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.

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.

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.

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.

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.

Simultaneously targeting nitrocellulose and antibody by a dual-headed protein.

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix - a protein that has a strong affinity to nitrocellulose membrane and protein A - a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.

Third-degree burn mouse treatment using recombinant human fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein that has major roles in wound healing, tissue repair, and regeneration. This therapeutic protein is widely used for burn treatment because it can stimulate cell proliferation and differentiation, angiogenesis, and extracellular matrix remodeling. In this study, we developed a simple method using a controlled heated brass rod to create a homogenous third-degree burn murine model and evaluated the treatment using recombinant human FGF-2 (rhFGF-2). The results indicated that the wound area was 0.83 ± 0.05 cm2 and wound depth was 573.42 ± 147.82 µm. Mice treated with rhFGF-2 showed higher rates of wound closure, granulation tissue formation, angiogenesis, and re-epithelialization than that of phosphate-buffered saline (PBS)-treated group. In conclusion, our lab-made rhFGF-2 could be a potentially therapeutic protein for burn treatment as well as a bioequivalent drug for other commercial applications using FGF-2.

Practical direct synthesis of N-aryl-substituted azacycles from N-alkyl protected arylamines using TiCl4 and DBU.

A novel transformation of N-alkyl protected arylamines and cyclic ethers into N-aryl substituted azacycles is described. Alkyl groups have been used for the protection of amines in organic syntheses. In this synthesis, N-alkyl protected arylamines were reacted with cyclic ethers in the presence of TiCl4 and DBU, crucial reagents affording five- and six-membered azacycles. In particular, utilization of the novel TiCl4/DBU-mediated reaction allows various N-alkyl protected arylamines such as N-methyl-, N-ethyl-, N-isopropyl, and N-tert-butyl arylamines to be readily converted into N-aryl substituted azacycles in high yields. This practical approach using various N-alkyl arylamines leads to the efficient preparation of azacycles.

Practical preparation of diphenylmethyl ethers from 2-diphenylmethoxypyridine using catalytic iron(iii) chloride.

A novel facile synthetic method for producing diphenylmethyl (DPM) ethers from 2-diphenylmethoxypyridine was developed. A variety of DPM ethers was successfully achieved with high yield via treatment of alcohols with 2-diphenylmethoxypyridine in the presence of catalytic FeCl3. The procedure is a practical and efficient synthetic procedure to protect various alcohols, and it can be applied to prepare bioactive compounds.

Encapsulation of tagitinin C in liposomes coated by Tithonia diversifolia pectin.

Tagitinin C, a sesquiterpene lactone compound from Tithonia diversifolia, is known to have various bioactivities including anticancer effects. The disadvantages of tagitinin C which make its therapeutic application limited are low water solubility and poor stability. In this work, we encapsulated tagitinin C in the form of liposomal nanoparticles to overcome its limitations and evaluated its anticancer activity. Liposomes were prepared using phosphatidylcholine, tween 80 and with/without T. diversifolia pectin. Tagitinin C liposomes exhibited small sizes, a range of 252-311 nm, and negative surface charges. Liposomes coated by T. diversifolia pectin resulted in a slightly slower release rate than pectin-uncoated liposomes. The cytotoxicity of tagitinin C in pectin-coated liposomes was more pronounced than that in liposomes without pectin. Tagitinin C in liposomes showed significantly increased toxicity against human lung cancer cells LU-1 in comparison with DMSO-tagitinin C. Therefore, tagitinin C liposomes demonstrate significant potential as delivery systems for cancer therapy.

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.

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.

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.

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.

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.

Measles virus modulates dendritic cell/T-cell communication at the level of plexinA1/neuropilin-1 recruitment and activity.

Measles virus (MV)-infected DC fail to promote T-cell expansion, and this could explain important aspects of measles immunosuppression. The efficiency of the immune synapse (IS) is determined by the formation of stable, stimulatory conjugates involving a spatially and timely controlled architecture. PlexinA1 (plexA1) and its co-receptor neuropilin (NP-1) have been implicated in IS efficiency, while their repulsive ligand, SEMA3A, likely acts in terminating T-cell activation. Conjugates involving MV-infected DC and T cells are unstable and not stimulatory, and thus we addressed the potential role of plexA1/NP-1 and semaphorins (SEMAs) in this system. MV does not grossly affect expression levels of plexA1/NP-1 on T cells or DC, yet prevents their recruitment towards stimulatory interfaces. Moreover, MV infection promoted early release of SEMA3A from DC, which caused loss of actin based protrusions on T cells as did the plexA4 ligand SEMA6A. SEMA3A/6A differentially modulated chemokinetic migration of T cells and conjugation with allogeneic DC. Thus, MV targets SEMA receptor function both at the level of IS recruitment, and by promoting a timely inappropriate release of their repulsive ligand, SEMA3A. To the best of our knowledge, this is the first example of viral targeting of SEMA receptor function in the IS.

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.

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.

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.

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.