linked immobilized sorbent assay (ALISA) method was used to detect PAT. We screened aptamer against PAT for developing an efficient detection method. The selected PAT specific aptamers, HRPA-05 and HRPA-07, showed the distinct target binding behaviors, and detected PAT protein by aptamer-linked immobilized sorbent assay method with high efficiency and selectivity.

To alleviate carbon dioxide (CO2) emission, alternative approaches have been considered, such as employing microorganisms capable of CO2-fixation using their own metabolic pathways. In this study, we introduced a synthetic CO2-fixation pathway into the industrial platform microorganism Escherichia coli to achieve immediate CO2 mitigation with a non-photosynthetic reaction. Specifically, the whole gene clusters (cbbI and cbbII operons) involved in the Calvin–Benson–Bassham (CBB) pathway in Rhodobacter sphaeroides (rCBB) were expressed heterogeneously in E. coli under a restrictive carbon source and dark conditions. The resulting strain with the whole rCBB was able to exhibit a higher level of endogenous CO2 recycling compared to the control strain. In addition, the electron requirements from the cathode and redox cofactors were increased following the activation of the CO2-fixation pathway without light irradiation. Exogenous CO2 from yeast fermentation was also mitigated through a simple gas-phase connection with the E. coli/rCBB culture system. This bacterial CO2 mitigation strategy has great potential to alleviate greenhouse gas emissions through biological processes to produce carbon neutral biofuels and value-added chemicals.

Background Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is a biomarker of hepatocellular carcinoma (HCC) progression. Aptamers specifically binding to target biomolecules have recently emerged as clinical disease diagnosis targets. Here, we describe 3D structure-based aptaprobe platforms for detecting GPC3, such as aptablotting, aptaprobe-based sandwich assay (ALISA), and aptaprobe-based imaging analysis. Results For preparing the aptaprobe–GPC3 platforms, we obtained 12 high affinity aptamer candidates (GPC3_1 to GPC3_12) that specifically bind to target GPC3 molecules. Structure-based molecular interactions identified distinct aptatopic residues responsible for binding to the paratopic nucleotide sequences (nt-paratope) of GPC3 aptaprobes. Sandwichable and overlapped aptaprobes were selected through structural analysis. The aptaprobe specificity for using in HCC diagnostics were verified through Aptablotting and ALISA. Moreover, aptaprobe-based imaging showed that the binding property of GPC3_3 and their GPC3 specificity were maintained in HCC xenograft models, which may indicate a new HCC imaging diagnosis. Conclusion Aptaprobe has the potential to be used as an affinity reagent to detect the target in vivo and in vitro diagnosing system. Graphical Abstract

Background mRNA vaccines hold great potential as therapeutic techniques against viral infections due to their efficacy, safety, and large-scale production. mRNA vaccines offer flexibility in development as any protein can be produced from mRNA without altering the production or application process. Objective This review highlights the iterative optimization of mRNA vaccine structural elements that impact the type, specificity, and intensity of immune responses leading to higher translational potency and intracellular stability. Results Modifying the mRNA structural elements particularly the 5' cap, 5'-and 3'-untranslated regions (UTRs), the coding region, and polyadenylation tail help reduce the excessive mRNA immunogenicity and consistently improve its intracellular stability and translational efficiency. Conclusion Further studies regarding mRNA-structural elements and their optimization are needed to create new opportunities for engineering mRNA vaccines.

Vacuoles are membrane vesicles in eukaryotic cells, the digestive system of cells that break down substances absorbed outside the cell and digest the useless components of the cell itself. Researches on anticancer and intractable diseases using vacuoles are being actively conducted. The practical application of the present study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 cells. This showed a significant increase in the production of nitric oxide (NO) produced by macrophage activity. Using Reactive Oxygen Species (ROS) assay, we identified that ROS is increased in a manner dependent on vacuole concentration. Western blot analysis showed that vacuole concentration-dependently increased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2). Therefore, iNOS expression was stimulated to induce NO production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin (IL) 6 (IL-6) and tumor necrosis factor (TNF) α (TNF-α). In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.

