Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Serine protease inhibitor 3 (Serpin3) from Penaeus vannamei selectively interacts with Vibrio parahaemolyticus PirAvp.

Acute Hepatopancreatic Necrosis Disease (AHPND) represents a significant challenge in the field of shrimp aquaculture. This disease is primarily caused by Vibrio parahaemolyticus strains harbouring the pVA1 plasmid encoding the PirAvp and PirBvp toxins. To combat this epidemic and mitigate its devastating consequences, it is crucial to identify and characterize the receptors responsible for the binding of these pathogenic toxins. Our studied discovered that Penaeus vannamei's Serine protease inhibitor 3 (PvSerpin3) derived from shrimp hepatopancreatic tissues could bind to recombinant PirAvp , confirming its role as a novel PirAvp -binding protein (PA BP). Through comprehensive computational methods, we revealed two truncated PirAvp -binding proteins derived from PvSerpin3 called Serpin3(13) and Serpin3(22), which had higher affinity to PirAvp than the full-length PvSerpin3. The PA BP genes were amplified from a cDNA library that was reversed from total RNA extracted from shrimp, cloned and expressed in Escherichia coli. Three PA BP inclusion bodies were refolded to obtain the soluble form, and the recovery efficacy was found to be 100% for Serpin3 and Serpin3(13), while Serpin3(22) had a recovery efficacy of roundly 50%. Co-Immunoprecipitation (co-IP) and dot blot assays substantiated the interaction of these recombinant PA BPs with both recombinant PirAvp and VPAHPND (XN89)-producing natural toxins.

Pentacyclic triterpenes from the leaves of Camellia hakodae Ninh.

Nine pentacyclic triterpene derivatives including new 3-O-cis-p-coumaroyl trichadenic acid B (1) and two new ursane-type triterpene derivatives, 11α,12-[1-(methyl)-2-(4-hydroxy-3-methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3ß-ol (2) and 11α,12-[2-(methyl)-1-(4-hydroxy-3-methoxyphenyl)ethane-1,2-dioxy]-urs-12-ene-3ß-ol (3) were isolated from the leaves of Camellia hakodae Ninh., along with six known compounds (4-9). This is the first report on pentacyclic triterpenoids from this species. New compounds 1-3 and compound 7 were tested for cytotoxic activity against four human cancer cell lines (KB; Hep-G2; Lu; MCF-7) using the MTT assay to show moderate activity.

Two new flavonoids with antimicrobial activity from the roots of Byttneria aspera Colebr. ex Wall (Malvaceae).

Two new flavonoids, 4',5,7-trihydroxy-5'-methoxy-6,8-dimethylisoflavone (1) and 2',5',7-trihydroxy-5-methoxy-6,8-dimethylflavanone (2) together with the known flavonoids 4´,5,7-trihydroxy-3´-methoxy-6.8-dimethylflavone (3), epigallocatechin (4), 4´-O-methylepicatechin (5) and quercetin (6) were isolated from the roots of Byttneria aspera. The structures of these compounds were determined by means of spectroscopic methods. Compounds 1-6 were submitted to cytotoxic activity assays against three cancer cell lines including KB, MCF7 and A549, as well as their antimicrobial activity against a panel of clinically significant microorganisms. Compound 6 showed moderate cytotoxic activity with IC50 values of 12.7, 56.9 and 17.5 µM against KB, MCF7 and A549. Interestingly, the new compounds 1 and 2 exhibits antimicrobial activity, with compound 1 displaying selective antifungal activity against Candida albicans giving an MIC value of 128 µg/mL, compared to cyclohexamide with 32 µg/mL, while compound 2 shows potent inhibition of the Gram-positive bacterium Enterococcus faecalis displaying an MIC of 64 µg/mL, compared to streptomycin with 256 µg/mL.

Construction of L-type lectin displaying Saccharomyces cerevisiae for Vibrio parahaemolyticus agglutination.

The utilization of Aga1P anchor protein in the display system for expressing heterologous proteins on the surface of Saccharomyces cerevisiae has been shown to be an ideal approach. This system has the ability to improve the expression of target proteins beyond the cell surface, resulting in increased activity and stability of the expression system. Recent studies have demonstrated that a new L-type lectin from Litopenaeus vannamei (LvLTLC1) has been found to possess the capability of agglutinating Vibrio parahaemolyticus, a pathogen responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, LvLTLC1 protein was designed to be expressed on the surface of S. cerevisiae via Aga1P anchor. The expression of LvLTLC1 protein on the surface of S. cerevisiae::pYIP-LvLTLC1-Aga1P was confirmed through the use of analytical techniques including SDS-PAGE, dot blot, and fluorescent immunoassay with LvLTC1-specific antibody. Subsequently, the newly generated yeast strain was evaluated for its ability to agglutinate V. parahaemolyticus and A. hydrophila. The obtained results indicated that S. cerevisiae expressing LvLTLC1 protein on its surface had the ability to agglutinate both AHPND-causing V. parahaemolyticus and A. hydrophila. This newly generated yeast strain could be served as a feed supplement for controlling bacteria in general and AHPND in particular.

