Improving water treatment using a novel antibacterial kappa-carrageenan-coated magnetite decorated with silver nanoparticles.

In this study, we aimed to fabricate an enhanced antibacterial agent to act against pathogenic bacteria in aqueous environments. To achieve this, silver nanoparticles (AgNPs) were inlaid on a kappa-carrageenan (KC) base and coated on Fe3O4 magnetic cores (Fe3O4@KC@Ag). Superparamagnetic Fe3O4 nanoparticles were designed at the center of the composite nanostructure, allowing magnetic recovery from aqueous media in the presence of a magnet. The synthesized nanoconjugate was characterized in each step using XRD, FT-IR, EDX, FE-SEM, TEM, DLS, VSM, and disk-diffusion antibacterial method. Results show that the nanocomposite system is formed, while the magnetic properties remain practically stable. The agglomeration of the AgNPs was decreased by the trap-like function of KC coating, which resulted in an improved antibacterial activity for the Fe3O4@KC@Ag formulation. These findings suggest that Fe3O4@KC@Ag nanocomposites could be promising agents for combating bacterial infections in aqueous environments.

Design and characterization of Persian gum/polyvinyl alcohol electrospun nanofibrous scaffolds for cell culture applications.

Biocompatible electrospun nanofiber scaffolds were fabricated in this study using Persian gum (PG) and poly (vinyl alcohol) (PVA) to build an artificial extracellular matrix for cell growth. The preparation procedure involves mixing various ratios of PG/PVA to be electrospun and seeded with L929 fibroblasts. Upon addition of PG up to 60% to the solutions, a 30% decrease to around 240 µs·cm-1 is found in electrical conductivity which is in the range of semi-conductive polymers, whereas the surface tension is increased to around 3%. The fabricated scaffolds were characterized by morphological, chemical, thermal and structural analyses including SEM, FTIR spectroscopy, DSC, XRD, and tensile stress. The results showed that incorporation of 50% PG to the polymer solutions causes the formation of nanofibers with the least bead-shaped segments. All ratios of nanofibers containing PG showed significant biocompatibility with the cultured cells, which is presumably due to the radical scavenging feature of PG. The MTT and SEM analyses demonstrated that the scaffolds containing 50% PG possess the optimal cell compatibility, adhesion and proliferation properties. The fabricated PG/PVA cell culture scaffolds are potentially appropriate for wound dressing and cell culture applications in biomedicine.

Tetracycline removal enhancement with Fe-saturated nanoporous montmorillonite in a tripartite adsorption/desorption/photo-Fenton degradation process.

The adsorption and photo-Fenton degradation of tetracycline (TC) over Fe-saturated nanoporous montmorillonite was analyzed. The synthesized samples were characterized using XRD, FTIR, SEM, and XRF analysis, and the adsorption and desorption of TC onto these samples, as well as the antimicrobial activity of TC during these processes, were analyzed at different pH. Initially, a set of adsorption/desorption experiments was conducted, and surprisingly, up to 50% of TC adsorbed was released from Mt structure. Moreover, the desorbed TC had strong antibacterial activity. Then, an acid treatment (for the creation of nanoporous layers) and Fe saturation of the montmorillonite were applied to improve its adsorption and photocatalytic degradation properties over TC. Surprisingly, the desorption of TC from modified montmorillonite was still high up to 40% of adsorbed TC. However, simultaneous adsorption and photodegradation of TC were detected and almost no antimicrobial activity was detected after 180 min of visible light irradiation, which could be due to the photo-Fenton degradation of TC on the modified montmorillonite surface. In the porous structures of modified montmorillonite high, ˙OH radicals were created in the photo-Fenton reaction and were measured using the Coumarin technique. The ˙OH radicals help the degradation of TC as proposed in an oxidation process. Surprisingly, more than 90% of antimicrobial activity of the TC decreased under visible light (after 180 min) when desorbed from nanoporous Fe-saturated montmorillonite compared to natural montmorillonite. To the best of our knowledge, this is the first time that such a high TC desorption rate from an adsorbent with the least residual antimicrobial activity is reported which makes nanoporous Fe-saturated montmorillonite a perfect separation substance of TC from the environment.

