Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate.

BACKGROUND: Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. RESULTS AND CONCLUSION: We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

Pan-Genomic Approaches in Lactobacillus reuteri as a Porcine Probiotic: Investigation of Host Adaptation and Antipathogenic Activity.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, reuterin-producing Lactobacillus reuteri is getting attention as an alternative to AGPs. In this study, we investigated genetic features of L. reuteri associated with host specificity and antipathogenic effect. We isolated 104 L. reuteri strains from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (group HS) and eight strains exhibiting the lower effect (group LS), were selected for genomic comparison. We generated draft genomes of the 16 isolates and investigated their pan-genome together with the 26 National Center for Biotechnology Information-registered genomes. L. reuteri genomes organized six clades with multi-locus sequence analysis, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein-coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host gastrointestinal epithelia and that these host-specific traits were acquired by horizontal gene transfer. We also identified differences between groups HS and LS in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. This study provides a deeper understanding of the relationship between probiotic properties and genomic features of L. reuteri.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study. RESULTS: We firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 µg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers. CONCLUSIONS: In this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.

Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding.

The emergence of highly pathogenic variant porcine epidemic diarrhea virus (PEDV) strains, from 2013 to 2014, in North American and Asian countries have greatly threatened global swine industry. Therefore, development of effective vaccines against PEDV variant strains is urgently needed. Recently, it has been reported that the N-terminal domain (NTD) of S1 domain of PEDV spike protein is responsible for binding to the 5-N-acetylneuraminic acid (Neu5Ac), a possible sugar co-receptor. Therefore, the NTD of S1 domain could be an attractive target for the development of subunit vaccines. In this study, the NTD spanning amino acid residues 25-229 (S25-229) of S1 domain of PEDV variant strain was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies (IBs). S25-229 IBs were solubilized in 20 mM sodium acetate (pH 4.5) buffer containing 8 M urea and 1 mM dithiothreitol with 95% yield. Solubilized S25-229 IBs were refolded by 10-fold flash dilution and purified by one-step cation exchange chromatography with >95% purity and 20% yield. The CD spectrum of S25-229 showed the characteristic pattern of alpha helical structure. In an indirect ELISA, purified S25-229 showed strong reactivity with mouse anti-PEDV sera. In addition, immunization of mice with 20 µg of purified S25-229 elicited highly potent serum IgG titers. Finally, mouse antisera against S25-229 showed immune reactivity with native PEDV S protein in an immunofluorescence assay. These results suggest that purified S25-229 may have potential to be used as a subunit vaccine against PEDV variant strains.

Influence of Flaxseed Oil on Fecal Microbiota, Egg Quality and Fatty Acid Composition of Egg Yolks in Laying Hens.

Although there have been many attempts to produce ω-3 fatty acid-rich eggs using alpha-linolenic acid (ALA) that is a popular fatty acid in the poultry feed industry, only limited knowledge about the effects of ALA-enriched diets on chicken fecal microbiota is currently available. Herein we examined the changes in the fecal microbiota composition, egg quality traits and fatty acid composition of the egg yolks of laying hens fed ALA-rich flaxseed oil for 8 weeks. The animals fed the experimental diets that contained 0 % (group C), 0.5 % (group T1), and 1.0 % (group T2) of flaxseed oil, respectively, and eggs and feces were obtained for the analyses. ω-3 fatty acids, including ALA, were increased in T1 and T2 compared with C. Furthermore, the freshness of eggs was improved with no side effects on the eggs. The diet also changed the fecal microbiota; Firmicutes was increased in T1 and T2 (48.6 to 83 and 79.6 %) and Bacteroidetes was decreased (40.2 to 8.8 and 4.2 %). Principal coordinate analysis revealed that Lactobacillus, among the 56 examined genera, was the most influenced bacterial group in terms of the fecal microbial community shifts. These results indicate that ALA-rich diets influenced both the egg and fecal microbiota in beneficial manners in laying hens although the association between the fatty acid composition of the egg yolk and the fecal microbiota was not clear. This study is a first step to understand the effect of flaxseed oil as well as intestinal microbiota of laying hens.

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant.

BACKGROUND: To initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer's patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant. RESULTS: For efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB. CONCLUSIONS: Our results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.

Combinatorial Approach of Antigen Delivery Using M Cell-Homing Peptide and Mucoadhesive Vehicle to Enhance the Efficacy of Oral Vaccine.

Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer's patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4(+) T cell immune responses but also generated strong CD8(+) T cell responses with enhanced production of IFN-γ in spleen. Given that M cells are considered a promising target for oral vaccination, this study could provide a new combinatorial method for the development of M-cell-targeted mucosal vaccines.

Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 µ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.

Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational way to prepare a suitable vector for targeted gene delivery to vimentin-expressing cells.

Heat-shock protein beta 1 regulates androgen-mediated bovine myogenesis.

