Progress on miRNA in giant panda (Ailuropoda melanoleuca).

MicroRNAs (miRNAs), a family of endogenous non-coding RNAs with a length of about 22 nucleotides, are widely found in eukaryotes. miRNAs can affect gene expression through specific bindings with mRNAs of target genes and participate in the regulation of a variety of biological processes. Giant panda is not only a unique rare animal in China, but also the focus of attention on wildlife preservation worldwide. In recent years, with the popularization of next-generation sequencing (NGS) technology, miRNAs in giant panda have been discovered and identified one after another. In this review, we focus on the research progress on miRNAs in giant panda, involved in immune response, mammary gland development, sperm freezing tolerance and other biological processes, and then discuss future research directions of miRNAs in giant panda, and thus providing the scientific references and new ideas for studying the regulatory mechanisms of miRNAs and promoting the breeding and protection of giant panda.

Total Synthesis of (±)-Clivonine via Diels-Alder Reactions of 3,5-Dibromo-2-pyrone.

New synthetic routes to (±)-clivonine were devised starting with the Diels-Alder cycloadditions of 3,5-dibromo-2-pyrone with styrene pinacol boronate dienophiles. In the first-generation synthesis, the pivotal perhydroindoline system including the C5-hydroxyl group was constructed via a reaction sequence involving the Eschenmoser-Claisen rearrangement and regio/stereoselective epoxide opening reaction. In the second-generation synthesis, a radical-mediated cyclization approach was employed for the rapid assembly of the pyrrolidine ring. In this route, the C5-hydroxyl group provided by the dienophile in a stereochemically defined form was preserved throughout the synthesis.

Transcriptome Sequencing Reveals the Differentially Expressed lncRNAs and mRNAs Involved in Cryoinjuries in Frozen-Thawed Giant Panda (Ailuropoda melanoleuca) Sperm.

Sperm cryopreservation and artificial insemination are important methods for giant panda breeding and preservation of extant genetic diversity. Lower conception rates limit the use of artificial insemination with frozen-thawed giant panda sperm, due to the lack of understanding of the cryodamaging or cryoinjuring mechanisms in cryopreservation. Long non-coding RNAs (lncRNAs) are involved in regulating spermatogenesis. However, their roles during cryopreservation remain largely unexplored. Therefore, this study aimed to identify differentially expressed lncRNAs and mRNAs associated with cryodamage or freeze tolerance in frozen-thawed sperm through high throughput sequencing. A total of 61.05 Gb clean reads and 22,774 lncRNA transcripts were obtained. From the sequencing results, 1477 significantly up-regulated and 1,396 significantly down-regulated lncRNA transcripts from fresh and frozen-thawed sperm of giant panda were identified. GO and KEGG showed that the significantly dysregulated lncRNAs and mRNAs were mainly involved in regulating responses to cold stress and apoptosis, such as the integral component of membrane, calcium transport, and various signaling pathways including PI3K-Akt, p53 and cAMP. Our work is the first systematic profiling of lncRNA and mRNA in fresh and frozen-thawed giant panda sperm, and provides valuableinsights into the potential mechanism of cryodamage in sperm.

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China.

BACKGROUND: Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China. RESULTS: Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species. CONCLUSIONS: These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-canids in China.

Developmental expression of three prmt genes in Xenopus.

Protein arginine methyltransferases (PRMTs) are involved in many cellular processes via the arginine methylation of histone or non-histone proteins. We examined the expression patterns of prmt4, prmt7, and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR). Xenopus prmt4 and prmt7 were expressed in the neural crest, brain, and spinal cord, and also detected in the eye, branchial arches, and heart at the tailbud stage. Specific prmt9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches. Quantitative RT-PCR indicated that the expressions of prmt4 and prmt7 were up-regulated during the neurula stage, whereas prmt9 maintained its low expression until the late tailbud stage, consistent with the whole-mount in situ hybridization results. Thus, the developmental expression patterns of these three prmt genes in Xenopus embryos provide a basis for further functional study of such genes.

Comparative Analysis of MicroRNA and mRNA Profiles of Sperm with Different Freeze Tolerance Capacities in Boar (Susscrofa) and Giant Panda (Ailuropodamelanoleuca).

