Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

BACKGROUND: The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. OBJECTIVE: We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. METHODS: B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. RESULTS: B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. CONCLUSION: B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies.

Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

BACKGROUND: Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. METHODS: The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. RESULTS: The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. CONCLUSIONS: Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma.

Native Pig and Chicken Breed Database: NPCDB.

Indigenous (native) breeds of livestock have higher disease resistance and adaptation to the environment due to high genetic diversity. Even though their extinction rate is accelerated due to the increase of commercial breeds, natural disaster, and civil war, there is a lack of well-established databases for the native breeds. Thus, we constructed the native pig and chicken breed database (NPCDB) which integrates available information on the breeds from around the world. It is a nonprofit public database aimed to provide information on the genetic resources of indigenous pig and chicken breeds for their conservation. The NPCDB (http://npcdb.snu.ac.kr/) provides the phenotypic information and population size of each breed as well as its specific habitat. In addition, it provides information on the distribution of genetic resources across the country. The database will contribute to understanding of the breed's characteristics such as disease resistance and adaptation to environmental changes as well as the conservation of indigenous genetic resources.

Early-life-trauma triggers interferon-ß resistance and neurodegeneration in a multiple sclerosis model via downregulated ß1-adrenergic signaling.

Environmental triggers have important functions in multiple sclerosis (MS) susceptibility, phenotype, and trajectory. Exposure to early life trauma (ELT) has been associated with higher relapse rates in MS patients; however, the underlying mechanisms are not well-defined. Here we show ELT induces mechanistic and phenotypical alterations during experimental autoimmune encephalitis (EAE). ELT sustains downregulation of immune cell adrenergic receptors, which can be attributed to chronic norepinephrine circulation. ELT-subjected mice exhibit interferon-ß resistance and neurodegeneration driven by lymphotoxin and CXCR2 involvement. These phenotypic changes are observed in control EAE mice treated with ß1 adrenergic receptor antagonist. Conversely, ß1 adrenergic receptor agonist treatment to ELT mice abrogates phenotype changes via restoration of immune cell ß1 adrenergic receptor function. Our results indicate that ELT alters EAE phenotype via downregulation of ß1 adrenergic signaling in immune cells. These results have implications for the effect of environmental factors in provoking disease heterogeneity and might enable prediction of long-term outcomes in MS.

Use of a bovine genome array to identify new biological pathways for beef marbling in Hanwoo (Korean Cattle).

BACKGROUND: Marbling (intramuscular fat) is a valuable trait that impacts on meat quality and an important factor determining price of beef in the Korean beef market. Animals that are destined for this high marbling market are fed a high concentrate ration for approximately 30 months in the Korean finishing farms. However, this feeding strategy leads to inefficiencies and excessive fat production. This study aimed to identify candidate genes and pathways associated with intramuscular fat deposition on highly divergent marbling phenotypes in adult Hanwoo cattle. RESULTS: Bovine genome array analysis was conducted to detect differentially expressed genes (DEGs) in m. longissimus with divergent marbling phenotype (marbling score 2 to 7). Three data-processing methods (MAS5.0, GCRMA and RMA) were used to test for differential expression (DE). Statistical analysis identified 21 significant transcripts from at least two data-processing methods (P < 0.01). All 21 differentially expressed genes were validated by real-time PCR. Results showed a high concordance in the gene expression fold change between the microarrays and the real time PCR data. Gene Ontology (GO) and pathway analysis demonstrated that some genes (ADAMTS4, CYP51A and SQLE) over expressed in high marbled animals are involved in a protein catabolic process and a cholesterol biosynthesis process. In addition, pathway analysis also revealed that ADAMTS4 is activated by three regulators (IL-17A, TNFα and TGFß1). QRT-PCR was used to investigate gene expression of these regulators in muscle with divergent intramuscular fat contents. The results demonstrate that ADAMTS4 and TGFß1 are associated with increasing marbling fat. An ADAMTS4/TGFß1 pathway seems to be associated with the phenotypic differences between high and low marbled groups. CONCLUSIONS: Marbling differences are possibly a function of complex signaling pathway interactions between muscle and fat. These results suggest that ADAMTS4, which is involved in connective tissue degradation, could play a role in an important biological pathway for building up marbling in cattle. Moreover, ADAMTS4 and TGFß1could potentially be used as an early biological marker for marbling fat content in the early stages of growth.

Identification of differentially expressed genes related to intramuscular fat development in the early and late fattening stages of hanwoo steers.