To elucidate the functional significance of heat-shock protein beta 1 (HSPB1) in androgen-mediated myogenesis of bovine cells, we conducted 'loss and gain of function of HSPB1' assays by siRNA inhibition and gene overexpression. siRNA inhibition of HSPB1 expression reduced the expression of desmin (a myogenic marker) and repressed the formation of myotubes in cells induced for myogenic differentiation. In contrast, overexpression of HSPB1 enhanced the expression of desmin and accelerated formation of myotubes. The loss and gain of HSPB1 function was closely associated with the expression level of androgen receptor (AR). Our findings suggest that HSPB1 mediates androgen signaling by binding directly to AR and then enhancing androgen-mediated myogenesis in myogenic cells.

Polyxylitol-based gene carrier improves the efficiency of gene transfer through enhanced endosomal osmolysis.

Endosomal escape is one of the important processes for efficient non-viral gene delivery. In this study, we synthesized a novel non-viral vector called polyxylitol-based gene carrier (XGC) through a Miachael addition reaction between xylitol diacrylate as a crosslinking agent and low molecular weight polyethylenimine (PEI 1.2kDa). The small amount of xylitol integrated into XGC (3.9% w/w) contributed 50% of the osmotic pressure of XGC, and enhaned the osmolysis of endosome cooperatively with the proton sponge effect, thus improving endosomal escape. Furthermore, XGC showed higher transfection efficiency in vivo in muscle tissue than pDNA alone or PEI 25kDa. In conclusion, our results show that XGC enhanced transfection efficiency compared with PEI 25kDa, the golden standard non-viral gene carrier, by enhancing endosomal escape without increasing the number of transfected cells. FROM THE CLINICAL EDITOR: Enhanced gene delivery methods would greatly facilitate the development of gene therapies. These authors demonstrate that a polyxylitol-based gene carrier enhanced the transfection efficiency compared with the gold standard non-viral gene carrier, as a result of enhancing endosomal escape without increasing the number of transfected cells, warranting further studies of this method.

Association of protein expression in isolated milk epithelial cells and cis-9, trans-11 conjugated linoleic acid proportions in milk from dairy cows.

BACKGROUND: The effects of the individual variation among dairy cows on the synthesis of cis-9, trans-11 conjugated linoleic acid (CLA) are still not well characterised. Therefore, the protein expression profiles of isolated milk epithelial cells (MECs) were detected by two-dimensional electrophoresis and their correlation with the various proportion of cis-9, trans-11 CLA were evaluated. RESULTS: Although animals were offered the same diet, the proportion of cis-9, trans-11 CLA in group High (1.02 ± 0.10%) was twice as high as that in group Low (0.59 ± 0.14%) (P < 0.05). MECs with the characteristics of native epithelial cells were successfully isolated from the milk and these cells had no obvious RNA degradation or were hardly contaminated with leucocytes or blood red cells. Moreover, the protein expression pattern of cathelicidin 5 in isolated MECs was positive, whereas annexin I (confirmed by real-time polymerase chain reaction), ZW10 interactor and κ-casein were negatively related to the proportion of cis-9, trans-11 CLA in the milk fat. CONCLUSION: The varied individual content of cis-9, trans-11 CLA in cows may be associated with annexin I. These findings may provide some theoretical basis for studies concerning the effects of the individual variation among dairy cows of the synthesis of cis-9, trans-11 CLA. © 2013 Society of Chemical Industry.

High-level soluble expression of bioactive porcine myostatin propeptide in E. coli.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 µg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 µg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 µg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated whether the administration of the MSTNpro can improve skeletal muscle growth in pigs via suppression of MSTN activity in vivo.

Effect of microencapsulation of Lactobacillus plantarum 25 into alginate/chitosan/alginate microcapsules on viability and cytokine induction.

Lactobacillus plantarum 25 (LP25) encapsulated into alginate/chitosan/alginate (ACA) microcapsules (LP25-ACA MCs) prepared by an extrusion methods were characterized to assess their efficacy in oral delivery. The particle sizes of LP25-ACA MCs were 1.11 +/- 0.32 mm. The loading content of LP25 was 1.11 x 10(7) colony forming unit (cfu)/microcapsule and encapsulation efficiency was above 98%. The viability of LP25 in ACA MCs was more than 65% in simulated gastric fluid (SGF, pH 2.0) and 75% in simulated small intestinal fluid (SIF, pH 7.2) up to 2 h. Encapsulated LP25 were completely released from LP25-ACA MCs in SIF and simulated colon fluid (SCF, pH 6.0) within 12 h and 8 h respectively. The viability of LP25 in ACA MCs till 5 weeks was above 58%, whereas viability of free LP25 stored at room temperature up to 5 weeks was zero. Besides, LP25-ACA MCs induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) from macrophages and dendritic cells showing the immunomodulatory effect of LP25. These findings demonstrate that the encapsulation of LP25 by ACA is a suitable strategy for oral delivery of probiotics.