Salmonella enterica ser. Typhimurium is a foodborne pathogen that causes salmonellosis. Symptoms of salmonellosis include fever, diarrhea, and gastroenteritis. Conventional culture methods for detecting foodborne bacterial pathogens require long incubation time, expensive immunoassay methods, and sample enrichment steps. The objective of this study was to develop an aptamer-based sandwich assay to detect S. enterica ser. Typhimurium. First, S. enterica ser. Typhimurium specific binding aptamers (S11 and S24) were obtained by whole-cell SELEX for high sensitivity and specificity detection using live S. enterica ser. Typhimurium. S11 and S24 aptamers were able to capture S. enterica ser. Typhimurium selectively and distinguish it from other species of Salmonella (S. enterica ser. Typhimurium, S. enterica ser. Choleraesuis, S. enterica ser. Dublin, and S. enterica ser. Enteritidis) and food-borne bacterial pathogens (Escherichia coli K12, Listeria monocytogenes, Shigella sonnei and Staphylococcus aureus) with KD values of 4.41×10-12 M and 3.75×10-11 M, respectively. S. enterica ser. Typhimurium. aptamer-based sandwich assay exhibited a linear response for sensing S. enterica ser. Typhimurium. cells at concentration ranging from 2×101 to 2×105 CFU/mL. This aptamer-based sandwich assay can be used for on-site detection of S. enterica ser. Typhimurium.

We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.

The yeast vacuole is functionally corresponding to vacuoles in eukaryote cells, it consists of a fusion protein that assists in the fusion of vacuoles and plays an important role in many processes. In addition, chemicals such as NH4Cl can reduce the size of vacuoles but as a side effect that also inhibits vacuoles making them inactive. In this study, to develop pre-treatments for extending the life of cut flowers, we constructed recombinant yeast using the fusion protein YPT7 and confirmed the activity of down-sized vacuoles. All the vacuoles of the recombinant yeast except vacuoles from recombinant yeast (MBTL-MYH-3) were found to be small vacuoles than mock (MBTL-MYH-0) and YPT7 overexpression model (MBTL-MYH-1). To confirm their activity, we conducted a test for antimicrobial activity. The results showed the other vacuoles of recombinant yeast had lower antimicrobial activity than the mock control, most of them showed about 60 % to 80 % of the antimicrobial activity. However, MBTL-MYH-3, whose vacuole did not change its size, showed antimicrobial activity lower than 40 %. Therefore, the cut flowers are better able to absorb smaller vacuoles after using the fusion protein YPT7. We expect that absorbing vacuoles more effective to senescence of cut flower than vacuolar enzymes.

Bacterial exopolymer Levan (β-(2,6) polyfructan) synthesized by levansucrase has attracted interest for various applications due to its low intrinsic viscosity compared with other polysaccharides. We report a novel levansucrase (Lsc) isolated from Sphingobium chunbukense DJ77 and verify its biochemical characteristics by comparative analysis of molecular docking analysis (MOE) and catalytic residue analysis. The complete sequence of the Lsc encoding gene ( lsc) was cloned under the direction of the T7 promoter and purified in an Escherichia coli BL21 (DE3) protein expression system. The enzyme activity analysis and ligand docking MOE study of S. chungbukense DJ77 Lsc revealed that Arg 77, Ser112, Arg 195, Asp196, Glu257, and Gln275 were involved in the sucrose binding and splitting as well as transfructosylation activity. A catalytic comparison of Lsc of S. chungbukense DJ77 with the results of site-directed mutational analysis indicated that Gln275 may coordinate a favorable substrate binding environment, offering broad pH resistance in the range of 5-10. The results suggest that the recombinant E. coli carrying S. chungbukense DJ77 Lsc might produce levan under the regular growth conditions with less need for pH manipulation.

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Aptamer-based paper strip sensor for detecting Vibrio fischeri was developed. Our method was based on the aptamer sandwich assay between whole live cells, V. fischeri and DNA aptamer probes. Following 9 rounds of Cell-SELEX and one of the negative-SELEX, V. fischeri Cell Aptamer (VFCA)-02 and -03 were isolated, with the former showing approximately 10-fold greater avidity (in the subnanomolar range) for the target cells when arrayed on a surface. The colorimetric response of a paper sensor based on VFCA-02 was linear in the range of 4 × 101 to 4 × 105 CFU/mL of target cell by using scanning reader. The linear regression correlation coefficient (R2) was 0.9809. This system shows promise for use in aptamer-conjugated gold nanoparticle probes in paper strip format for in-field detection of marine bioindicating bacteria.

We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein-aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody-aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.