Design of polysaccharidicAloe veragel incorporated PVA/tetracycline electrospun cell culture scaffolds for biomedical applications.

In this study, hybrid nanofibrous 3D scaffolds containingAloe vera(AV), polyvinyl alcohol (PVA) and tetracycline hydrochloride (TCH) are fabricated by electrospinning for cell culture applications. The role of polysaccharides present in AV gel is found to enhance the biocompatibility of the nanofibrous scaffolds. Different combinations of the polymers were selected to produce homogenous nanofibers with favorable mean fiber diameter and tensile strength. The surface morphology of the products was studied by SEM and it is found that the mean fiber diameter is decreased to about 188 nm upon addition of the AV component. The electrospun scaffolds were investigated by FT-IR spectroscopy to reveal the chemical structure of the samples and their crystallinity was studied by XRD. The hydrophilicity of the scaffolds was tested by optical contact angle measurements and their mechanical strength was examined by tensile strength tests. It is found that PVA is the main component contributing the mechanical stability of the scaffold structure. The fabricated scaffolds presented a more pronounced inhibitory effect against Gram-positive bacterial strains ofS. aureusandB. cereus. Cell culture experiments using fibroblast L929 murine cells reveals that the AV/PVA/TCH scaffolds are promising for cell growth and the cells are capable of achieving a proper cell adhesion and proliferation. The cell viability experiment by MTT assay exhibits the contributing role of AV gel to L929 cell viability on the AV/PVA/TCH scaffolds.

Biocompatibility of electrospun cell culture scaffolds made from balangu seed mucilage/PVA composites.

Synthesis of Balangu (Lallemantia royleana) seed mucilage (BSM) solutions combined with polyvinyl alcohol (PVA) was studied for the purpose of producing 3D electrospun cell culture scaffolds. Production of pure BSM nanofibers proved to be difficult, yet integration of PVA contributed to a facile and successful formation of BSM/PVA nanofibers. Different BSM/PVA ratios were fabricated to achieve the desired nanofibrous structure for cell proliferation. It is found that the optimal bead-free ratio of 50/50 with a mean fiber diameter of ≈180 nm presents the most desirable scaffold structure for cell growth. The positive effect of PVA incorporation was approved by analyzing BSM/PVA solutions through physiochemical assays such as electrical conductivity, viscosity and surface tension tests. According to the thermal analysis (TGA/DSC), incorporation of PVA enhanced thermal stability of the samples. Successful fabrication of the nanofibers is verified by FT-IR spectra, where no major chemical interaction between BSM and PVA is detected. The crystallinity of the electrospun nanofibers is investigated by XRD, revealing the nearly amorphous structure of BSM/PVA scaffolds. The MTT assay is employed to verify the biocompatibility of the scaffolds. The cell culture experiment using epithelial Vero cells shows the affinity of the cells to adhere to their nanofibrous substrate and grow to form continuous cell layers after 72 h of incubation.

Production and characterization of biocompatible nanofibrous scaffolds made ofß-sitosterolloaded polyvinyl alcohol/tragacanth gum composites.

Electrospun polyvinyl alcohol (PVA) and tragacanth gum (TG) were used to develop nanofibrous scaffolds containing poorly water-solubleß-Sitosterol (ß-S). Different concentrations and ratios of the polymeric composite includingß-S (10% w v-1) in PVA (8% w v-1) combined with TG (0.5 and 1% w v-1) were prepared and electrospun. The synthesis method includes four electrospinning parameters of solution concentration, feeding rate, voltage, and distance of the collector to the tip of the needle, which are independently optimized to achieve uniform nanofibers with a desirable mean diameter for cell culture. The collected nanofibers were characterized by SEM, FTIR, and XRD measurements. A contact angle measurement described the hydrophilicity of the scaffold. MTT test was carried out on the obtained nanofibers containing L929 normal fibroblast cells. The mechanical strength, porosity, and deterioration of the scaffolds were well discussed. The mean nanofiber diameters ranged from 63 ± 20 nm to 97 ± 46 nm. The nanofibers loaded withß-S were freely soluble in water and displayed a remarkable biocompatible nature. The cultured cells illustrated sheet-like stretched growth morphology and penetrated the nanofibrous pores of the PVA/ß-S/TG scaffolds. The dissolution was related to submicron-level recrystallization ofß-S with sufficient conditions for culturing L929 cells. It was concluded that electrospinning is a promising technique for poorly water-solubleß-S formulations that could be used in biomedical applications.

