Transient Co-Expression of Bioactive Murine Interferon-Gamma and HBsAg in Tobacco and Lettuce Leaves.

Background: The synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-γ) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model. Methods: Monocistronic and bicistronic cassettes, carrying genes encoding mIFN-γ and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5' leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-γ in different constructs and HBsAg was tested by ELISA. Murine IFN-γ was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow. Results: Extracts of agroinfiltrated leaves contained recombinant mIFN-γ and HBsAg proteins at about 14 unit/mg and 50 ng/mg of soluble protein, respectively. Subsequently, mIFN-γ was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence. Conclusion: The co-expression of recombinant HBsAg and mIFN-γ using TEV 5' leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-γ in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications.

High prevalence of antibiotic resistance and biofilm formation in Salmonella Gallinarum.

Background and Objectives: Antibiotic resistance is an indicator of the passively acquired and circulating resistance genes. Salmonella Gallinarum significantly affects the poultry food industry. The present study is the first study of the S. Gallinarum biofilm in Iran, which is focused on the characterization of the S. Gallinarum serovars and their acquired antibiotic resistance genes circulating in poultry fields in central and northwestern Iran. Materials and Methods: Sixty isolates of S. Gallinarum serovar were collected from feces of live poultry. The bacteria were isolated using biochemical tests and confirmed by Multiplex PCR. Biofilm formation ability and the antibacterial resistance were evaluated using both phenotypic and genotypic methods. The data were analyzed using SPSS software. Results: According to Multiplex PCR for ratA, SteB, and rhs genes, all 60 S. Gallinarum serovars were Gallinarum biovars. In our study, the antibiotic resistance rate among isolated strains was as follows: Penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). All isolates were susceptible to imipenem, ertapenem, ceftriaxone, ceftazidime, and ceftazidime+clavulanic acid. All sixty isolates did not express the resistance genes IMP, VIM, NDM, DHA, blaOXA48, and qnrA. On the other hand, they expressed GES (85%), qnrB (75%), Fox M (70%), SHV (60%), CITM (20%), KPC (15%), FOX (10%), MOXM (5%), and qnrS (5%). All S. Gallinarum isolates formed biofilm and expressed sdiA gene. Conclusion: Considering that the presence of this bacteria is equal to the death penalty to the herd, the distribution of resistance genes could be a critical alarm for pathogen monitoring programs in the region. This study showed a positive correlation between biofilm formation and 50% of tested resistance genes. Also, it was found that the most common circulating S. gallinarum biovars are multidrug-resistant.

Antibacterial and Virucidal Evaluation of Ultrafine Wire Arc Sprayed German Silver Coatings.

Copper and its alloys are known as antimicrobial agents that can be used in public places; however, pure copper has a low wear resistance and tends to lose its gloss relatively fast and stainless steel is still more desirable because of its mechanical properties and stable appearance. In this research, German silver coatings, a copper-nickel alloy, are studied as a superior alternative for pure copper coatings. German silver coating on mild steel substrates and stainless steel with two different surface roughnesses was prepared and placed into water bath up to 6 months to investigate the corrosion and exposure effects on the antibacterial behavior. A range of techniques was used to study the microstructure, surface morphology and mechanical properties such as microhardness, coating bonding adhesion, surface roughness and wettability of the coating. Colony count method was used to measure the antibacterial properties, and samples were tested against influenza A virus to evaluate the virucidal activity. The coating thickness was around 130 µm and contained 15% pores and oxides with splats forming inside the coating structure. Inside each splat, columnar grains could be seen with an average of 700 nm width and 4 µm length. The bonding strength of the coating was about 15 MPa, the hardness of coatings was about 180 HV, and the average surface roughness of the as-sprayed samples was about 10 µm. German silver coatings can destroy both Staphylococcus aureus and Escherichia coli by more than 90% after 6 h of exposure time, and it also has a high-level of virucidal activity against influenza A virus after 2 h exposure time. Antibacterial behavior did not show any significant changes after 6 months of immersing samples in water bath. Thus, thermally sprayed German silver coatings exhibited silvery color for a long period of time, while its antimicrobial efficiency was comparable to pure copper coatings. Supplementary Information: The online version contains supplementary material available at 10.1007/s11666-022-01528-4.