Direct transformation of benzyl esters into esters, amides, and anhydrides using catalytic ferric(III) chloride under mild conditions.

A facile one-pot transformation of benzyl esters into esters, amides, and anhydrides is described. α,α-Dichlorodiphenylmethane and FeCl3 were employed as the chlorinating agent and catalyst respectively to convert benzyl esters into acid chloride intermediates, which directly reacted with alcohols, amines, and carboxylic acids. Various esters, amides, and anhydrides were readily obtained with high yields under mild conditions. This method is promising for the practical synthesis of esters, amides, and anhydrides from benzyl esters.

Tin(II) Chloride-Catalyzed Direct Esterification and Amidation of tert-Butyl Esters Using α,α-Dichlorodiphenylmethane Under Mild Conditions.

A practical one-pot synthesis of esters and amides from tert-butyl esters via acid chloride was developed. Reactions of tert-butyl esters with α,α-dichlorodiphenylmethane as the chlorinating agent and SnCl2 as catalyst-generated acid chloride intermediates in situ were subsequently used in reactions with a variety of alcohols and amines to afford the corresponding esters and amides in high yields under mild reaction conditions. This catalytic synthetic procedure offers an effective strategy for the facile esterification and amidation of tert-butyl esters.

Investigation of variants in genetics and virulence of Porcine Epidemic Diarrhea Virus after serial passage on Vero cells.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal virus. However, the current PEDV vaccine, which is produced from classical strain G1, offers low protection against recently emerged strain G2. This study aims to develop a better vaccine strain by propagating the PS6 strain, a G2b subgroup originating from Vietnam, on Vero cells until the 100th passage. As the virus was propagated, its titer increased, and its harvest time decreased. Analysis of the nucleotide and amino acid variation of the PS6 strain showed that the P100PS6 had 11, 4, and 2 amino acid variations in the 0 domain, B domain, and ORF3 protein, respectively, compared to the P7PS6 strain. Notably, the ORF3 gene was truncated due to a 16-nucleotide deletion mutation, resulting in a stop codon. The PS6 strain's virulence was evaluated in 5-day-old piglets, with P7PS6 and P100PS6 chosen for comparison. The results showed that P100PS6-inoculated piglets exhibited mild clinical symptoms and histopathological lesions, with a 100% survival rate. In contrast, P7PS6-inoculated piglets showed rapid and typical clinical symptoms of PEDV infection, and the survival rate was 0%. Additionally, the antibodies (IgG and IgA) produced from inoculated piglets with P100PS6 bound to both the P7PS6 and P100PS6 antigens. This finding suggested that the P100PS6 strain was attenuated and could be used to develop a live-attenuated vaccine against highly pathogenic and prevalent G2b-PEDV strains.

Molecular Dynamics Simulations Reveal Novel Interacting Regions of Human Prion Protein to Brucella abortus Hsp60 Protein.

The distinctive morphology characteristics of microfold cells (M cells) allow the vaccine antigen not only to interact with immune cells directly, but also to effectively stimulate mucosal immune responses via receptors on its apical surface. Human prion protein, a transmembrane receptor for Brucella abortus Hsp60, is highly expressed on the M cell surface. Nonetheless, this protein tends to express in inclusion body in prokaryotic hosts. In this study, the shorter interacting regions of human prion protein were identified via computational methods such as docking and molecular dynamics simulations to minimize its aggregation tendency. The computational calculations revealed three novel human prion protein-interacting regions, namely PrP125, PrP174, and PrP180. In accordance with in silico prediction, the biologically synthesized peptides fusing with GST tag demonstrated their specific binding to Hsp60 protein via pull-down assay. Hence, this finding laid the groundwork for M-cell targeting candidate validation through these newly identified interacting regions.

Flagellin from Salmonella enteritidis Enhances the Immune Response of Fused F18 from Enterotoxigenic Escherichia coli.

F18 plays an important role in helping Enterotoxigenic Escherichia coli (ETEC) binds to specific receptors on small intestinal enterocytes, followed by secreting of toxins causing diarrhea in post-weaning piglets (post-weaning diarrhea, PWD). However, the F18 subunit vaccine is not sufficient to stimulate an immune response that can protect weaning pigs from F18-positive ETEC (F18+ETEC). Recently, a body of evidence shows that flagellin protein (FliC) helps to increase the immunity of fused proteins. Therefore, in this study, we combined FliC with F18 to enhance the immune response of F18. The f18 gene was obtained from F18+ETEC, then was fused with the fliC gene. The expression of recombinant FliC-F18 protein was induced by Isopropyl-beta-D-Thiogalactopyranoside (IPTG). The purified protein was tested in vivo in mouse models to evaluate the immunostimulation. Results showed that the fusion of FliC and F18 protein increased the production of anti-F18 antibodies. Besides, the anti-F18 antibody in the collected antiserum specifically identified F18+ETEC. This result provides proof-of-concept for the development of subunit vaccine to prevent PWD using F18 antigen.

Visible-light-driven SAQS-catalyzed aerobic oxidative dehydrogenation of alkyl 2-phenylhydrazinecarboxylates.

Azo compounds are useful molecules with a wide range of applications in organic chemistry. Here, a novel visible-light-driven oxidative dehydrogenation of alkyl 2-phenylhydrazinecarboxylates is used for the synthesis of azo compounds. This synthetic method was conducted under an aerobic environment with mild reaction conditions. Sodium anthraquinone sulfonate (SAQS) was employed as the crucial organic photocatalyst in a visible-light-driven reaction to generate various azo compounds in high yields. In addition, aerobic transformation of hydrazobenzenes to azobenzenes using visible light was successfully carried out under SAQS-mediated reaction conditions. This procedure is a practical and promising synthetic approach to produce useful azo compounds.

Fabrication of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND) is a contagious disease for the shrimp cultivation, thus early detection of disease is an unmet need. This present study documented for the first time a simple lateral flow immunoassay (LFIA) strip using polyclonal antibodies was created for the rapid detection both of PirAvp and PirBvp protein simultaneously. LFIA method based on the principle of sandwich format. The label is the colloidal gold. The polyclonal antibody was conjugated with the colloidal gold acting as biorecognition element and coated onto the conjugate pad. The rabbit anti-Pirvp, anti-PirBvp antibodies, and goat anti-rabbit IgG antibody were separately sprayed onto a nitrocellulose membrane to form two test lines and one control line, respectively. The appearance of red bands at the control line and the test line indicated a positive result. A single coloured band at control area indicated a negative result. The limit of detection of LFIA was found to be 125 ng, which could be visually detected by naked eye within 15 min. There was no cross-reactivity observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. The developed test strip could be a game changer for early and in situ diagnosis of AHPND.

Recombinase-free cloning (RFC) protocol for gene swapping.

Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC). This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in E. coli host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories.

Facile tin(II)-catalyzed synthesis of N-heterocycles from dicarboxylic acids and arylamines.

A novel efficient transformation reaction of dicarboxylic acids into N-aryl-substituted azacycles is described. In this synthetic procedure, both catalytic SnCl2 and phenylsilane were used as crucial reagents for reaction of arylamines with dicarboxylic acids to produce the desired azacycles. Using this SnCl2-catalyzed synthetic method, various N-aryl-substituted azacycles were successfully prepared from arylamines with dicarboxylic acids in high yield. This practical synthetic method using catalytic SnCl2 can provide a useful approach for preparation of the desired azacycle products from many available dicarboxylic acid starting materials.

Prokaryotic expression of chimeric GFP-hFc protein as a potential immune-based tool.

GFP is an old-yet-powerful protein marker, which has been widely used in molecular biotechnology due to its capacity of exhibiting bright green fluorescence when exposed to ultraviolet light. The hFc region of IgG antibodies is a specific binding ligand of expressed receptors on immune cells with well-known cellular-associated functions like opsonization and phagocytosis. In this present study, we proceeded to fuse gfp-hfc gene into pET-28a to create a recombinant pET-28a-gfp-hfc vector. The expression of GPF-hFc was induced by IPTG and confirmed using SDS-PAGE and followed by Western blot probed with 6xHis antibodies. This chimeric protein was utilized in specific binding experiments with protein A/G-coated magnetic beads using a fluorescence microscope. Due to its fluorescence and binding ability, GFP-hFc could be used as a model molecule for monitoring molecule detection studies, tracking nanoparticle migration and distribution, or stimulating immune responses.

Production of polyclonal antibody against the recombinant PirBvp protein of Vibrio parahaemolyticus.

BACKGROUND: Acute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirAvp and PirBvp encoded in pVA1 plasmid. The polyclonal antibodies against PirBvp protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND. RESULTS: In this study, PirBvp gene was amplified, cloned, and expressed in E. coli. The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirBvp (rPirBvp) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirBvp, and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot. CONCLUSION: This study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.

Generation and evaluation of polyclonal antibodies specific for ToxA from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp.

Acute Hepatopancreatic Necrosis Disease (AHPND) is a newly emerging shrimp disease with mortality up to 100 percent caused by Vibrio parahaemolyticus which carries a plasmid encoding for two toxins, ToxA and ToxB. In 2013, the Global Aquaculture Alliance (GAA) estimated shrimp farming decline in Asia accounted for 1-billion US dollar lost. Currently, diagnosis using PCR method does not meet the demand of in situ detection, which is based on antigen-antibody interaction, has not been developed yet. In this present study, we proceeded to create the toxin and its antibody for lateral flow development. First, recombinant toxin ToxA was generated by gene manipulation. After that, purified ToxA was used to immunize rabbits. Finally, antisera from rabbits and protein-A purified antibodies were evaluated for titer, specificity, and detection threshold. Results showed that recombinant ToxA was overexpressed in soluble fraction at 37oC with 1mM IPTG. Purification by affinity chromatography was able to isolate recombinant ToxA with the purity up to 94.49%. In ELISA experiment, the immunized antisera reached a titer of up to 1/5,210,000 with 1µg/ml of antigen, and detection threshold was 100ng recombinant toxin. After purification, the detection threshold of purified polyclonal antibodies was 25ng toxin per dot. These results laid a groundwork for the development of AHPND detection kit based on antigen - antibody interactions.

Improving silymarin oral bioavailability using silica-installed redox nanoparticle to suppress inflammatory bowel disease.

Chronic inflammatory diseases such as inflammatory bowel diseases (IBD), which are strongly related to the overproduction of reactive oxygen species (ROS), have become more threatening to health. Silymarin is an active compound with the effect of expressing anti-inflammatory activity; however, it exhibits poor bioavailability due to the rapid metabolism and secretion, low permeability across the intestinal epithelial cells, and poor water solubility. In this study, we developed silica-containing redox nanoparticles (siRNP) with 50-60 nm in diameter to improve the bioavailability of silymarin by improving its uptake into the bloodstream and delivery to the targeted tissues of the colon. Silymarin-loaded siRNP (SM@siRNP) significantly increased the antioxidant capacity and anti-inflammatory efficacy in vitro by scavenging 2,2-diphenyl-1-picrylhydrazyl free radical and suppressing nitric oxide and pro-inflammatory cytokines as compared to the other treatments such as free silymarin, siRNP, and silymarin-loaded si-nRNP (the control nanoparticle without ROS scavenging property). Orally administered SM@siRNP significantly improved the bioavailability of silymarin and its retention in the colonic mucosa. The anti-inflammatory effects of SM@siRNP were also investigated in dextran sodium sulfate (DSS)-induced colitis in mice and it was observed that SM@siRNP treatment significantly improved the damage in the colonic mucosa of DSS colitis mice as compared to the other treatments. The results in this study indicate that SM@siRNP is a promising nanomedicine for enhancing the anti-inflammatory activity of silymarin and has a high potential for the treatment of IBD.

Recombinant Human SCARB2 Expressed in Escherichia coli and its Potential in Enterovirus 71 Blockage.

Hand, foot and mouth disease is a common viral infectious disease caused by enteroviruses, including coxsackie A16 (CVA16) and enterovirus 71 (EV71). HFMD can cause severe symptoms in children which can be fatal. Human scavenger receptor class B member 2 (SCARB2) is a cellular receptor for EV71 and CVA16, providing a potential approach for preventing EV71 infection and transmission. In this present study, we constructed and assessed the potential of recombinant SCARB2, using E. coli expression system. To generate this construct, scarb2 gene was cloned into pET22b vector and expressed in E. coli BL21 (DE3). The expression of SCARB2 was induced by 0.1 mM IPTG and analyzed using SDS-PAGE, followed by Western blot. Expressed SCARB2 was in inclusion bodies and refolded to obtain the soluble form with recovery efficacy of 100%. This recombinant protein was then validated for binding with EV71 via indirect ELISA in two different pHs (7.4 and 5.5), which partially revealed the mechanism of virus-receptor interaction. These results envisaged potential applications for utilizing recombinant SCARB2 in preventing the virus transmission.

Practical one-pot amidation of N-Alloc-, N-Boc-, and N-Cbz protected amines under mild conditions.

A facile one-pot synthesis of amides from N-Alloc-, N-Boc-, and N-Cbz-protected amines has been described. The reactions involve the use of isocyanate intermediates, which are generated in situ in the presence of 2-chloropyridine and trifluoromethanesulfonyl anhydride, to react with Grignard reagents to produce the corresponding amides. Using this reaction protocol, a variety of N-Alloc-, N-Boc-, and N-Cbz-protected aliphatic amines and aryl amines were efficiently converted to amides with high yields. This method is highly effective for the synthesis of amides and offers a promising approach for facile amidation.