Quince seed mucilage coated iron oxide nanoparticles for plasmid DNA delivery.

This study investigates the potential of iron oxide nanoparticles (Fe3O4) and quince seed mucilage as combined genetic carriers to deliver plasmid DNA (pDNA) through the gastrointestinal system. The samples are characterized by x-ray diffraction (XRD), zeta potential, dynamic light scattering, FT-IR spectroscopy, field emission scanning electron microscopy and vibrating sample magnetometry. The stability of pDNA loading on the nanocarriers and their release pattern are evaluated in simulated gastrointestinal environments by electrophoresis. The XRD patterns reveal that the nanocarriers could preserve their structure during various synthesis levels. The saturation magnetization (Ms) of the Fe3O4cores are 56.48 emu g-1without any magnetic hysteresis. Not only does the loaded pDNA contents experience a remarkable stability in the simulated gastric environment, but also, they could be released up to 99% when exposed to an alkaline environment similar to the intestinal fluid of fish. The results indicate that the synthesized nanoparticles could be employed as efficient low-cost pDNA carriers.

Flooding or drought which one is more offensive on pepper physiology and growth?

Both extreme usage of water in agriculture i.e., drought and flooding affect physiological and growth aspects of the plant as well as gene expression undertaken in water absorption. These affect depend on the stress duration i.e., shock or gradual stress exposer. The factorial experiment based on CRD with 10 replicates was conducted to investigate the physiological and water relation as well as aquaporin expression in (Capsicum annuum L.). Drought stress was applied gradually from - 2, - 3, - 4 to - 5 MPa during 8 days but in shock stress - 5 MPa applied at one time. The gradual flooding stress adjusted with changing the aeration duration from 15 to 0 min gradually every 2 days and for the shock- flooding, peppers keep in a nutrient solution without aeration in a sealed container. Results showed that both extreme water stress had a deleterious effect on the growth and physiological parameter of pepper for a longer duration. Antioxidant, proline, fluorescence chlorophyll stimulate in the gradual period except for ABA content, which is higher in shock stress. PIP1expression showed a reverse effect in leaf and root at flooding i.e., PIP1expression raised in root while it was reduced in leaf at shock-flooding. The highest PIP1expression was observed in gradual-drought of root and gradual duration of drought and flooding stress in leaf. In the physiological aspect of plant response to stress in pepper, results showed an enhanced in proline and phenol content to help osmotic adjustment and keep water status in moderate condition. Conclusively, shocked stress first, motivated these defense systems, and then in the next step, the other adaptive mechanism like gene expression activated to help pepper face stress. On the other hand, shock stress showed down-regulation, but when the stress lasted for a longer time results in up-regulation.

Transient expression of the full-length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration.

The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO4  + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 µg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

Fermented soybean meal affects the ruminal fermentation and the abundance of selected bacterial species in Holstein calves: a multilevel analysis.