Marbling of cattle meat is dependent on the coordinated expression of multiple genes. Cattle dramatically increase their intramuscular fat content in the longissimus dorsi muscle between 12 and 27 months of age. We used the annealing control primer (ACP)-differential display RT-PCR method to identify differentially expressed genes (DEGs) that may participate in the development of intramuscular fat between early (12 months old) and late fattening stages (27 months old). Using 20 arbitrary ACP primers, we identified and sequenced 14 DEGs. BLAST searches revealed that expression of the MDH, PI4-K, ferritin, ICER, NID-2, WDNMI, telethonin, filamin, and desmin (DES) genes increased while that of GAPD, COP VII, ACTA1, CamK II, and nebulin decreased during the late fattening stage. The results of functional categorization using the Gene Ontology database for 14 known genes indicated that MDH, GAPD, and COP VII are involved in metabolic pathways such as glycolysis and the TCA cycle, whereas telethonin, filamin, nebulin, desmin, and ACTA1 contribute to the muscle contractile apparatus, and PI4-K, CamK II, and ICER have roles in signal transduction pathways regulated by growth factor or hormones. The final three genes, NID-2, WDNMI, and ferritin, are involved in iron transport and extracellular protein inhibition. The expression patterns were confirmed for seven genes (MDH, PI4-K, ferritin, ICER, nebulin, WDNMI, and telethonin) using real-time PCR. We found that the novel transcription repressor ICER gene was highly expressed in the late fattening stage and during bovine preadipocyte differentiation. This information may be helpful in selecting candidate genes that participate in intramuscular fat development in cattle.

Growth hormone-releasing hormone (GHRH) polymorphisms associated with carcass traits of meat in Korean cattle.

BACKGROUND: Cold carcass weight (CW) and longissimus muscle area (EMA) are the major quantitative traits in beef cattle. In this study, we found several polymorphisms of growth hormone-releasing hormone (GHRH) gene and examined the association of polymorphisms with carcass traits (CW and EMA) in Korean native cattle (Hanwoo). RESULTS: By direct DNA sequencing in 24 unrelated Korean cattle, we identified 12 single nucleotide polymorphisms within the 9 kb full gene region, including the 1.5 kb promoter region. Among them, six polymorphic sites were selected for genotyping in our beef cattle (n = 428) and five marker haplotypes (frequency > 0.1) were identified. Statistical analysis revealed that -4241A>T showed significant associations with CW and EMA. CONCLUSION: Our findings suggest that polymorphisms in GHRH might be one of the important genetic factors that influence carcass yield in beef cattle. Sequence variation/haplotype information identified in this study would provide valuable information for the production of a commercial line of beef cattle.

Confirming single nucleotide polymorphisms from expressed sequence tag datasets derived from three cattle cDNA libraries.

Using the Phred/Phrap/Polyphred/Consed pipeline established in the National Livestock Research Institute of Korea, we predicted candidate coding single nucleotide polymorphisms (cSNPs) from 7,600 expressed sequence tags (ESTs) derived from three cDNA libraries (liver, M. longissimus dorsi, and intermuscular fat) of Hanwoo (Korean native cattle) steers. From the 7,600 ESTs, 829 contigs comprising more than two EST reads were assembled using the Phrap assembler. Based on the contig analysis, 201 candidate cSNPs were identified in 129 contigs, in which transitions (69%) outnumbered transversions (31%). To verify whether the predicted cSNPs are real, 17 SNPs involved in lipid and energy metabolism were selected from the ESTs. Twelve of these were confirmed to be real while five were identified as artifacts, possibly due to expressed sequence tag sequence error. Further analysis of the 12 verified cSNPs was performed using the program BLASTX. Five were identified as nonsynonymous cSNPs, five were synonymous cSNPs, and two SNPs were located in 3'-UTRs. Our data indicated that a relatively high SNP prediction rate (71%) from a large EST database could produce abundant cSNPs rapidly, which can be used as valuable genetic markers in cattle.

Characterization and cardiac differentiation of chicken spermatogonial stem cells.

Spermatogonial stem cells (SSCs), a unique population of germline stem cells in adult testis, have the capability to self-renew and produce daughter cells destined to differentiate into spermatozoa throughout the life of the bird. Chicken SSCs were successfully isolated from testicular cells and subsequent analysis was performed to identify pluripotent cells by investigation with cytochemical reagents including Periodic acid-Schiff (PAS), alkaline phosphatase (AP), and antibodies to germline cell specific (DAZL or VASA) and stage-specific embryonic antigens (Oct4, SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81). Results confirmed these as germline cells with the expression of DAZL (Deleted in Azoospermia-Like) and VASA genes in isolated cells. Immunochemistry results showed that multipotent germline stem cells (mGSCs) expressed these gene markers related to embryonic stem cells (ESCs) and could spontaneously differentiate into three embryonic germ (EG) layers in vitro. The mGSC-derived cardiomyocytes expressed cardiac-specific markers such as sarcomeric alpha actinin, alpha-cardiac actinin; conexin-43, the major protein of gap junctions which are thought to have an important role in the synchronized contraction of the heart and in embryonic development; and cardiac troponin T, the tropomyosin binding subunit of the troponin complex which regulates muscle contraction. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) results indicated that the genes related to cardiac transcription factors were expressed following differentiation. Results of the present study strongly contribute to the information related to the ability of chicken mGSCs to differentiate into cells such as contraction cardiomyocytes similar to ESCs and may provide a new source of cardiomyocytes for basic research and potential therapeutic application in various cardiac degenerative diseases of birds and other animals.

