Advances in genome-wide association studies for important traits in sheep and goats.

Genome-wide association study (GWAS), an effective strategy to identify genetic variants associated with complex traits, has been used to study candidate genes of economical traits in animals. With the recent completion of sheep and goat genomes, single nucleotide polymorphism (SNP) chips of different densities are developed and commercialized. All these advances have enlarged the collection of molecular markers and also shed new light on the genetics of traits of interest in sheep and goats. In this review, we focus on the adoption of GWAS for important traits in sheep and goats, such as horn types, wool, dairy, growth and meat, reproduction and disease types, etc., and summarize the populations, major statistical methods and results of the GWAS analysis. Moreover, we also discuss the current state of GWAS, aiming to provide a reference for further studies on the genetic background of the important traits of sheep and goats by GWAS.

[Inhibitory effects of human umbilical cord-derived mesenchymal stem cells on proliferation of peripheral blood mononuclear cells from spondyloarthritis patients].

OBJECTIVE: To explore the inhibitory effects of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on the proliferation of peripheral blood mononuclear cells (PBMC) from spondyloarthritis (SpA) patients. METHODS: A total of 12 SpA patients at Chinese PLA General Hospital were recruited from May 2012 to October 2012. Information on demographic characteristics, disease and functional activity was collected. Isolated PBMC were stimulated by phytohemagglutinin (PHA, 1 µg/ml) in the presence or absence of hUCMSC.The proliferation of hUCMSC was suppressed by irradiation with Co60 (30 Gy) before co-culturing with PBMC. The proliferation of PBMC was determined by Cell Counting Kit-8 (CCK-8). Cell cycle profiles of PBMC were analyzed by flow cytometry. The association of inhibitory effect of hUCMSC with the disease and functional activity of SpA patients was examined. RESULTS: After coculturing with hUCMSC by cell-to-cell contact for 5 days, the proliferation of PBMC stimulated by PHA (1 µg/ml) was significantly inhibited by hUCMSC in a dose-dependent manner.The inhibition rate of the proliferation of PBMC cocultured with hUCMSC by cell-to-cell contact was higher than that by Transwell culture (57% ± 17% vs 32% ± 12%, P < 0.01). Compared to PBMC cultured alone, a larger number of PBMC cocultured with hUCMSC were in phase G1 (86% ± 3% vs 68% ± 5%, P < 0.01) while a lower number of cells in phases S and G2 (8% ± 3% vs 26% ± 5%, P < 0.01). No association was found between the inhibitory effect of hUCMSC and the disease and functional activity. CONCLUSION: The proliferation of PBMC from SpA patients may be inhibited by hUCMSC. And hUCMSC have therapeutic potentials for SpA patients.

[Influence of environmental factors on DNA methylation].

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs) that transfer a methyl group from S-adenyl methionine (SAM) to the fifth carbon of a cytosine residue to form 5mC. DNA methylation affects the interaction between the histone and DNA, which changes the chromosome structure and has reverse relationship with gene expression in general. Up to now, more and more studies have confirmed that environmental factors can alter epigenetic modifications, which do not involve in changing DNA sequence. So it can explain the phenotype of creature in a certain degree. This article focused on the influence of environmental factors, such as temperature, nutrient supply, heavy metal, early stress and radiation, on DNA methylation change. As a matter of fact, it does not only change the DNA methylation in parents and offspring but also their behavior and phenotype. Overall, this review will help us get better understanding of the relationship between environmental factors and gene expression regulation.

Dictionary learning LASSO for feature selection with application to hepatocellular carcinoma grading using contrast enhanced magnetic resonance imaging.