The effect of soybean meal (SBM) replacement with fermented SBM (FSBM) on ruminal fermentation and bacterial abundance in Holstein calves was investigated in this study. Thirty nine calves were randomized to: (1) control: 27% SBM + 0% FSBM (FSBM0, n = 13); (2) 18% SBM + 9% FSBM (FSBM9, n = 13); and (3) 13.5% SBM + 13.5% FSBM (FSBM13, n = 13). SBM contained a greater amount of large peptides containing 3 to 10 amino acids (AAs), while FSBM had a greater amount of ammonia nitrogen (NH3-N), free AAs, and small peptides containing 2 to 3 AAs. The calves fed FSBM13 had the lowest acetic acid, NH3-N, and the ratio of acetate to propionate, with the greatest concentration of caproic acid, valeric acid and isovaleric acid in ruminal fluid. Compared to those fed FSBM9 or FSBM13, the calves fed FSBM0 had the greatest proportion of Butyrivibrio fibrisolvens and Ruminococcus albus in rumen fluid. However, the ruminal abundance of Prevotella ruminicola in calves fed FSBM13 was greater than in calves fed FSBM0. Network analysis showed that the abundance of the Ruminococcus albus was associated with large peptides, and butyric acid was correlated with small peptide. Taken together, our findings suggest that FSBM may have the potential to boost calf performance by changing the fermentation products and the relative abundance of some members of the bacterial community in the rumen.

Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences.

Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an iterative method for in vitro selection of aptamers from a random synthetic oligonucleotide library. Successful retrieving of aptamers by SELEX relies on optimization of various steps including target immobilization, aptamer partitioning, amplification, and ssDNA generation, which all require spending considerable effort and cost. Furthermore, due to the random nature of the initial library, SELEX may redirect toward the selection of low-affinity aptamers that are over-represented in the ssDNA population due to PCR bias. Thus, precise monitoring of the SELEX process is crucial to ensure the selection of target-specific aptamers. In the present study, we investigated the reliability and simplicity of different direct and indirect monitoring methods including UV-Vis spectroscopy, real-time PCR quantification and melt-curve analysis, electrophoretic mobility shift assay (EMSA) and enzyme-linked oligonucleotide assay (ELONA) for selection of DNA aptamers for a protein target. All the examined methods were capable of illustrating the gradual evolution of specific aptamers by the progression of SELEX and showed almost similar results regarding the identification of the enriched round of selection. Moreover, we describe the use of melt-curve analysis in the colony real-time PCR method as a simple, robust, and repeatable tool for pre-sequencing separation of distinct aptamer clones.

Preparation of cell culture scaffolds using polycaprolactone/quince seed mucilage.

In the present study, the polymer obtained from Cydonia oblonga Miller seeds (quince seed mucilage (QSM)) in combination with polycaprolactone (PCL) was used for producing hybrid electrospun scaffolds as three-dimensional (3D) cell culture platforms. Various PCL/QSM ratios were tested to obtain a uniform product with an appropriate mean fiber diameter for cell growth. The chemical structures of the scaffolds were studied by FT-IR spectroscopy and their crystallinity was investigated by XRD. Fiber morphology studies by SEM revealed that the fiber mean diameters decrease upon increasing the QSM component of the electrospun compound. The PCL/QSM ratio of 60/40 exhibited desirable uniform bead-free nanofibers. The hydrophilicity of the scaffolds were explored by contact angle measurements, which indicated that more hydrophilic fibers are produced as the PCL/QSM ratio is decreased. Porosity and strength of the scaffolds were also studied, which showed the key role of PCL in mechanical strength of the nanofibers. The results of cell culture experiments using epithelial Vero cells on the scaffolds displayed their high capacity in cell growth and proliferation. It was revealed that the electrospun PCL/QSM based scaffolds with 3D structures and 75-150 nm mean fiber diameters are able to maximize adhesion and growth of Vero cells.

Determination of phenolic compounds, antioxidant and anticancer activity of Chrozophora tinctoria accessions collected from different regions of Iran.

The present study was conducted to determine the total phenolics, flavonoids, flavonols, anthocyanin, and carotenoids as well as the antioxidant activity of 10 different Crozophora tinctoria accessions originated from Iran. The highest contents of total phenolics, flavonoids, flavonols, anthocyanin, and carotenoids were detected in C.t (2), C.t (10), C.t (10), C.t (9), and C.t (3) accessions, respectively. The highest antioxidant activities of the samples, evaluated using 1,1-diphenyl-2-picrylhydrazyl and phosphomolibdate assays, were detected in C.t (8) and C.t (3), respectively. The highest phenyl aminolyase activity was observed in C.t (4). Ten phenolic compounds, including quercetin, catechin, ellagic acid, rosmarinic acid, hesperetin, eugenol, hesperidin, carvacrol, thymol, and kaempferol were identified and quantified using high-performance liquid chromatography (HPLC) technique. The best anticancer activity against HeLa cell lines was observed at C.t (8) accession, originated from Qazvin Region. This study confirmed that the C. tinctoria accessions with rich phenolics compounds may be recommended as a novel source of natural antioxidants for future aims in medicinal fields. PRACTICAL APPLICATIONS: According to this findings there was high variation among phenolics compounds of C. tinctoria which is revealed by HPLC analysis. This study identified phenolic compounds with antioxidant and anticancer activity in Iranian accessions of C. tinctoria. With an increase in the rate of cancer disease, superior accessions of C. tinctoria may be recommended as a novel source of natural antioxidants for ethno pharmacological aims as cancer therapy, and also in nutraceutical fields.

Molecular evolution and selection pressure analysis of infectious hematopoietic necrosis virus (IHNV) revealed the origin and phylogenetic relationship of Iranian isolates in recent epidemics in Iran.

Infectious hematopoietic necrosis virus (IHNV) is the causative agent for a lethal salmonid disease. In this study, we surveyed the IHNV's epidemiology, diversity and the origin of infection in Iran. Phylogenetic analysis revealed that Iranian isolates belonged to one of the two lineages of E genogroup. Subsequently, a combination of phylogenetic, antigenic and structural analysis was performed to investigate the evolution of E genogroup lineages. Site-specific analysis of the viral glycoprotein showed different co-evolving and positively selected sites in each lineage. Most of these sites were mapped to the predicted antigenic patches of the glycoprotein. Further characterization revealed E lineages can be differentiated, in part, by specific mutations at positions 91 and 130, which are located in the structurally flexible regions of the glycoprotein, suggesting a key adaptative role for these sites. These data may assist in better monitoring the emerging isolates in regions infected to IHNV from E genogroup.

A Novel Approach to Deliver a Mycobacterium avium subsp. paratuberculosis Antigen in Eukaryotic Cells.

This study was aimed to express and deliver a Mycobacterium avium subsp. paratuberculosis antigen to macrophages using salmonella as carrier. The coding sequence of a fibronectin attachment protein which is expressed by Mycobacterium avium subsp. paratuberculosis was cloned into pcDNA3.1 (+) plasmid. The construct was introduced into the attenuated Salmonella typhimurium strain SL7207 (ΔhisG, ΔaroA) as carrier. In order to evaluate the delivery capacity of Salmonella and gene expression by antigen-presenting cells, the THP-1 derived macrophages were infected with the salmonella carrier. SDS-PAGE and western blot analysis showed the successful delivery and expression of targeted gene in THP-1 cell line. Although, in vitro stimulation of peripheral blood mononuclear cells with Salmonella containing plasmid did not trigger IFNγ production significantly. But it seems that this carrier can increase plasmid uptake and antigen expression by host intestinal antigen-presenting cells after mucosal administration. So, the construct can be used for further in vivo studies on the Salmonella carrier's efficiency in mycobacterial DNA vaccines.

Effect of dietary date seed meal as an alternative carbohydrate source on immune-related gene expression of Pacific white shrimp Penaeus vannamei.

Current study aimed to examine the effect of dietary date seed meal (DSM) as an alternative carbohydrate ingredient on immune-related genes expression of Pacific white shrimp (Penaeus vannamei). A total number of 750 shrimp (4.0 ±â€¯0.1 g) were randomly assigned into five experimental treatments, each with three replicates. Experimental diets contained 0, 50, 100, 150 and 230 g/kg DSM substituted for both wheat and rice flour. Shrimp were fed at a rate of 5% body weight three times daily and the mRNA expression of α2-macroglobulin (α2-M), lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP), integrin ß, and peroxinectin (PX) was studied on five shrimp per treatment after 8 weeks feeding trial. The results showed that replacement of DSM up to 100 g/kg in the diet of P. vannamei caused no significant changes in the expression of α2-M and integrin ß, however both of these genes showed lower expression in shrimp fed diets containing 150 and 230 g/kg DSM compared to control group. Moreover, no significant differences were observed in the expression of PX and LGBP between shrimp fed diets containing different amounts of DSM and control group. These findings demonstrated that as an inexpensive source of carbohydrate, DSM could be substituted up to 100 g/kg for wheat and rice flour in the diet of farmed shrimp without suppressive effects on the immune status.

