Structural stability for surface display of antigen 43 and application to bacterial outer membrane vesicles production.

Antigen 43 (Ag43) proteins, found on the outer membrane of Escherichia coli, are ß-sheets that fold into a unique cylindrical structure known as a ß-barrel. There are several known structural similarities between bacterial Ag43 autotransporters and physical components; however, the factors that stabilize the barrel and the mechanism for Ag43 passenger domainmediated translocation across the pore of the ß-barrel remain unclear. In this study, we analyzed Ag43ß-enhanced green fluorescent protein chimeric variants to provide new insights into the autotransporter Ag43 ß-barrel assembly, focusing on the impact of the α-helical linker domain. Among the chimeric variants, Ag43ß700 showed the highest surface display, which was confirmed through extracellular protease digestion, flow cytometry, and an evaluation of outer membrane vesicles (OMVs). The Ag43ß700 module offered reliable information on stable barrel folding and chimera expression at the exterior of the OMVs.

In vitro and in vivo validation of the antiviral effect of hCypA against SARS-CoV-2 via binding to the RBD of spike protein.

The novel coronavirus disease 2019 has stimulated the rapid development of new biological therapeutics to inhibit SARS-CoV-2 infection; however, this remains a challenging task. In a previous study using structural analysis, we revealed that human cyclophilin A inhibits the entry of SARS-CoV-2 into host cells by interfering with the interaction of the receptor-binding domain of the spike protein with angiotensin-converting enzyme 2 on the host cell surface, highlighting its potential for antiviral therapy. For a comprehensive experimental validation, in this study, we verified the antiviral effects of human cyclophilin A against SARS-CoV-2, including its variants, using in vitro assays and experiments on an in vivo mouse model. Human cyclophilin A demonstrated a highly effective antiviral effect, with an 85% survival rate upon SARS-CoV-2 infection. It also reduced viral titers, inflammation in the lungs and brain, and cytokine release in the serum, suggesting a controlled immune response and potentially faster recovery. Overall, our study provides insights into the potential of human cyclophilin A as a therapeutic agent against SARS-CoV-2, which should guide future clinical trials that might provide an additional therapeutic option for patients.

Correction: The emerging role of medical foods and therapeutic potential of medical food-derived exosomes.

[This corrects the article DOI: 10.1039/D3NA00649B.].

Synergistic vesicle-vector systems for targeted delivery.

With the immense progress in drug delivery systems (DDS) and the rise of nanotechnology, challenges such as target specificity remain. The vesicle-vector system (VVS) is a delivery system that uses lipid-based vesicles as vectors for a targeted drug delivery. When modified with target-probing materials, these vesicles become powerful vectors for drug delivery with high target specificity. In this review, we discuss three general types of VVS based on different modification strategies: (1) vesicle-probes; (2) vesicle-vesicles; and (3) genetically engineered vesicles. The synthesis of each VVS type and their corresponding properties that are advantageous for targeted drug delivery, are also highlighted. The applications, challenges, and limitations of VVS are briefly examined. Finally, we share a number of insights and perspectives regarding the future of VVS as a targeted drug delivery system at the nanoscale.

Pathogenic Factors and Recent Study on the Rapid Detection of Shiga Toxin-Producing Escherichia coli (STEC).

This comprehensive review delves into the pathogenicity and detection of Shiga Toxin-Producing Escherichia coli (STEC), shedding light on its various genetic and clinical manifestations. STEC originating from E. coli acquires pathogenicity through mobility and genetic elements. The pathogenicity of STEC is explored in terms of clinical progression, complications, and key toxins such as Shiga toxin (Stx). Stx1 and Stx2 are two distinct Stx types exhibiting different toxicities, with Stx2 often associated with severe diseases. This review also delves into Subtilase cytotoxin, an additional cytotoxin produced by some STEC strains. Pathogenic mechanisms of STEC, such as attaching and effacing intestinal lesions, are discussed, with a focus on roles of genetic factors. Plasmids in STEC can confer unique pathogenicity. Hybridization with other pathogenic E. coli can create more lethal pathogens. This review covers a range of detection methods, ranging from DNA amplification to antigen detection techniques, emphasizing the need for innovative approaches to improve the sensitivity and speed of STEC diagnosis. In conclusion, understanding diverse aspects of STEC pathogenicity and exploring enhanced diagnostic methods are critical to addressing this foodborne pathogen effectively. Pathology of Shiga toxin toxicity. STEC-derived Shiga toxin consists of one A subunit and five B subunits. Pathological symptoms of the disease can progress to HUS within two weeks after the onset of diarrhea. Shiga toxin intoxication is also associated with many complications, such as neurological and cardiac complications. This figure was reconstructed based on data from Bruyand et al.