Introduction: The successful use of machine learning (ML) for medical diagnostic purposes has prompted myriad applications in cancer image analysis. Particularly for hepatocellular carcinoma (HCC) grading, there has been a surge of interest in ML-based selection of the discriminative features from high-dimensional magnetic resonance imaging (MRI) radiomics data. As one of the most commonly used ML-based selection methods, the least absolute shrinkage and selection operator (LASSO) has high discriminative power of the essential feature based on linear representation between input features and output labels. However, most LASSO methods directly explore the original training data rather than effectively exploiting the most informative features of radiomics data for HCC grading. To overcome this limitation, this study marks the first attempt to propose a feature selection method based on LASSO with dictionary learning, where a dictionary is learned from the training features, using the Fisher ratio to maximize the discriminative information in the feature. Methods: This study proposes a LASSO method with dictionary learning to ensure the accuracy and discrimination of feature selection. Specifically, based on the Fisher ratio score, each radiomic feature is classified into two groups: the high-information and the low-information group. Then, a dictionary is learned through an optimal mapping matrix to enhance the high-information part and suppress the low discriminative information for the task of HCC grading. Finally, we select the most discrimination features according to the LASSO coefficients based on the learned dictionary. Results and discussion: The experimental results based on two classifiers (KNN and SVM) showed that the proposed method yielded accuracy gains, compared favorably with another 5 state-of-the-practice feature selection methods.

[Differential expression analysis of proteins in neutrophils between clinical mastitis and healthy dairy cows].

Neutrophils provide the first line of defense against invading pathogens and have been reported to play a key role in bovine mammary immune. To examine the differential expression of proteins in neutrophils between clinical mastitis and healthy dairy cows, a 2-dimensional electrophoresis gel map with high repeatability was constructed for bovine neutrophils. From this map, seven differentially expressed proteins were identified by MALDI-TOF MS which are believed to be involved in pathways such as cell metabolism, oxidative stress, and inflammatory reaction. The differentially expressed proteins identified in this study may provide the basis for bovine mastitis resistance breeding research.

[Animal gene pyramiding in cross populations].

Gene pyramiding aims at producing individuals with one superior economic trait according to the optimal breeding scheme involving selection of favorable target alleles or linked markers after crossing basal populations and pyramiding them into a single individual. In consideration of animal traditional cross program along with the features of animal segregating population, four types of cross programs and two types of selection strategies for gene pyramiding are performed from practice perspective of view, two population cross for pyramiding two genes (denoted II), three populations cascading cross for pyramiding three genes (denoted III), four population symmetrical (denoted IV-S) and cascading cross for pyramiding four genes (denoted IV-C), and various schemes (denoted cross program-A-E) were designed for each cross program with different levels of initial favorable allele frequencies, basal population sizes, and trait heritabilities. The process of gene pyramiding for various schemes were simulated and compared based on the population hamming distance, average superior genotype frequencies, and average phenotypic values. By simulation, the results showed that larger base population size and higher initial favorite allele frequency resulted in higher efficiency of gene pyramiding. The order of parent crossing was shown to be the most important factor in cascading cross, but had no significant influence on the symmetric cross. The results also showed that genotypic selection strategy was superior to phenotypic selection in accelerating gene pyramiding. The method and corresponding software would be used to compare different cross schemes and selection strategies. Moreover, our study would help to build the optimal gene pyramiding simulation platform.

[Diagnostic value of MRI in testicular tumor].

OBJECTIVE: To study the MRI manifestation of testicular tumor and the value of MRI in the diagnosis of the disease. METHODS: We retrospectively analyzed 23 cases of pathologically confirmed testicular tumor, and observed the morphological characteristics, signals and surrounding conditions of the tumor using plain and enhanced MRI scanning. RESULTS: Of the 23 cases, seminoma was identified in 7, mixed germinoma in 3, teratoma in 3, endodermal sinus tumor in 2, epidermoid in 1, Leydig cell tumor in 1, leucoma in 1, nonspecific inflammatory mass in 3, and tuberculosis in 2. MRI revealed the precise locations and specific characteristics of CONCLUSION: Based on MRI findings and clinical manifestation, most testicular tumors can be diagnosed correctly.

The Co-Expression of Melanoma-Antigen Family a Proteins and New York Esophageal Squamous Cell Carcinoma-1 in Breast Cancer: A Pilot Study.