Post-thawed sperm quality parameters vary across different species after cryopreservation. To date, the molecular mechanism of sperm cryoinjury, freeze-tolerance and other influential factors are largely unknown. In this study, significantly dysregulated microRNAs (miRNAs) and mRNAs in boar and giant panda sperm with different cryo-resistance capacity were evaluated. From the result of miRNA profile of fresh and frozen-thawed giant panda sperm, a total of 899 mature, novel miRNAs were identified, and 284 miRNAs were found to be significantly dysregulated (195 up-regulated and 89 down-regulated). Combined analysis of miRNA profiling of giant panda sperm and our previously published data on boar sperm, 46, 21 and 4 differentially expressed (DE) mRNAs in boar sperm were believed to be related to apoptosis, glycolysis and oxidative phosphorylation, respectively. Meanwhile, 87, 17 and 7 DE mRNAs in giant panda were associated with apoptosis, glycolysis and oxidative phosphorylation, respectively. Gene ontology (GO) analysis of the targets of DE miRNAs showed that they were mainly distributed on membrane related pathway in giant panda sperm, while cell components and cell processes were tied to the targets of DE miRNAs in boar sperm. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DE mRNAs indicated that most of these DE mRNAs were distributed in membrane signal transduction-related pathways in giant panda sperm, while those in boar sperm were mainly distributed in the cytokine-cytokine receptor interaction pathway and inflammatory related pathways. In conclusion, although the different freezing extenders and programs were used, the DE miRNAs and mRNAs involved in apoptosis, energy metabolism, olfactory transduction pathway, inflammatory response and cytokine-cytokine interactions, could be the possible molecular mechanism of sperm cryoinjury and freeze tolerance.

Complete mitochondrial genome sequence of Jianmen-guan phasianus colchicus (Aves, Gallifoemes, phasianidae) and its phylogenetic analysis.

Jianmen-guan phasianus colchicusare one of the most important and be cherished uncultivated animals, although, origins of the most J. phasianus colchicus remain unknown. Therefore, the complete mitochondrial genome of the J. phasianus colchicus (also named seven-colours J. phasianus colchicus, one of the oldest wild birds in China known for their preciousness) was obtained for the first time, the mitogenome is 166,786 bp in length, and it harbours 2rRNA genes, 13 protein-coding genes, 22tRNA genes, and a D-loop region. According to the phylogenetic tree, we can assume that Jianmen-guan and Xianju are within the same lineage, and J. phasianus colchicus is a different group with Red Jungle.

Inhibition of NF-κB Pathway and Modulation of MAPK Signaling Pathways in Glioblastoma and Implications for Lovastatin and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) Combination Therapy.

Glioblastoma is a common malignant brain tumor and it is refractory to therapy because it usually contains a mixture of cell types. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in a range of tumor cell types. Previously, we found that two human glioblastoma cell lines are resistant to TRAIL, while lovastatin sensitizes these glioblastoma cells to TRAIL-induced cell death. In this study, we investigated the mechanisms underlying the TRAIL-induced apoptosis in human glioblastoma cell lines by lovastatin. Furthermore, we have confirmed the anti-tumor effect of combination therapy with lovastatin and TRAIL in the subcutaneous brain tumor model. We showed that lovastatin significantly up-regulated the expression of death receptor 5 (DR5) in glioblastoma cell lines as well as in tumor-bearing mice with peri-tumoral administration of lovastatin. Further study in glioblastoma cell lines suggested that lovastatin treatment could inhibit NF-κB and Erk/MAPK pathways but activates JNK pathway. These results suggest that lovastatin sensitizes TRAIL-induced apoptosis by up-regulation of DR5 level via NF-κB inactivation, but also directly induces apoptosis by dysregulation of MAPK pathway. Our in vivo study showed that local peri-tumoral co-injection of lovastatin and TRAIL substantially reduced tumor growth compared with single injection of lovastatin or TRAIL in subcutaneous nude mice model. This study suggests that combined treatment of lovastatin and TRAIL is a promising therapeutic strategy to TRAIL-resistant glioblastoma.

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

BACKGROUND: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. METHODS: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. RESULTS: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. CONCLUSION: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

The Surgical Management of H-Type Rectovestibular Fistula: A Novel Modification.

Introduction H-type rectovestibular fistulas could be rare anorectal malformations or acquired diseases secondary to perianal infection. Various surgical procedures have been described in the literature, however the problem of recurrence still remains to be solved. We describe a novel modified surgical procedure and outcome in the management of these patients. Methods From 1999 to 2014, 14 patients who had an H-type rectovestibular fistula underwent the same surgical procedure performed by the same surgical team. Rectal-vestibular pull-through inside-out and endorectal mucosal advancement flap was used, including circumferential incision of the fistula from the opening on the rectal side, pulling the fistula inside-out, ligating the fistula, and mobilizing a rectal mucosal flap to cover the internal opening. Results All the patients have been followed-up for 12 months to 15 years with no recurrences and no incontinence. Conclusion Our surgical management is a simple, safe, and probable choice for the treatment of H-type rectovestibular fistula with favorable outcomes.

Analysis of the cytochrome c oxidase subunit 1 (COX1) gene reveals the unique evolution of the giant panda.

As the rate-limiting enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX) plays a crucial role in biological metabolism. "Living fossil" giant panda (Ailuropoda melanoleuca) is well-known for its special bamboo diet. In an effort to explore functional variation of COX1 in the energy metabolism behind giant panda's low-energy bamboo diet, we looked at genetic variation of COX1 gene in giant panda, and tested for its selection effect. In 1545 base pairs of the gene from 15 samples, 9 positions were variable and 1 mutation leaded to an amino acid sequence change. COX1 gene produces six haplotypes, nucleotide (pi), haplotype diversity (Hd). In addition, the average number of nucleotide differences (k) is 0.001629±0.001036, 0.8083±0.0694 and 2.517, respectively. Also, dN/dS ratio is significantly below 1. These results indicated that giant panda had a low population genetic diversity, and an obvious purifying selection of the COX1 gene which reduces synthesis of ATP determines giant panda's low-energy bamboo diet. Phylogenetic trees based on the COX1 gene were constructed to demonstrate that giant panda is the sister group of other Ursidae.

[Metabolic profiling of the short-term responses of Nicotiana tabacum leaves cultivated under different LED lights].

The physiologically mature tobacco (Nicotiana tabacum) leaves was exposed to different light-emitting diode (LED) lights, i.e. ultraviolet A (UV-A), blue, green, yellow, red, white, to investigate their short-term response. Results showed that: 1) 68 GC/MS-stable metabolites were detected by non-targeted method. In the PLS-DA score plot, tobacco leaf samples showed clear grouping in each light cultivating condition. 61 metabolites were identified in mass spectra library. Besides, 45 metabolites, mainly including organic acids, carbohydrates, TCA cycle intermediate metabolites and amino acids, showed significant differences among the six light treatments. Hierarchical cluster analysis (HCA) and heat map showed that differential metabolites could be divided into five groups, and there were significant differences among the six treatments, especially under red and blue lights. Except for the metabolites of group B, almost all other metabolites contents in tobacco leaves treated with red light were higher than those under blue light. 2) Contents of solanesol, 3 alkaloids and 5 polyphenols were measured with targeted method. 4 alkaloids, including nicotine detected by non-targeted method, showed similar variation among all treatments, of which red and yellow light increased alkaloid accumulation significantly. The kaempferol-3-O-rutinoside and rutin showed similar variation among the six treatments, with the lowest content under blue light and the highest content under yellow light, nevertheless, 3 other polyphenols were differently affected by light qualities. The aolanesol accumulation was significantly repressed by yellow light, but showed highest content under blue light. In conclusion, light quality affected many metabolic pathways significantly in tobacco, such as fatty acid metabolism, glycometabolism, alkaloid metabolism, amino acid metabolism, tricarboxylic acid cycle and shikimate pathway.

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

Analysis of the genetic diversity of the nematode parasite Baylisascaris schroederi from wild giant pandas in different mountain ranges in China.

BACKGROUND: Baylisascaris schroederi is one of the most common nematodes of the giant panda, and can cause severe baylisascarosis in both wild and captive giant pandas. Previous studies of the giant pandas indicated that this population is genetically distinct, implying the presence of a new subspecies. Based on the co-evolution between the parasite and the host, the aim of this study was to investigate the genetic differentiation in the B. schroederi population collected from giant pandas inhabiting different mountain ranges, and further to identify whether the evolution of this parasite correlates with the evolution of giant pandas. METHODS: In this study, 48 B. schroederi were collected from 28 wild giant pandas inhabiting the Qinling, Minshan and Qionglai mountain ranges in China. The complete sequence of the mitochondrial cytochrome b (mtCytb) gene was amplified by PCR, and the corresponding population genetic diversity of the three mountain populations was determined. In addition, we discussed the evolutionary relationship between B. schroederi and its host giant panda. RESULTS: For the DNA dataset, insignificant Fst values and a significant, high level of gene flow were detected among the three mountain populations of B. schroederi, and high genetic variation within populations and a low genetic distance were observed. Both phylogenetic analyses and network mapping of the 16 haplotypes revealed a dispersed pattern and an absence of branches strictly corresponding to the three mountain range sampling sites. Neutrality tests and mismatch analysis indicated that B. schroederi experienced a population expansion in the past. CONCLUSIONS: Taken together, the dispersed haplotype map, extremely high gene flow among the three populations of B. schroederi, low genetic structure and rapid evolutionary rate suggest that the B. schroederi populations did not follow a pattern of isolation by distance, indicating the existence of physical connections before these populations became geographically separated.

A sensitive and specific PCR assay for the detection of Baylisascaris schroederi eggs in giant panda feces.

Baylisascaris schroederi is one of the most common intestinal nematodes in giant pandas. It can cause severe baylisascariasis which is highly infectious in its natural hosts. A rapid and reliable diagnosis of parasite infections is crucial to protect giant pandas, as well as for environmental monitoring and disease surveillance. Here, we established a specific PCR assay for B. schroederi detection which was targeting a 331-bp long fragment of the mitochondrial cytochrome c oxidase subunit II (COII) gene. Fifty fresh fecal samples collected from captive giant pandas were tested by the established PCR assay and the traditional flotation technique. DNA extracted from a single B. schroederi egg could be successfully amplified, while no cross-reactivity was found with DNA from Ancylostoma caninum eggs. The detection rate of the PCR assay was 68%, which was higher than that of the traditional egg flotation (46%). Our findings demonstrated that the PCR assay is sensitive and specific for the detection and identification of B. schroederi eggs. Therefore, it could become a useful tool for the investigation of B. schroederi infections in giant pandas.

Molecular diagnosis of Baylisascaris schroederi infections in giant panda (Ailuropoda melanoleuca) feces using PCR.

The helminth Baylisascaris schroederi is one of the most harmful parasites infecting giant pandas (Ailuropoda melanoleuca). It is therefore important to develop an exact diagnostic technique to detect this parasite. Using a known number (1, 2, 3, 4, 5, 10, 25, 50, 100) of feces-isolated B. schroederi egg and adult DNA, we developed a PCR to detect a portion of the mitochondrial 12S rRNA and applied it to giant panda fecal samples. The method was sufficiently sensitive to detect B. schroederi DNA from isolated eggs in a fecal sample with a detection threshold of one egg. We detected B. schroederi in 88% of fecal samples, 30% higher than the conventional flotation technique. No cross-reactivity with other common nematode DNA was detected. Our PCR assay may constitute a valuable alternative for the diagnosis of B. schroederi infections.

Down syndrome critical region protein 5 regulates membrane localization of Wnt receptors, Dishevelled stability and convergent extension in vertebrate embryos.

The Glypican family of heparan sulfate proteoglycans regulates Wnt signaling and convergent extension (CE) in vertebrate embryos. They are predicted to be glycosylphosphatidylinositol (GPI)-tethered membrane-bound proteins, but there is no functional evidence of their regulation by the GPI synthesis complex. Down syndrome critical region protein 5 (Dscr5, also known as Pigp) is a component of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex, and is associated with specific features of Down syndrome. Here we report that Dscr5 regulates CE movements through the non-canonical Wnt pathway. Both dscr5 overexpression and knockdown impaired convergence and extension movements. Dscr5 functionally interacted with Knypek/Glypican 4 and was required for its localization at the cell surface. Knockdown of dscr5 disrupted Knypek membrane localization and caused an enhanced Frizzled 7 receptor endocytosis in a Caveolin-dependent manner. Furthermore, dscr5 knockdown promoted specific Dishevelled degradation by the ubiquitin-proteosome pathway. These results reveal a functional link between Knypek/Glypican 4 and the GPI synthesis complex in the non-canonical Wnt pathway, and provide the new mechanistic insight that Dscr5 regulates CE in vertebrate embryos by anchoring different Wnt receptors at the cell surface and maintaining Dishevelled stability.