The emerging role of medical foods and therapeutic potential of medical food-derived exosomes.

Medical food is consumed for the purpose of improving specific nutritional requirements or disease conditions, such as inflammation, diabetes, and cancer. It involves partial or exclusive feeding for fulfilling unique nutritional requirements of patients and is different from medicine, consisting of basic nutrients, such as polyphenols, vitamins, sugars, proteins, lipids, and other functional ingredients to nourish the patients. Recently, studies on extracellular vesicles (exosomes) with therapeutic and drug carrier potential have been actively conducted. In addition, there have been attempts to utilize exosomes as medical food components. Consequently, the application of exosomes is expanding in different fields with increasing research being conducted on their stability and safety. Herein, we introduced the current trends of medical food and the potential utilization of exosomes in them. Moreover, we proposed Medi-Exo, a exosome-based medical food. Furthermore, we comprehensively elucidate various disease aspects between medical food-derived exosomes (Medi-Exo) and therapeutic natural bionanocomposites. This review highlights the therapeutic challenges regarding Medi-Exo and its potential health benefits.

Increase CO2 recycling of Escherichia coli containing CBB genes by enhancing solubility of multiple expressed proteins from an operon through temperature reduction.

IMPORTANCE: In a previous study, we successfully engineered Escherichia coli capable of endogenous CO2 recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO2 fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO2 recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO2 recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.

Synergistic outcomes of Chlorella-bacterial cellulose based hydrogel as an ethylene scavenger.

Increasing the freshness of vegetables requires the elimination of ethylene, which can be done through chemical methods. However, the development of eco-friendly approaches is required for environmental reasons. Chlorella vulgaris (C. vulgaris) was selected as a new biological material for demonstrating an excellent performance in ethylene removal. To support C. vulgaris, bacterial cellulose (BC) produced by Gluconacetobacter hansenii (G. hansenii) was chosen due to its high water content and biodegradability. To increase BC productivity, UV-induced mutant G. hansenii was isolated, and they produced high yields of BC (9.80 ±â€¯0.52 g/L). Furthermore, comparative transcriptome analysis revealed metabolic flux changes toward UDP-glucose accumulation and enhanced BC production. BC-based hydrogels (BC hydrogels) were successfully prepared using a 2.4 % carboxymethyl cellulose (CMC) and 1 % agar mixture. We used Chlorella-BC hydrogels as an ethylene scavenger, which reduced 90 % of ethylene even when the immobilized C. vulgaris was preserved for 14 days at room temperature without media supplementation. We demonstrated for the first time the potential of BC hydrogels to integrate C. vulgaris as a sustainable ethylene absorber for green food packaging and biomass technology.

Inducing a Proinflammatory Response with Bioengineered Yeast Vacuoles with TLR2-Binding Peptides (VacT2BP) as a Drug Carrier for Daunorubicin Delivery.

Immune adjuvants have roles in immune activation for cancer therapy, and adjuvants derived from microbes have been applied. In this study, we propose the use of bioengineered vacuoles, derived from recombinant yeast with acute myeloid leukemia (AML) specificity and having a TLR-2-binding peptide (VacT2BP) on their surface, to induce a proinflammatory response as a dual-function nanomaterial for daunorubicin (DNR) delivery. Our results demonstrate that nanosized, isolated VacT2BP induced HL-60 cell-specific DNR delivery and apoptosis. Furthermore, we observed the selective release of high-mobility group box 1 from apoptotic HL-60 cells by DNR@VacT2BP. We concluded that DNR@VacT2BP exhibited target selectivity, and the indiscriminate occurrence of damage-associated molecular patterns (DAMPs) was inhibited by the VacT2BP carrier. The therapeutic efficacy of DNR@VacT2BP was confirmed in AML xenograft mice, with about 82% tumor growth inhibition. Following drug delivery, apoptotic cells and DAMPs with residual VacT2BP (apopDNR@VacT2BP) upregulated the proinflammatory immune response of macrophages. In addition, apopDNR@VacT2BP enhanced phagocytosis activity. Macrophages stimulated by apopDNR@VacT2BP suppressed cancer proliferation by about 40%. In summary, our results suggest that dual-functional vacuoles with a target-specific peptide can be a potential strategy for selective drug delivery and construction of an immune environment to fight cancer, thereby improving prognosis.

Bovine Colostrum Exosomes Are a Promising Natural Bacteriostatic Agent against Staphylococcus aureus.

Bioactive molecules and immune factors in the bovine colostrum (BC) are important elements of passive immunity that prevent bacterial infection. However, the mechanisms underlying the antimicrobial activity of BC are not fully understood. We assessed the antibacterial properties of BC-derived exosomes (BC-Exo) and found that they had bacteriostatic, anti-hemolytic, and biofilm-eradication effects on Staphylococcus aureus. Moreover, cell surface deformation and reduced ATP production were observed following BC-Exo treatment. The most reasonable explanation for this finding is that BC-Exo has a strong inhibitory effect on the oxidative phosphorylation pathway in S. aureus. We demonstrated, for the first time, that BC-Exo can exhibit clear antimicrobial activity against S. aureus. Our findings constitute an important basis for future antibiotic discovery.

Distinction of Male and Female Trees of Ginkgo biloba Using LAMP.

Ginkgo biloba is utilized as food, medicine, wood, and street trees among other things. The objective of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for gender distinction of G. biloba. Male-specific SCAR gene can be utilized to identify G. biloba gender using LAMP. The optimized LAMP conditions, temperature 60 °C, 2-mM MgSO4, and [F3/B3]:[FIP/BIP] primer ratio of 1:4 were selected as final conditions. The G. biloba SCAR LAMP displayed a sensitivity of 10 ng when amplified by concentration under the optimum conditions. Additionally, it demonstrated a particular response in male with SYBR Green I in LAMP analysis that can be a more powerful tool for field and scale-up applications. Our work represents a first attempt to identify G. biloba gender using LAMP and offers an efficient and reliable tool for roadside landscaping.

Cardiac biomarkers and detection methods for myocardial infarction.

Background: 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. Objective: We propose a biomarker and a method that can be used for the diagnosis of myocardial infarction, and an aptamer-based approach. Results: 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. Conclusion: 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. Purpose of Review: 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. Recent Findings: 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.

Structure based innovative approach to analyze aptaprobe-GPC3 complexes in hepatocellular carcinoma.

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.

Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency.

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.

Cyclophilin A-mediated mitigation of coronavirus SARS-CoV-2.

Human cyclophilin A (hCypA) is important for the replication of multiple coronaviruses (CoVs), and cyclosporine A inhibitors can suppress CoVs. The emergence of rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has sparked concerns that mutations affect the binding ability of the spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) cell receptor, affecting the severity of coronavirus disease (COVID-19). Far-western blotting and surface plasmon resonance (SPR) results revealed that hCypA interacts strongly with the viral SARS-CoV-2 receptor-binding domain (RBD), with a binding affinity of 6.85 × 10-8 M. The molecular interaction between hCypA and the viral protein interface was shown using three-dimensional structural analysis, which revealed the blocking of key residues on the RBD interface by hCypA. The RBD facilitates binding to the ACE2 receptor. The hCypA-S protein complex suppressed the binding of RBD to the ACE2 receptor, which a required event for CoV entry into the host cell. The reliability of this postulated blocking mechanism of the hCypA-SARS-CoV2 RBD complex with ACE was confirmed by SPR and molecular interaction lateral flow (MILF) strip assay, which offers the immunochromatographic signal read-outs. The emergence of new SARS-CoV-2 variants with key mutations in RBD had a negligible effect on the binding of the RBD variants to hCypA, indicating an effective mitigation strategy for SARS-CoV-2 variants. The MILF strip assay results also highlight the neutralizing effect of hCypA by effectively blocking RBD (wild type and its variants) from binding ACE2. Given the importance of hCypA in viral entry regulation, it has the potential to be used as a target for antiviral therapy.

Enhanced immune response by vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 macrophages.

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.

Enhanced Immune Response by Vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 Macrophages.

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 anti-cancer and intractable diseases using vacuoles are being actively conducted. The practical application of this study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from S. cerevisiae in RAW264.7 cells. This showed a significant increase in the production of nitric oxide 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 Nitric oxide (NO) production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin 6 and tumor necrosis factor -α. In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.

Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77.

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.

YPT7's deletion regulates yeast vacuoles' activity.

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.

Docking Simulation and Sandwich Assay for Aptamer-Based Botulinum Neurotoxin Type C Detection.

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-1. These results show that docking simulations are a useful strategy for screening aptamers that bind to specific targets.