OBJECTIVE: The aim of this study was to quantify the expression of melanoma-antigen family A proteins (MAGE-A) and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in breast cancer and establish the prognosis of breast cancer patients with MAGE-A and NY-ESO-1 co-expression. METHODS: A total of 122 patients with breast cancer were recruited for this study. Their clinicopathological data were collected retrospectively, and the MAGE-A and NY-ESO-1 expressions in paraffin-embedded specimens from the 122 patients were evaluated using immunohistochemical analysis. In addition, the survival states of the patients were recorded. RESULTS: Fifty-four patients (44.26%) were MAGE-A positive and 46 (37.70%) were NY-ESO-1 positive. Interestingly, 22 of the 46 NY-ESO-1-positive cases co-expressed MAGE-A. The expression of MAGE-A was positively associated with estrogen-receptor status (χ2 = 4.026, p = 0.045) and human epidermal growth factor receptor 2 status (χ2 = 5.482, p = 0.019), while the expression of NY-ESO-1 was positively associated with p53 expression (χ2 = 4.541, p = 0.033). Of the 122 patients, the lowest survival rate was observed in patients with NY-ESO-1 (+)/MAGE-A (+), with a 5-year survival rate of 59.09% and a median survival of 97 months. CONCLUSION: The results showed that MAGE-A and NY-ESO-1 were frequently expressed in breast cancer patients. The co-expression of MAGE-A and NY-ESO-1 occurred in about 18% of these patients, and it may indicate a poor prognosis.

Directed differentiation of chick embryonic germ cells into neural cells using retinoic acid induction in vitro.

Embryonic germ cells (EG) are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Like embryonic stem cells (ES), EG are also capable of proliferation and self-renewal and have the capacity to differentiate in vitro into all cell types. To date, it has been proven that ES are capable of directed differentiation into neural precursors and progenies in mammals. However, similar studies on EG in mammals and other species are few. This investigation aimed to induce chick EG to differentiate into neural cells and compare the difference of efficiency between directed differentiation and spontaneous differentiation. EG were isolated and identified from 5.5-day chick gonadal PGC, incubated and passaged in conditioned medium. After the formation of embryoid bodies (EB), EB were grown in suspension and induced by retinoic acid (RA), using a protocol named 4-/4+, to make the formation of neurospheres and progenies. RT-PCR and immunocytochemistry analysis demonstrated that neural-specific markers can be detected after directed induction. Moreover, EG differentiated into neural lineage cells using 4-/4+ protocol much more efficiently than that in the spontaneous differentiation with fluorescence-activated cell sorting (FACS) analysis. The results revealed that RA can obviously promote the directed differentiation of chick EG into neural lineage.

[Optimizing the expression of Mx gene in Escherichia coli based on rare codon and mRNA structure].

Rare codon and mRNA secondary structure of translation initiation region were analyzed. Four bioengineered bacterium BL21(DE3)/pET-Mx, Rosseta (DE3)/pET-Mx, BL21(DE3)/pGEX-Mx, and Rosseta (DE3)/pGEX-Mx were obtained. Through optimizing the rare codon and mRNA secondary structure of translation initiation region, Mx protein was expressed in Rosseta (DE3)/pET-Mx, and Rosseta (DE3)/pGEX-Mx. The specified product of 75 kDa was detected by means of Western blotting analysis. The result showed that Rosetta (DE3) strain expressing some rare codon used in our experiment can obtain Mx protein expression, and the lower energy of mRNA structure can improve the expression level of Mx protein. This is the first report on the expression of the full open reading frame of Mx gene.

[Cloning and characterization of CAST transcript 2 and 4 in sheep].

As an endogenous inhibitor of the calpain system activated by Ca2+, calpastatin (CAST) plays a regulatory role in muscle proteolysis. Based on the bovine mRNA sequences, part of cDNA fragments of sheep CAST transcript 2 and 4 were obtained by RT-PCR. Bioinformatic analysis showed that sheep CAST transcript 2 was 4 358 bp in length with an open reading frame (ORF) 2 361 bp long and encoded 786 amino acids, while sheep CAST transcript 4 was 1 467 bp in length with 1 317 bp ORF encoding 438 amino acids. It was predicted that CAST type II contained four conserved domains and CAST type IV contained three conserved domains, and their secondary structures were rich in both hydrophobic regions and helical regions, with certain conserved phosphorylation sites and phosphorylation sites of protein kinase C (PKC). RT-PCR was conducted to analyze the expression patterns of CAST transcript 2 and transcript 4. CAST transcript 2 was ex-pressed in ten tissues detected while CAST transcript 4 only in testis.

