Screening potential SSR markers of the anadromous fish Coilia nasus by de novo transcriptome analysis using Illumina sequencing.

RNA-Seq technology has been widely applied to transcriptomics, genomics, molecular marker development, and functional gene studies. In the genome, microsatellites are simple sequence repeats (SSR) with a high degree of polymorphism that are used as DNA markers in many molecular genetic studies. Using traditional methods such as magnetic bead enrichment, only a few microsatellite markers have been isolated. Coilia nasus is an anadromous, small-to-moderately sized fish species that is famous as an important fishery resource. Here, we have identified a large number of microsatellites from the fish brains by using Illumina sequencing. About 20 million Illumina reads were assembled into 148,845 unigenes. A total of 13,038 SSR motifs were identified via analysis of 3,958,293,117 (3.96 Gb) nucleotides to produce a comprehensive transcript dataset for the C. nasus brain, including mono-, di-, tri-, tetra-, and penta-repeat motifs. The most abundant type of repeat motif was di-nucleotide (42.97%), followed by mono-nucleotide (38.86%), tri-nucleotide (16.21%), tetra-nucleotide (1.83%), and penta-nucleotide (0.05%) repeat units, which is similar to the results obtained in studies in other species. These data provide a base of sequence information to improve molecular-assisted markers to study C. nasus genetic diversity.

Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma.

Alteration of human leukocyte antigen (HLA) expression, such as decreased HLA I (HLA-A, -B and -C) antigens and elevated nonclassical HLA I antigens (HLA-E, -F and -G), was reported to have an unfavorable prognosis in various cancers. In our study, HLA-F expression in 105 primary esophageal squamous cell carcinoma (ESCC) lesions and 62 case-matched adjacent normal tissues, and HLA I antigens among 68 cases were analyzed by immunohistochemistry. Data revealed that HLA-F expression was observed in 58.1% (61/105) of the ESCC lesions and in 54.8% (34/62) of the normal esophageal tissues. Among the 62 case-matched samples, HLA-F expression (lesion vs. normal tissue) was upregulated, unchanged and downregulated in 13 (21.0%), 6 (9.6%) and 43 (69.4%) cases, respectively. Patients with HLA-F positive had a worse survival than those with HLA-F negative (p = 0.040). Patients with upregulated HLA-F expression (lesion vs. normal tissue) had significantly worse survival than those with HLA-F unchanged and downregulated (p = 0.010). Furthermore, decreased HLA I expression was observed in 41.2% (28/68) patients and was with worse prognosis in comparison to those with preserved HLA I expression (p = 0.001). Multivariate analysis using Cox's proportional hazards model revealed that upregulated HLA-F expression (p = 0.026) and downregulated HLA I expression (p = 0.013) could be an independent unfavorable prognostic factor. In conclusion, our study provided the evidence that alteration of HLA I and HLA-F antigen expression was associated with survival in patients with ESCC.

VNTR polymorphism of human IL1RN in Chinese Han and She ethnic populations.

Interleukin 1 receptor antagonist (IL-1Ra) is an important anti-inflammatory molecule encoded by the IL1RN gene. The polymorphism of IL1RN characterized by variable numbers of an 86 bp tandem repeat (VNTR) sequence in intron 2 has been described. Moreover, frequencies of the IL1RN alleles vary among different ethnics. In the present study, we analysed the IL1RN polymorphism in intron 2 in 256 Chinese Han and 252 Chinese She individuals. Four alleles including IL1RN*1, *2, *3 and IL1RN*4 were identified in this study. Data revealed that the distribution of the IL-1RN genotypes and allele was significantly different between the two Chinese populations (P < 0.001). Among them, 66.8% (171/256) and 86.5% (218/252) were homozygous for the allele IL-1RN*1 in Chinese Han and She individuals respectively. Homozygosity for allele IL-1RN*2 was only observed in Chinese Han with the percentage of 0.8% (2/256). Heterozygosity for IL-1RN*1/2, IL1RN*1/3 and IL1RN*1/4 was 30.9% (79/256), 0.4% (1/256) and 1.2% (3/256) in Chinese Han, whereas only heterozygosity for IL-1RN*1/2 was found in Chinese She (13.5%, 34/252). Frequencies of the most common allele IL-1RN*1 and IL-1RN*2 were 83.0% and 16.2% for Chinese Han and 93.3% and 6.7% for Chinese She respectively. The rare allele IL-1RN*3 and IL-1RN*4 was only observed in the Chinese Han population with the frequency of 0.2% and 0.6% respectively. Our findings suggested that the ethnic background plays an important role in IL-1Ra gene variation in different populations.

Polymorphism of human CD1a, CD1d, and CD1e in exon 2 in Chinese Han and She ethnic populations.

CD1 molecules are the major histocompatibility complex (MHC)-like glycoproteins specialized in capturing and presenting a variety of glycolipid to antigen-specific T-cells. There are five closely linked CD1 genes termed as CD1a, CD1b, CD1c, CD1d, and CD1e. CD1 gene features limited the polymorphism in exon 2 which encodes for the alpha1 domain. Few investigations on the allele frequencies of the CD1 genes have been reported to date; however, variation of CD1 allele frequency in different ethnics has been observed. In the current study, the CD1a, CD1d, and CD1e gene polymorphisms in exon 2 (alleles 01 and 02) in a group of normal Chinese Han and She individuals were analyzed. Similar allele prevalence was observed between the two populations. The CD1e allele frequency was 37.1% (allele 01); 62.9% (allele 02) and 39.3% (allele 01); 60.7% (allele 02) for Han and She populations, respectively. CD1e was the only polymorphic gene with a genotype frequency for Chinese Han (01/01, 11.0%; 01/02, 52.2%; 02/02, 36.8%) and She (01/01, 13.2%; 01/02, 52.1%; 02/02, 34.7%) individuals, respectively. No CD1a allele 01 and CD1d allele 02 were observed in either population. Our findings indicate that the polymorphism of CD1a, CD1d, and CD1e genes in exon 2 is very limited in the Chinese Han and She ethnics.

Ethnic variation of the HLA-G*0105N allele in two Chinese populations.

Unlike high polymorphic classical human leukocyte antigen (HLA) class I molecules, the genetic polymorphism of HLA-G is very limited. However, the prevalence of HLA-G alleles among different ethnic populations varied dramatically. The HLA-G null allele (HLA-G*0105N) is defined by a cytosine deletion (Delta C) at position 1597 in exon 3, which disrupts the reading frame and alters the expression of HLA-G proteins. The HLA-G*0105N allelic frequency was investigated in previous studies and possible roles were addressed. In the current study, a total of 310 Chinese Han and 260 Chinese She ethnic minority population had been genotyped for the G*0105N polymorphism. Marked difference was observed that the G*0105N allelic frequency in Chinese Han was 1.61%, while no copy of the null allele was observed in the Chinese She minority population (P(c) = 0.0073). Data also revealed that no homozygote of HLA-G*0105N allele exists in this Chinese Han population. Furthermore, significant difference was found for the frequencies of HLA-G*0105N both in Chinese Han and in Chinese She populations when compared with other ethnic populations. Taken together, our results indicated that ethnic variation of the HLA-G*0105N polymorphism among different ethnic populations is possibly the result of evolution. However, the advantages of the selection of this allele are necessary to be further investigated.

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma.

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.