In silico design and in vivo evaluation of two multi-epitope vaccines containing build-in adjuvant with chitosan nanoparticles against uropathogenic Escherichia coli.

BACKGROUND: Urinary pathogenic Escherichia coli (UPEC) is one of the most important bacterial causes of urinary tract infections (UTIs). Rising antimicrobial resistance and serious clinical challenges such as persistent and recurrent UTIs make it a serious public health concern. Therefore, preventative approaches such as vaccinations are required. METHODS: In this study, we selected three conserve and protective antigens (FdeC, Hma and UpaB) and also subunit B of cholera toxin (as build-in adjuvant) to design two multi-epitope vaccines (construct B containing B cell epitopes and construct T containing T epitopes) using different bioinformatics methods. The expression of the recombinant protein was performed using the BL21(DE3)/pET28 expression system and purified through a Ni-NTA column. Vaccine proteins were encapsulated in chitosan nanoparticles (CNP) based on ionic gelation via a microfluidic system. Mice were immunized intranasally with different vaccine formulations. Antibody responses and also cytokine expression (IFN-γ and IL-4) were measured by ELISA and real-time PCR respectively. The effectiveness of immune responses was assessed by bladder challenge. RESULTS: Based on the in silico study, construct B and construct T have high confidence value and stable structure in vivo. High yield expression of both constructs was confirmed by SDS-PAGE and western blot assay. Immunization of mice with construct B induced strong Th2 (IgG1 and IL4) responses and construct T shift immune responses to Th1 (IFNγ and IgG2a). Vaccine protein-encapsulated CNP elicited higher levels of antibodies and cell-mediated responses than the vaccine proteins alone. CONCLUSIONS: The results of this study suggest that intranasal administration of the construct B has the potential to enhance humoral immunity and construct T has the potential to stimulate cellular immunity. In addition, the combination of CTB as a build-in adjuvant and CNP can be proposed as a potent adjuvant for the development of a novel vaccine against UTI.

In Silico Design and In Vitro Evaluation of Some Novel AMPs Derived From Human LL-37 as Potential Antimicrobial Agents for Keratitis.

The human body produces two classes of antimicrobial peptides (AMPs), namely defensins and cathelicidins. In this study, a novel decapeptide (Catoid) and its dimer (Dicatoid) based on human cathelicidin (LL-37) have been designed by bioinformatics tools to be used in the treatment of bacterial keratitis. After the selection and synthesis of peptide sequences, their antimicrobial activities against the standard and resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus were evaluated. This test was performed with LL-37, gentamicin, ciprofloxacin, amikacin, and penicillin for a more accurate comparison. Furthermore, the cytotoxicity levels of the specified compounds on fibroblast cells and bovine corneal endothelial cells were investigated. The results demonstrated that the designed peptides had a superior antimicrobial activity on P. aeruginosa, compared to LL-37; however, Catoid had a better effect on the S. aureus strain. Additionally, a significant achievement is the very low toxicity level of Catoid and Dicatoid on the human skin fibroblast cell line and bovine corneal endothelial cells, compared to that of LL-37 as the initial design model.

Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.

Time-dependent behavior of the Staphylococcus aureus biofilm following exposure to cold atmospheric pressure plasma.