[Identification of SNPs located in putative microRNA tar-get region of six functional genes in chickens through bioinformatic analysis].

GDF-8, IGF-I, IGF-III, IGF2R, IGFBP2, and GHR are candidate genes affecting important economic trait in chicken. The putative microRNA target sites of 3' untranslated region of these genes were identified by means of specialized algorithms (miRanda and TargetScan), and the candidate SNPs located in the microRNA target region were also identified. Approximately 125 candidate SNPs were found throughout the 26 putative microRNA target regions in six gene 3'-UTR. Among the 125 SNPs, 47 were located in the microRNA targets, 44 and 35 were located in 5'and 3'flanking regions which equalled to the size of the given target. Twelve of the 47 candidates were located in the match of the microRNA seed. These SNPs, which were located in the match of the microRNA seed and 3 ' flanking regions may affect microRNA regulation and contribute to poultry phenotypic variation.

[Progress on the research of microRNAs and its function in humans and animals].

MicroRNAs, a class of noncoding RNA of approximately 22 nucleotides, can regulate gene expression by binding to the region of 3' UTR of the target mRNAs. To date, it has been demonstrated for organism that miRNAs play an important role in growth, development,and occurrence of disease. This paper introduces the character and the active mechanism of miRNAs, and the newest progress in the research on the function of microRNA, the identification of microRNA gene and the prediction of target gene also are summarized here.

[Components and assembly of RNA-induced silencing complex].

Degradation of homologous RNA in RNA interference is carried out by functional RNA-induced silencing complex (RISC). RISC contains Dicer, Argonaute proein, siRNA and other components. Researching structures and functions of these components is primary important for understanding assembly and functional mechanism of RISC, as well as the whole RNAi pathway. Recent research works showed that Dicer, containing RNaseIII domain, is responsible for production of siRNA at the beginning of RNAi, and guarantees the stability of RISC intermediate in assembly process. As the core component of RISC, Argonaute protein functions as slicer to cleave target RNA and offers the binding site of siRNA in RISC assembly, which are depended on PIWI domain and PAZ domain separately. Although there is only one strand of siRNA that is the guider of RISC, the double stranded structural character of siRNA is determinant of RNAi. Except those, there are still other components with unknown functions in RISC. The knowledge about RISC components and assembly now, is basis of a presumed RISC assembly model.

[Biological characteristics of the 5' regulatory region of FSHR gene in sheep].

The 5' transcriptional promoter regulation region of FSHR gene was cloned and analyzed in Little-tailed Han sheep, Tan sheep and Australian Merino sheep. Comparison of 15 transcriptional regulatory elements in the FSHR gene showed no difference in the regulatory region of FSHR gene among the three breeds. The results showed no association between sheep breeds and 5' transcriptional promoter regulation region and eliminated the possibility that the mutation of transcriptional regulation region can affect the transcription of FSHR gene in sheep.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. METHODS: A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. RESULTS: This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. CONCLUSION: This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

[Relationship of T-->A mutation in the promoter region of myostatin gene with growth traits in swine].

A T-->A mutation in the promoter region of porcine myostatin (MSTN) gene has been identified in previous work. Associations of the myostatin genotypes with growth traits are unknown in swine. The present study attempts to analyze the relationship of the mutation with the growth traits which included body weight at 60 d (BW60), average daily gain from 25 kg to 60 kg(ADG1), average daily gain from 60 kg to 100 kg (ADG2) and average daily gain from 25 kg to 100 kg (ADG). Data from 165, 275, 276 and 276 unrelated individuals respectively were collected from three different swine breeding companies. Detections of the mutation were carried out by PCR-RFLP approach. The effect of MSTN genotypes (TT and TA) on growth traits was estimated by GLM procedure. The results showed that for ADG2, individuals with TA genotype were higher than those of TT genotype (P = 0.052), indicating a positive effect for A allele. For BW60, ADG1 and ADG, the effect of porcine MSTN genotype was non-significant (P > 0.1). Studies are still necessary for examining the effects in "double-muscled" pigs.