High gene transfer by the osmotic polysorbitol-mediated transporter through the selective caveolae endocytic pathway.

Cationic polymers have been the subject of intense research as nonviral gene delivery systems due to several advantages in comparison with viral vectors. However, the nonsimultaneous combination of high transfection efficiency and low cytotoxicity of nonviral vectors for gene delivery has long been an issue for scientists looking into ways to deliver genes into cells. Toward this goal, we designed, synthesized, and evaluated a safe and accelerated gene transfer system through polysorbitol-mediated transporter (PSMT) based on sorbitol diacrylate (SDA) and low molecular weight polyethylenimine (LMW PEI). The PSMT formed stable complexes with plasmid DNA in serum. The nano sizes and spherical shapes of PSMT/DNA complexes are not toxic, even at a high concentration of PSMT. The higher transfection efficiency of PSMT compared to PEI 25K was observed both in vitro, despite the existence of many hydroxyl groups, and in vivo. These improvements presumably stem from the osmotic property of polysorbitol and endosomal buffer capacity of PEI in PSMT. Most importantly, we confirmed that the selective cavaeolae endocytic pathway played a role in high transfection efficiency by osmotic PSMT-mediated gene delivery. We propose that PSMT is a promising nonviral carrier for the effective gene delivery to cancer cells via synergistic effects derived from rapid cellular uptake through the caveolae endocytic pathway and a high endosomal buffering capacity.

Identification of tissue-specific targeting peptide.

Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

Differentially expressed proteins associated with myogenesis and adipogenesis in skeletal muscle and adipose tissue between bulls and steers.

The objective of this study was to identify some proteins associated with testosterone-related differences in myogenesis and adipogenesis between bulls and steers. Global proteins were monitored in skeletal muscle and adipose tissue from bulls (n = 20) and steers (n = 20), respectively. We identified four differentially expressed (twofold or more) proteins in skeletal muscle from bulls, myosin light chain 1 (MLC1), ankyrin repeat domain-containing protein 1 (ANKRD1) and heat shock protein beta 1 (HSPB1) that were up-regulated and cofilin 2 (CFL2) that was down-regulated, and also identified two down-regulated proteins in adipose tissue, transaldolase 1 (TALDO1) and L: -lactate dehydrogenase B chain (LDHB). In vitro, after myogenic differentiation of a bovine cell line, the mRNA expression of HSPB1 not only increased approximately tenfold in response to differentiation but threefold in response to testosterone addition, respectively, but that of ANKRD1 and CFL2 did not significantly change in response to myogenic differentiation or testosterone addition. Likewise, after adipogenic differentiation of a bovine cell line, the mRNA expression of TALDO1 and LDHB did not significantly vary in response to adipogenic differentiation or testosterone addition. Therefore, we suggest that HSPB1 could have an important role during testosterone-related myogenesis.

Effect of microencapsulation of Lactobacillus salivarus 29 into alginate/chitosan/alginate microcapsules on viability and cytokine induction.

Harsh gastric condition causes low bioavailability of probiotics when supplied orally. Polymeric encapsulation has successfully protected bacteria from harsh gastric condition and ultimately increased persistency and multiplication at the targeted region. In this study, we encapsulated LS29 into ACA microcapsules and characterized them. The survivability and release of LS29 from LS29-loaded ACA microcapsules in SGF and SIF were studied. Encapsulation efficiency of LS29 in ACA microcapsules was 99.9%. Approximately 70% of bacteria survived at pH 2 by 120 min after encapsulation. Although not much difference of the survivability of LS29 encapsulated into ACA and FDACA was observed, freeze-drying improved the controlled-release of LS29 in SIF and also showed better storage survivability at 4°C for 8 weeks. Furthermore, investigation of in vitro production of cytokines in RAW264.7 showed high level of induction of TNF-α and IL-10. These in vitro results support that the LS29 might have a balanced immunomodulatory effect.

Machine learning study for the prediction of transdermal peptide.

In order to develop a computational method to rapidly evaluate transdermal peptides, we report approaches for predicting the transdermal activity of peptides on the basis of peptide sequence information using Artificial Neural Network (ANN), Partial Least Squares (PLS) and Support Vector Machine (SVM). We identified 269 transdermal peptides by the phage display technique and use them as the positive controls to develop and test machine learning models. Combinations of three descriptors with neural network architectures, the number of latent variables and the kernel functions are tried in training to make appropriate predictions. The capacity of models is evaluated by means of statistical indicators including sensitivity, specificity, and the area under the receiver operating characteristic curve (ROC score). In the ROC score-based comparison, three methods proved capable of providing a reasonable prediction of transdermal peptide. The best result is obtained by SVM model with a radial basis function and VHSE descriptors. The results indicate that it is possible to discriminate between transdermal peptides and random sequences using our models. We anticipate that our models will be applicable to prediction of transdermal peptide for large peptide database for facilitating efficient transdermal drug delivery through intact skin.