Optimization of polygalacturonase production from a newly isolated Thalassospira frigidphilosprofundus to use in pectin hydrolysis: statistical approach.

The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20 °C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.

Generation of leukemia inhibitory factor-dependent induced pluripotent stem cells from the Massachusetts General Hospital miniature pig.

The generation and application of porcine induced pluripotent stem cells (iPSCs) may enable the testing for safety and efficacy of therapy in the field of human regenerative medicine. Here, the generation of iPSCs from the Massachusetts General Hospital miniature pig (MGH minipig) established for organ transplantation studies is reported. Fibroblasts were isolated from the skin of the ear of a 10-day-old MGH minipig and transduced with a cocktail of six human factors: POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28. Two distinct types of iPSCs were generated that were positive for alkaline phosphatase activity, as well as the classical pluripotency markers: Oct4, Nanog, Sox2, and the surface marker Ssea-1. Only one of two porcine iPSC lines differentiated into three germ layers both in vitro and in vivo. Western blot analysis showed that the porcine iPSCs were dependent on LIF or BMP-4 to sustain self-renewal and pluripotency. In conclusion, the results showed that human pluripotent factors could reprogram porcine ear fibroblasts into the pluripotent state. These cells may provide a useful source of cells that could be used for the treatment of degenerative and genetic diseases and agricultural research and application.

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult.

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences. The weights of clones at 6 and 12 months were slightly heavier than that of the donor. A horn pattern analysis revealed that seven clones had exactly the same horn pattern as the donor cow, whereas three were different. Although similarities such as general appearance can often be used to identify individual cloned animals, no study has characterized noseprint patterns for this end. A noseprint pattern analysis of all surviving clones showed that all eight animals had distinct noseprints. Four were similar to the donor, and the remaining four had more secondary-like characteristics.

The Pig Genome Database (PiGenome): an integrated database for pig genome research.

We established the Pig Genome Database (PiGenome) for pig genome research. The PiGenome integrates and analyzes all publicly available genome-wide data on pigs, including UniGenes, sequence tagged sites (STS) markers, quantitative trait loci (QTLs) data, and bacterial artificial chromosome (BAC) contigs. In addition, we produced 69,545 expressed sequence tags (ESTs) from the full-length enriched cDNA libraries of six tissues and 182 BAC contig sequences, which are also included in the database. QTLs, genetic markers, and BAC end-sequencing information were collected from public databases. The full-length enriched EST data were clustered and assembled into unique sequences, contigs, and singletons. The PiGenome provides functional annotation, identification of transcripts, mapping of coding sequences, and SNP information. It also provides an advanced search interface, a disease browser, alternative-splicing events, and a comparative gene map of the pig. A graphical map view and genome browser can map ESTs, contigs, BAC contigs (from the National Institute of Animal Science), Sino-Danish Pig Genome Project transcripts, and UniGene onto pig genome sequences which include our 182 BAC contigs and publically available BAC sequences of the Wellcome Trust Sanger Institute. The PiGenome is accessible at http://pigenome.nabc.go.kr/ .

Identification of marbling-related candidate genes in M. longissimus dorsi of high- and low marbled Hanwoo (Korean Native Cattle) steers.

This study was conducted to identify marbling-related candidate genes in M. longissimus dorsi of high- and low-marbled Hanwoo. The longissimus dorsi muscles were selected for gene expression from eight Hanwoo steer carcasses based on crude fat content. In the analysis of variance, gene expression of five candidate genes, FABP4, SCD, PPARgamma, Titin and Nebulin was determined to be significantly different between high- and low-marbled Hanwoo steers (P < 0.0001). The Pik-4 and CaMK II genes were also shown to have a significant effect on crude fat content (P < 0.01). In the analysis of the differential expression between high- and low marbled groups, FABP4 gene expression was approximately 2 times higher in the high marbled group relative to the low marbled group. However, the PPARgamma and SCD gene were highly expressed in the low marbled group. In addition, Titin and Nebulin were highly expressed in the low marbled group when placed under relatively high shear force. Finally, the Pik-4 and CaM K II gene also displayed a high expression pattern in the low marbled group.

Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6.

On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21-q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5' UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K(a)/K(s) ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs.