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.

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.

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.

Pneumatically Driven Microfluidic Platform and Fully Automated Particle Concentration System for the Capture and Enrichment of Pathogens.

In this study, we developed a pneumatically driven microfluidic platform (PDMFP) operated by a fully automated particle concentration system (FAPCS) for the pretreatment of micro- and nano-sized materials. The proposed PDMFP comprises a 3D network with a curved fluidic chamber and channel, five on/off pneumatic valves for blocking fluid flow, and a sieve valve for sequential trapping of microbeads and target particles. Using this setup, concentrated targets are automatically released into an outlet port. The FAPCS mainly comprises solenoid valves, glass reservoirs, a regulator, pressure sensor, main printed circuit board, and liquid crystal display touch panel. All pneumatic valves in the microfluidic platform as well as the working fluids in the glass reservoirs are controlled using FAPCS. The flow rate of the working fluids is measured to demonstrate the sequential programed operation of the proposed pretreatment process using FAPCS. In our study, we successfully achieved rapid and efficient enrichment using PDMFP-FAPCS with fluorescence-labeled Escherichia coli. With pretreatment-10 min for the microbead concentration and 25 min for target binding-almost all the target bacteria could be captured. A total of 526 Gram-negative bacteria were attached to 82 beads, whereas Gram-positive bacteria were attached to only 2 of the 100 beads. Finally, we evaluated the PDMFP-FAPCS for SARS-CoV-2 receptor-binding domain (RBD)-based outer membrane vesicles (OMVs) (RBD-OMVs). Specific probes involved in PDMFP-FAPCS successfully isolated RBD-OMVs. Thus, PDMFP-FAPCS exhibits excellent enrichment of particles, including microbes and nanovesicles, and is an effective pretreatment platform for disease diagnosis and investigation.

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.

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.

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP).

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

Multifaceted effects of milk-exosomes (Mi-Exo) as a modulator of scar-free wound healing.

Scar-free treatment is complex involving many cells in the human body but a very elaborate reaction. This process demands regulation of various growth factors on behalf of TGFß3 around the damaged tissue, and it is also important to protect cells from inflammatory reactions and oxidative stress to avoid abnormalities. Here, we focused on bovine derived milk exosomes (Mi-Exo) and their scar-free healing potential. The physiological properties (size and shape), biological markers (TSG101 and Bta-miR2478) and stability on storage of Mi-Exo were analyzed. Mi-Exo exhibited significant NP (number of Mi-Exo particles)-dependent scavenging activity in ABTS assay. In addition, Mi-Exo suppressed the expression of pro-inflammatory mediators, IL-6 and TNFα, and pro-inflammatory chemokines, COX-2 and iNOS. This study showed that cell migration was significantly inhibited in a Mi-Exo NP-dependent manner. We also evaluated the expression of TGFß1 and TGFß3 on the basis of mRNA and protein levels. Furthermore, the role of functional behavior of Mi-Exo in TGFß1 maturation was explored. This is the first study to demonstrate that Mi-Exo may target the TGFß signaling pathway, which plays important roles in scar-free wound healing.

Zika virus lateral flow assays using reverse transcription-loop-mediated isothermal amplification.

Owing to the global spread of the Zika virus (ZIKV) infection, field-ready diagnostics are urgently warranted. In this study, we sought to detect ZIKV using reverse transcription loop-mediated isothermal amplification (RT-LAMP). Briefly, we performed and optimized ZIKV RT-LAMP for the analysis of biological samples (PBS, urine, and plasma). Based on our findings, this method could detect ZIKV RNA in 40 min at 63 °C without any off-target amplification. After performing specificity tests using BtsI restriction enzyme digestion, the feasibility of ZIKV RT-LAMP was determined via end-point detection with different sample matrices. Thereafter, a lateral flow assay (LFA) was conducted to directly detect the ZIKV RT-LAMP products. Based on the LFA reaction, hybridization occurred between the AuNPs:polyadenylated (polyA10)-ZIKV probe and the LAMP amplicons. Subsequently, we optimized the assay parameters, including the concentration of AuNPs and migration matrices (glass fiber and nitrocellulose membrane). By employing a specific AuNP:polyA10-ZIKV LAMP probe, we could demonstrate the purpose and utility of primary and secondary antibodies. Owing to LFA, the resultant ZIKV RT-LAMP products were rapidly and simply assayed in less than 5 min. Further, no preparation step was required to achieve LAMP-probe hybridization, highlighting the utility of this method for field-ready ZIKV diagnosis. Collectively, our findings suggest that ZIKV RT-LAMP combined with LFA could serve as a rapid, accurate, and independent point-of-care detection method for preventing ZIKV outbreaks.

Poly-adenine-Coupled LAMP Barcoding to Detect Apple Scar Skin Viroid.

Apple Scar Skin Viroid (ASSVd), a nonprotein coding, circular RNA pathogen is relatively difficult to detect by immunoassay. We report here a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to improve selectivity for diagnostic use in detecting ASSVd in plants. ASSVd RT-LAMP was accelerated using loop primers and was found to be highly sensitive with a detection limit of 104 copies of cDNA-ASSVd within 30 min. Real-time LAMP and melting curve analysis could differentiate between the true-positive LAMP amplicons and false-positive nonspecific primer amplification products. The optimized RT-LAMP was then followed by the addition of nonthiolated AuNP:poly-adenine (A10)-ASSVd LAMP barcodes, showing a high authentication capacity with colorimetric changes. This type of barcoding assay is a potential alternative for rapid and multiple viroid diagnosis, providing for visible sensing in the field that can be applied to viroid-free planting.