Aptamers are biomaterials that bind to a target molecule through a unique structure, and have high applicability in the diagnostic and medical fields. To effectively utilize aptamers, it is important to analyze the structure of the aptamer binding to the target molecule, however, there are difficulties in experimentally identifying this structure. In the modern pharmaceutical industry, computer-driven docking simulations that predict intermolecular binding models are used to select candidates that effectively bind target molecules. Botulinum toxin (BoNT) is the most poisonous neurotoxin produced from the Clostridium botulinum bacteria, and BoNT/C, one of the eight serotypes, causes paralysis in livestock. In this study, the aptamers that bound to BoNT/C were screened via the systematic evolution of ligands by exponential enrichment, and the binding affinity analysis and binding model were evaluated to select optimal aptamers. Based on surface plasmon resonance analysis and molecular operating environment docking simulation, a pair of aptamers that had high binding affinity to BoNT/C and were bound to different BoNT/C sites were selected. A sandwich assay based on this aptamer pair detected the BoNT/C protein to a concentration as low as ~0.2 ng Ml&minus, 1. These results show that docking simulations are a useful strategy for screening aptamers that bind to specific targets.

Proteome profiling is most commonly based on a two-dimensional gel electrophoresis to separate and visualize proteins for identification. The success of a proteomic experiment is critically dependent on the sample preparation. The two most effective and widely used lysis buffers for extracting proteins from cells and tissues are Radio-Immunoprecipitation Assay buffer (RIPA buffer) and Urea lysis buffer. These buffers are rapid and highly efficient for cell lysis and good solublization of a wide range of proteins. In this study, the proteome profile of Rabbit muscle and tendon was analysed by 2-D gel electrophoresis using RIPA buffer and Urea lysis buffers. The variations in lysis buffers for extracting proteins clearly improve the resolution of protein identification spots as compared to conventional methods.

Osteoporosis is a common disorder of bone remodeling, caused by the imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Recently, we reported that matrix metalloproteinase-9 (MMP-9)-dependent histone H3 proteolysis is a key event for proficient osteoclast formation. Although it has been reported that several MMP-9 inhibitors, such as tetracycline and its derivatives, show an inhibitory effect on osteoclastogenesis, the molecular mechanisms for this are not fully understood. Here we show that tetracycline analogs, especially tigecycline and minocycline, inhibit osteoclast formation by blocking MMP-9-mediated histone H3 tail cleavage. Our molecular docking approach found that tigecycline and minocycline are the most potent inhibitors of MMP-9. We also observed that both inhibitors significantly inhibited H3 tail cleavage by MMP-9 in vitro. These compounds inhibited receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast formation by blocking the NFATc1 signaling pathway. Furthermore, MMP-9-mediated H3 tail cleavage during osteoclast differentiation was selectively blocked by these compounds. Treatment with both tigecycline and minocycline rescued the osteoporotic phenotype induced by prednisolone in a zebrafish osteoporosis model. Our findings demonstrate that the tetracycline analogs suppress osteoclastogenesis via MMP-9-mediated H3 tail cleavage, and suggest that MMP-9 inhibition could offer a new strategy for the treatment of glucocorticoid-induced osteoporosis.

Grinding is an indispensible step in preparation of espresso coffee. The purpose of this study was to evaluate physicochemical and sensory characteristics of espresso according to the grinding grades of coffee beans. Specifically, physicochemical characteristics according to the grinding grades of coffee beans were tested based on foam index, persistence of foam, total solids, concentration, extraction yield, pH, viscosity, total soluble solid content and density. With regard to sensory characteristics, QDA were carried out by 10 trained panels. Persistence of foam, total solids, concentration, extraction, pH and viscosity in physicochemical test showed significant differences among the three categories of the samples. In other words, the finer the size of particles was, the higher value was, at the p=0.001 value. Meanwhile, the value of foam index and total soluble solid content to the grinding grades was the highest in'fine, at the p=0.001 value. Tiger-skin …K effect in crema, an important attribute for excellent espresso coffee, was perceived by all of the panels in'fine. Also, intensity of flavor was …K perceived as to the strongest in'fine. Moreover,'fine had the sweetness to balance the acids and bitters. In conclusion, according to the …K …K grinding grades, it was identified that there were differences in the physicochemical and sensory characteristics. Furthermore, when the size of particles was'fine, it brought to the most positive result. …K