Green synthesis of silver nanoparticles using Glaucium corniculatum (L.) Curtis extract and evaluation of its antibacterial activity.

The metal nanoparticles, due to interesting features such as electrical, optical, chemical and magnetic properties, have been investigated repeatedly. Also, the mentioned nanoparticles have specific uses in terms of their antibacterial activity. The biosynthesis method is more appropriate than the chemical method for producing the nanoparticles because it does not need any special facilities; it is also economically affordable. In the current study, the silver nanoparticles (AgNPs) were obtained by using a very simple and low-cost method via Glaucium corniculatum (L.) Curtis plant extract. The characteristics of the AgNPs were investigated using techniques including: X-ray diffraction, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy. The SEM and TEM images showed that the nanoparticles had a spherical shape, and the mean diameter of them was 53.7 and 45 nm, respectively. The results of the disc diffusion test used for measuring the anti-bacterial activity of the synthesised nanoparticles indicated that the formed nanoparticles possessed a suitable anti-bacterial activity.

Green synthesis of silver nanoparticles with the Arial part of Dorema ammoniacum D. extract by antimicrobial analysis.

Silver nanoparticles (SNPs) were synthesised by using the Arial part extract of Dorema ammoniacum D. and characterised by employing UV-visible spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction techniques. Transmission electron microscopy and field emission scanning electron microscopy were applied to investigate the morphological structure of the bio-synthesised SNPs. The antimicrobial activity of SNPs was studied against Gram positive (Bacillus cereus and Staphylococcus aureus) and Gram-negative (Escherichia coli and Salmonella typhimurium) bacteria by employing the disk diffusion agar process. An extremely antimicrobial effect was observed for SNPs. Utilising D. ammoniacum D. as a mediator for the synthesis of SNPs helped to save time and cost.

Combined effects of dietary low molecular weight sodium alginate and Pediococcus acidilactici MA18/5M on growth performance, haematological and innate immune responses of Asian sea bass (Lates calcalifer) juveniles.

Six weeks feeding trial was conducted to examine the effects of dietary administration of low molecular weight sodium alginate (LMWSA) and Pediococcus acidilactici MA 18/5M (PA) on humoral and mucosal immune responses, haematological parameters and growth performance, of Lates calcarifer juveniles. Fish (12.0 ±â€¯0.2 g) were fed experimental diets as follows: Control (diet 1, basal diet), 5 g kg-1 LMWSA (diet 2), 10 g kg-1 LMWSA (diet 3), 0.9 × 107 CFU g-1 PA (diet 4), 5 g kg-1 LMWSA + 0.9 × 107 CFU g-1 PA (Diet 5), and 10 g kg-1 LMWSA + 0.9 × 107 CFU g-1 PA (Diet 6). Results indicated a significant (P < 0.05) increase in innate immune parameters including serum lysozyme, bactericidal, hemolytic and respiratory burst activities as well as mucosal immune responses including lysozyme and bactericidal activities, when diet was supplemented with immunostimulants. Moreover, the combined effects of LMWSA with PA resulted in more pronounced immunological responses compared to the control and singular administration. Red and white blood cell counts significantly increased with either singular or combined administration of LMWSA and PA compared with the control group (P < 0.05). The singular administration of PA and combined supplementation of 5 g kg-1 LMWSA with PA significantly increased growth performance and feed intake compared with other experimental groups (P < 0.05). These results indicated that combined administration of LMWSA and PA can be considered as beneficial feed additive and immunostimulant in L. calcarifer juveniles.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.