[Studies of BMPR-IB and BMP15 as candidate genes for fecundity in little tailed han sheep].

BMPR-IB gene which controls the fecundity of Booroola Merino and BMP15 gene which affects the ovulation of Invedale and Hanna were studied as candidate genes on the fecundity of Little Tailed Han Sheep, and their mutations and genetic effects were analyzed. The results showed that there was a same mutation in BMPR-IB gene (A746G) of Little Tailed Han Sheep as that of Booroola Merino. The BB mutation genotype was superior in prolific Little Tailed Han Sheep, and the ewes with genotype BB had 0.97(P < 0.05) and 1.5(P < 0.01) lambs more than those with genotype + + in the first parity and later parities, respectively. It could be inferred that BMPR-IB gene was related with the major gene that controls the high prolificacy of Little Tailed Han Sheep. While there was not mutation of V31D or Q23Ter in BMP15 gene of Little Tailed Han Sheep, it showed that the fecundity mechanism of Little Tailed Han Sheep was different from that of Romney sheep. Then it was ruled out the possibility that the ovulation of Little Tailed Han Sheep was affected by the mutation of BMP15 gene.

[Analysis of genetic diversity of Shandong indigenous chicken breeds using microsatellite marker].

Microsatellite marker is one of the frequently used molecular markers. It has been used in the genotype identification, pedigree analysis and estimation of genetic distance. In this paper, five microsatellite markers with high polymorphisms were selected to detect the genetic diversity of seven chicken breeds. The alleles frequencies, polymorphism information content (PIC) and average heterozygosity within each population, and DA genetic distance among breeds were analyzed. The application of microsatellite polymorphisms to the detection of genetic variability and relationship among populations was discussed. Altogether, forty alleles were found in this experiment, and among them the most alleles (10) were detected by ADL0136 and the least (5) were detected by ADL0146. The distribution of alleles was not balanced, each locus having one or more dominant alleles. The average heterozygosity in the Shouguang chicken was the lowest (0.3327), and that in other breeds was also less than 0.4. It can be seen then that microsatellite polymorphisms can be used to reveal the variability within population through calculation of average heterozygosity. The PIC values ranged from 0.6169 (Shouguang chicken) to 0.7027 (Laiwu Black chicken). UPGMA tree was completed through analysis of DA genetic distance. In the tree, the Rizhao Pockmarked and the Jining Hundred chicken were first grouped together with a bootstrap value of 92%, before they were grouped with the Laiwu Black and the Shouguang chickens. The Anoka Yellow and the Guangxi Yellow chicken were grouped together with a bootstrap value of 80%, but the Luxi Fighting chicken had its own branch. In summary, the UPGMA tree well reflected the evolutionary and breeding history of the seven breeds.

[Research on Alu element inserted mutation in porcine FSH beta subunit gene].

The inserted fragment of FSH beta subunit gene in Laiwu pigs, an excellent local pig breed in North China, Duli pigs and Landrace pigs was amplified, cloned and sequenced. Sequence analysis showed that the length of the fragments inserted between +809 bp and +810 bp of the published sequences (D00621) were 275 bp, 277 bp and 274 bp in Laiwu, Duli and Landrace pigs, respectively. And the poly (A)s in these inserted fragments were 17, 19 and 16 adenines, respectively. They were all shorter than those in Taihu pigs(292 bp and 32 adenines, respectively) reported formerly. According to the RNA polymerase III promoter structure and Alu I restriction enzyme site in the inserted fragment, it should be regarded as an Alu element. FSH beta subunit gene was considered as one candidate gene of pig litter size trait, while RNA polymerase III promoter could promote the transcription of the neighboring chromosome sequence so as to control the expression of FSH beta subunit gene or other genes. As a result, the major difference of the inserted fragment among different pig breeds was originated from the length of poly (A) end. So it was assumed that the poly (A) structure in the inserted fragment could influence the pig litter size. Because Laiwu pigs with genotype AA had 1.2 litter size more than those with genotype BB, it could be concluded that FSH beta subunit gene was related with pig litter size traits or linked with the genes of which, and the poly(A) structure in this Alu element played a critical role.