OBJECTIVES: Formation of Staphylococcus aureus biofilm leads to persistent infection in tissue or on exter-nal and indwelling devices in patients. Cold atmospheric plasma (CAP) is used for eradication of bacterial biofilms and it has diverse applications in the healthcare system. However, there is not sufficient information on the behavior of biofilms during the CAP exposure period. MATERIALS AND METHODS: Pre-established S. aureus biofilms were exposed to CAP for 0 to 360 sec, then subjected to washing steps and sonication. Subsequently, biomass, number of colonies, vitality of bacteria, structure of colonies, size of produced particles, and viability of bacteria were evaluated by different assays including crystal violet, colony-forming unit, MTT, scanning electron mi-croscopy, confocal laser scanning microscopy, and dynamic light scattering assays. RESULTS: The results showed that the strength of biomass increased in the first 60 sec, then decreased to less than no-CAP treated controls. Moreover, short CAP exposure (≤60 sec) ehances the fusion of the biofilm extracellular matrix and other components, which results in preservation of bacteria during ultra-sonication and washing steps compared with control biofilms. The S. aureus biofilm structure only breaks down following more CAP exposure (> 90 sec) and demolition. Interestingly, the 60 sec CAP exposure could cause the fusion of biofilm compo-nents, and large particles are detectable. CONCLUSION: According to this study, an inadequate CAP exposure period prevents absolute eradication of biofilm and enhances the preservation of bacteria in stronger biofilm compartments.

Identification and characterization of the type II toxin-antitoxin systems in the carbapenem-resistant Acinetobacterbaumannii.

Carbapenem -resistant A. baumannii (CRAB) is a major cause of both community-associated and nosocomial infections that are difficult to control and treat worldwide. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The functional diversity and ubiquitous distribution in bacterial genomes are causing significant attention toward TA systems in bacteria. However, there is no enough information on the prevalence and identity of TA systems in CRAB clinical isolates. This study aimed to identify type II toxin-antitoxin systems in carbapenem-resistant A. baumannii (CRAB) isolates. A total of 80 A. baumannii isolates were collected from different clinical samples. Antibiotic resistance patterns of A. baumannii isolates were evaluated phenotypically and genetically. The frequency of type II TA genes was evaluated in CRAB isolates using PCR. Moreover, the expression level of the most prevalent TA encoding genes in some clinical isolates were evaluated by RT-qPCR. To determine whether the SplT and SplA are functional, the growth of E. coli BL21 cells (DE3/pLysS) harboring pET28a, pET28a-splTA, and pET28a-splT were analyzed by kill-rescue assay. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin and levofloxacin, whereas, 72%, 81% and 87% were resistant to amikacin, carbapenems and tetracycline, respectively. The cheTA in 47 isolates (72.5%) and splTA in 39 isolates (60%) of 65 isolates were the most common genes encoding type II TA among CRAB isolates. RT-qPCR demonstrated that cheTA and splTA transcripts are produced in the clinical isolates. There was a significant correlation between the presence of splTA genes and blaOXA-24 in CRAB isolates. Over-expression of the splT gene in E. coli results in inhibition of bacterial growth, whereas co-expression of splTA effectively restores the growth. This study presents the first identification of the type II TA systems among the carbapenem -resistant A. baumannii isolates, in Iran.

Expression of BMP2-Hydrophobin fusion protein in the tobacco plant and molecular dynamic evaluation of its simulated model.

Plants are one of the ideal models for therapeutic protein production, however the recombinant protein purification problems in them must be overcome. Bone Morphogenetic Protein2 (BMP2) is employed for the restoration and construction of bone tissues. Hydrophobin is a fungal based protein with high hydrophobic characteristics. Due to this specificity, it is suitable for the purification of chimer protein from complex solutions when is fused to a protein utilizing an aqueous two-phase (A2P) technique. The plant optimized mature human BMP2 gene was designed and evaluated by in silico method. This process involves simulating molecular dynamics using the RMSD, RMSF and Gyration radius indexes. The synthesized Hyd-BMP2 gene was cloned into a pTRAkc-ERH plasmid and Transferred into Agrobacterium (Gv3101). The Nicotiana benthamiana plant leaves were co-agroinfiltrated with HA-Hyd-BMP2 and P19-pCambia1304 containing silencing suppressor. After purification of plant extract utilizing the A2P method, the sample was subjected to SDS-PAGE and Western-blot. By in silico study, the simulated fusion protein profitably shows reasonable protein compactness and the effect of amino acid substitution on protein-protein interaction is not remarkable. Western-blotting using anti HA tag has shown that the A2P technique partially purified the two 22 kDa and 44 kDa forms of Hydrophobin-BMP2. These results confirmed the presence of monomer and dimer forms of Hydrophobin-BMP2 proteins. Moreover, the expression level of the protein using P19 silencing suppressor increased six times and to 0.018% as shown by ELISA. This study presents a fast and easy technique for the purification of transient expressed pharmaceutical proteins from plants.

Immobilization of recombinant lysostaphin on nanoparticle through biotin-streptavidin conjugation technology as a geometrical progressed confrontation against Staphylococcus aureus infection.

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.

In silico Analysis of Pasteurella multocida PlpE Protein Epitopes As Novel Subunit Vaccine Candidates.

Background: Pasteurella multocida is a Gram-negative, non-motile, non-spore forming, and aerobic/anaerobic cocobacillus known as the causative agent of human and animal diseases. Humans can often be affected by cat scratch or bite, which may lead to soft tissue infections and in rare cases to bacteremia and septicemia. Commercial vaccines against this agent include inactivated, live attenuated, and non-pathogenic bacteria. Current vaccines have certain disadvantages such as reactogenicity or reversion to virulence. Therefore, the aim of this study was to reach a multi-epitope vaccine candidate that could be serotype independent and covers most incident serotypes of P. multocida. Methods: In this study, reverse vaccinology strategy was used to identify potentially immunogenic and protective epitopes. First, multiple alignments of different sequences of Pasteurella lipoprotein E (PlpE) from various serotypes of P. multocida were analyzed to identify the conserved regions. Bioinformatics tools were then applied to predict and select epitopes for further studies. Results: Three different conserved immunogenic regions were selected according to the selected criteria, and their various sequential orders were evaluated structurally by in silico tools to find the best order. Conclusion: In searching the epitopes of PlpE to design a new vaccine candidate against pasteurellosis, we found the region 1 + region 2 + region 3 (without any linker between regions) of epitope, including the regions of PlpE protein of P. multocida, as the appropriate serotype independent vaccine candidate against pasteurellosis.

PlpE Epitopes of Pasteurella multocida Fusion Protein as Novel Subunit Vaccine Candidates.

BACKGROUND: Pasteurella multocida is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate. MATERIALS AND METHODS: In this study, the fusion protein (PlpE1 + 2 + 3) and full PlpE genes (PlpE-Total) were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 Escherichia coli. Overlap polymerase chain reaction (PCR) using different primers for 5' and 3' end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning. RESULTS: Cloning of both PlpE1 + 2 + 3 and PlpE-Total into the pET28a vector and their transform into the BL21 (DE3) E. coli host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl ß-d-1-thiogalactopyranoside) concentration. CONCLUSION: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 E. coli have resulted in sufficient expression, which shows that Top10 E. coli may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens.

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM. The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.

Pasteurella multocida Vaccine Candidates: A Systematic Review.

Pasteurella multocida (P. multocida) is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by P. multocida include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome.

Biomedical applications of yeasts - a patent view, part two: era of humanized yeasts and expanded applications.

INTRODUCTION: Yeast humanization, ranging from a simple point mutation to substitution of yeast gene(s) or even a complete pathway by human counterparts has enormously expanded yeast biomedical applications. AREAS COVERED: General and patent-oriented insights into the application of native and humanized yeasts for production of human glycoproteins (gps) and antibodies (Abs), toxicity/mutagenicity assays, treatments of gastrointestinal (GI) disorders and potential drug delivery as a probiotic (with emphasis on Saccharomyces bulardii) and studies on human diseases/cancers and screening effective drugs. EXPERT OPINION: Humanized yeasts cover the classical advantageous features of a 'microbial eukaryote' together with advanced human cellular processes. These unique characteristics would permit their use in the production of functional and stable therapeutic gps and Abs in lower prices compared to mammalian (CHO) production-based systems. Availability of yeasts humanized for cytochrome P450 s will expand their application in metabolism-related chemical toxicity assays. Engineered S. bulardii for expression of human proteins might expand its application by synergistically combining the probiotic activity with the treatment of metabolic diseases such as phenylketonuria via GI-delivery. Yeast models of human diseases will facilitate rapid functional/phenotypic characterization of the disease-producing mutant genes and screening of the therapeutic compounds using yeast-based high-throughput research techniques (Yeast one/two hybrid systems) and viability assays.

A preliminary computational outputs versus experimental results: Application of sTRAP, a biophysical tool for the analysis of SNPs of transcription factor-binding sites.

BACKGROUND: In the human genome, the transcription factors (TFs) and transcription factor-binding sites (TFBSs) network has a great regulatory function in the biological pathways. Such crosstalk might be affected by the single-nucleotide polymorphisms (SNPs), which could create or disrupt a TFBS, leading to either a disease or a phenotypic defect. Many computational resources have been introduced to predict the TFs binding variations due to SNPs inside TFBSs, sTRAP being one of them. METHODS: A literature review was performed and the experimental data for 18 TFBSs located in 12 genes was provided. The sequences of TFBS motifs were extracted using two different strategies; in the size similar with synthetic target sites used in the experimental techniques, and with 60 bp upstream and downstream of the SNPs. The sTRAP (http://trap.molgen.mpg.de/cgi-bin/trap_two_seq_form.cgi) was applied to compute the binding affinity scores of their cognate TFs in the context of reference and mutant sequences of TFBSs. The alternative bioinformatics model used in this study was regulatory analysis of variation in enhancers (RAVEN; http://www.cisreg.ca/cgi-bin/RAVEN/a). The bioinformatics outputs of our study were compared with experimental data, electrophoretic mobility shift assay (EMSA). RESULTS: In 6 out of 18 TFBSs in the following genes COL1A1, Hb cá´ª, TF, FIX, MBL2, NOS2A, the outputs of sTRAP were inconsistent with the results of EMSA. Furthermore, no p value of the difference between the two scores of binding affinity under the wild and mutant conditions of TFBSs was presented. Nor, were any criteria for preference or selection of any of the measurements of different matrices used for the same analysis. CONCLUSION: Our preliminary study indicated some paradoxical results between sTRAP and experimental data. However, to link the data of sTRAP to the biological functions, its optimization via experimental procedures with the integration of expanded data and applying several other bioinformatics tools might be required.

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.

Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37.

PURPOSE: Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. METHODS: In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. RESULTS: The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. CONCLUSION: This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.

Construction of bicistronic cassette for co-expressing hepatitis B surface antigen and mouse granulocyte-macrophage colony stimulating factor as adjuvant in tobacco plant.

Context: The co-delivery of adjuvant and antigen has shown to be more effective for targeting the immune response than antigen alone. Therefore, designing an efficient bicistronic system is more assuring for production of both elements in the same tobacco cells as a plant model system. Objective: Comparing the efficient transient co-expression of hepatitis B surface antigen (HBsAg) and mouse granulocyte macrophage colony stimulating factor (mGM-CSF) in tobacco leaves by designing either mono or bicistronic cassettes. Materials and methods: Four expression cassettes containing tobacco etch virus (TEV) leader sequence were constructed with and without above genes in different orders. The cassettes were transferred into tobacco, Nicotiana tabacum L. (Solanaceae), leaves by agroinfiltration technique. The expression levels were compared using ELISA and western blotting and bioactivity of cytokine was assessed by in vitro proliferation of mouse GM-CSF-responsive progenitor cells. Results: Agroinfiltrated leaves contained recombinant HBsAg protein at 20-50 ng/mg and mGM-CSF at 0.2-4 ng/mg in both nonglycosylated and glycosylated forms. The highest expression obtained in HBsAg and mGM-CSF monocistronic co-agroinfiltrated leaves. The expression of mGM-CSF was 1.1 and 0.2 ng/mg in two different orders of bicistronic cassettes. The growth frequency of GM progenitors was approximately 1/187 cells for standard rGM-CSF and 3.2 times less activity for the plant produced. Discussion and conclusions: The recombinant mGM-CSF was produced less in bicistronic cassette than other forms; however, co-presenting of both vaccine candidate and adjuvant is confirmed and could be promising for amelioration of plant expression system as a means for vaccine production.