Optimization of microsatellite genotyping system used for genetic diversity evaluation of Ailuropoda melanoleuca.

Microsatellite DNA is one of the most widely used genetic markers of giant panda, especially in population size estimation, paternity testing, and genetic diversity analysis. However, there are few reports on the physical locations of microsatellite markers on the chromosomes of the giant panda (Ailuropoda melanoleuca) and research on the performance of microsatellite in genotyping system and the PCR amplification conditions. In this study, we analyzed the chromosomal locations and evaluated the application values of 34 microsatellite markers, based on the giant panda genome reference sequence (ASM200744v2). We optimized the PCR reaction systems and amplification procedures for these 34 microsatellite markers. We found the low value of the microsatellite marker of Ame-µ10 in genetic application, and the necessity in redesigning the primers for gpz-6. Our research helps to improve the reproducibility and reliability of genotyping results and is of great significance for promoting the establishment and standardized application of the "A Regulation for Giant Panda Population Genetic Archives" in giant panda conservation.

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda. RESULTS: By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What's more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system. CONCLUSION: The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Prognostic effect of adjuvant chemoradiotherapy for patients with gastric cancer: an updated evidence of randomized controlled trials.

The prognostic effect of chemoradiotherapy in gastric cancer has been evaluated for decades while the results are still in debate and heterogeneous. We thus comprehensively updated the evidence through systematic review and meta-analysis to evaluate chemoradiotherapy in gastric cancer to determine its effect. Pubmed, EMBASE, and Cochrane Library from the earliest possible year to April 2017 were searched. Randomised controlled trials (RCTs) that assessed the effects of combined chemoradiotherapy for patients with gastric cancer compared with that of single chemotherapy were included. The main outcome measure was 5-year overall survival (OS) and the second was disease-free survival (DFS) or recurrence-free survival (RFS). Fifteen RCTs involving 3347 patients were included into this meta-analysis. Compared with single chemotherapy, the relative risk (RR) for 5-year OS for chemoradiotherapy was 1.05 (95% CI 0.88 to 1.25), with moderate heterogeneity across eligible trials (I2 = 55.7%, p = 0.016). Subgroup analyses and sensitivity analyses confirmed the consistent findings. We found that significant survival benefit for 5-year DFS/RFS for chemoradiotherapy over single chemotherapy (RR 0.89 95% CI 0.81 to 0.98) for patients with gastric cancer. This updated meta-analysis does not provide strong evidence for a 5-year survival benefit of chemoradiotherapy over chemotherapy alone in patients with gastric cancer. A clear advantage of chemoradiotherapy over chemotherapy has not been established. Further larger RCTs should be conducted to determine its true effect.

Erratum: MiR-4295 promotes cell growth in bladder cancer by targeting BTG1.

[This corrects the article on p. 4892 in vol. 8, PMID: 27904689.].

MiR-4295 promotes cell growth in bladder cancer by targeting BTG1.

microRNAs (miRNAs) have been demonstrated to contribute to tumor progression and metastasis, and have been proposed to be key regulators of diverse biological processes. In this study, we report that miR-4295 is deregulated in bladder cancer tissues and cell lines. To characterize the role of miR-4295 in bladder cancer cells, we performed functional assays. The overexpression of miR-4295 significantly promoted bladder cancer cell proliferation, colony formation, and migration. Moreover, its downregulation induced cell cycle arrest and apoptosis of bladder cancer cells. Furthermore, a luciferase reporter assay and rescue experiment indicated that miR-4295 directly targets BTG1 by binding its 3'UTR. In conclusion, these results demonstrate that miR-4295 acts as an oncogene and may be a potential biomarker for bladder cancer diagnosis and treatment.

[Enrichment of giant panda microsatellite markers using dynal magnet beads].

The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

[Pedigree analysis of captive giant panda].

Pedigree analysis of captive giant panda was conducted by Sparks Ver1.4 Software. The result shows that genetic drift has a strong effect on the loss of genetic diversity. And the dispersal of captive giant panda is an acute dangerous factor when all of these small populations would be declined if inbreeding happened. For this reason, all these small populations should be managed as a whole unit to approach the goal for long-term conservation.

[Paternity assignment of giant panda by microsatellite genotyping].

We used genotype data across 18 microsatellite loci to establish the paternity of giant panda cubs at the Chengdu Zoo and the Chengdu Research Base of Giant Panda Breeding. The results demonstrate that the combined exclusion probability using these 18 microsatellite loci is 0.999921 while confidence is 95 % when the mother is known; if mother is unknown then the exclusion probability is 0.994109, but the confidence of this is less than 80%. Since the mother-offspring relationship is known in captive populations, the results could resolve unknown paternities in the Chengdu ex situ populations. However, to establish accurately the genetic relationships of wild giant pandas,more microsatellite loci may be required.

Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.