首例大熊猫睾丸精原细胞瘤的病理学诊断

<正>大熊猫(Ailuropoda melanoleuca),是我国濒危珍稀的国家一级保护动物,其疾病的研究与防控受到高度关注。目前在寄生虫疾病、病毒性疾病、细菌性疾病(李玉欢等,2013;杨虎田,2014; Jin et al.,2017)等方面进行了大量研究,对大熊猫保护起到了重要作用。肿瘤作为一种严重影响人类与动物健康的疾病一直是人类医学与动物医学研究的重要内容。目前,报道圈养野生动物肿瘤疾病的文献比例较低(张成林等,2013;曹霞,2019),此类疾病对患病个体伤害极大。近年来,     

大熊猫初生胎儿及幼仔组织器官的病理学研究

To elucidate the cause of prenatal and early postnatal death in giant panda, pathological studies have been carried out on paraffin-fixed tissue sections from two fetuses and four cubs. The fetuses appeared to have classical atrophic changes in lung and hemorrhage in multiple organs, whereas the cubs showed purulent inflmnmation in various organs, most profound in lung and umbilicus. Localized infusion of bacteria and neutrophils were identified in the focus. Acute enteritis and hepatitis were also observed, as well as purulent encephalitis in one case. Variable degrees of congestion, hemorrhage, denaturalization and putrescence were evident in heart, liver, spleen, kidney, intestines, and lymph nodes. The results indicated that the fetuses died from suffocation whereas the cubs died from infection.     

唐家河自然保护区大熊猫种群数量及栖息地的移动

本文根据大熊猫季节性活动规律特点,应用海拔高度路线调查法,全面收集唐家河自然保护区大熊猫的比较新鲜粪便,计获１２４个粪样材料,提取ＤＮＡ作ＤＮＡ指纹图,经微机检测,保护区有大熊猫３７只,个体数量与粪样采集量之比为１∶３３５１４,有６个家系,雌兽２１只,雄兽１６只。并搞清了保护区生态环境质量,箭竹遭受病虫害的程度,洪水对栖息地的影响,特别对引起大熊猫数量减少及栖息地移动等原因,作了较为详细的分析。     

基于野外痕迹点和GIS技术定量评估步道对大熊猫活动的影响

道路建设不仅直接导致野生动物死亡,还能对栖息地形成阻碍效应,导致小种群出现或隔离,增加物种灭绝的风险。生态学家在道路对野生动物影响研究中的一个重要进展是道路影响域(road-effectzone)的提出,但影响域既不能反映道路影响的变化性,也难以满足栖息地评估对数据的要求。为此,我们以大熊猫(Ailuropoda melanoleuca)为例来探讨道路影响的定量评估方法。在佛坪和长青保护区内选择了3条步道,获取了步道周边1,042个大熊猫的痕迹点数据,通过GIS计算各痕迹点到步道的距离,统计距离步道每20m内的痕迹点数量,以此作为其活动频率。在距步道每100m处设置检测点,通过非参数检验比较检测点前后活动频率分布的变化,寻找道路对大熊猫活动影响的突变点,确定影响变化的阈值距离和评价标准。研究发现在距离步道1,000m内,随距离的增加,大熊猫活动频率逐渐增大,大熊猫有明显的回避效应;在距步道500m、1,000m处发现活动频率发生了显著变化,为影响的阈值距离。本研究基于痕迹点和阈值距离的评估方法可以反映道路影响连续、渐变的特点,使定量、准确评估其影响成为可能。     

大熊猫尸体组织LDH同工酶盘电泳观察

大熊猫是冰川时期残存至今的古老、稀有的珍贵动物,有“活化石”之称。对大熊猫的研究除野外调查(王朗自然保护区大熊猫调查组,1974)、形态解剖(张鹤宇等,1959;冯文和等,1984)、人工饲养繁殖(北京动物园,1974;冯文和等,1983;1984)等外,尚有生化技术和免疫学等研究方法(Sarich,1973;潘文石等,1982;罗昌容等,1984)。     

Endophytic fungi and silica content of different bamboo species in giant panda diet

The giant panda (Ailuropoda melanoleuca), one of the most threatened mammalian species in the world, has adapted to herbivorous diet consisting mainly of bamboo (Poaceae: Bambusoidea). The most acute threats to the survival of the giant panda are habitat loss and fragmentation. However, changes in habitat may influence also the quality of giant panda diet through the bamboo species composition as well as their symbiotic leaf endophytes and plant chemical properties. Here we explore species composition and frequency of endophytic fungi and silica content in different bamboo species in the range of giant panda habitat in relation to panda food preference. Silica content of the bamboos varied from 3.7 g/kg to 45.7 g/kg and did not correlate with panda preference and altitudinal gradient. Systemic and vertically in seeds transmitted fungal endophytes or bacterial endophytes were not detected in bamboo leaves. Nearly half of the identified endophytic fungi belonged to genus Arthrinium. Pandas preferred bamboo species naturally occurring in higher altitudes. Furthermore, the total amount of endophytes tended to be lower in samples collected from bamboos in higher altitudes. This draws attention to the importance of more detailed studies on the endophytic fungi-bamboo-panda trophic interactions and the effect of land use and climate change on conservation programs of giant panda.     

An improved,chromosome-level genome of the giant panda (Ailuropoda melanoleuca)

BackgroundThe iconic giant panda (Ailuropoda melanoleuca), as both a flagship and umbrella species endemic to China, is a world famous symbol for wildlife conservation. The giant panda has several specific biological traits and holds a relatively small place in evolution. A high-quality genome of the giant panda is key to understanding the biology of this vulnerable species.FindingsWe generated a 2.48-Gb chromosome-level genome (GPv1) of the giant panda named “Jing Jing” with a contig N50 of 28.56 Mb and scaffold N50 of 134.17 Mb, respectively. The total length of chromosomes (n = 21) was 2.39-Gb, accounting for 96.4% of the whole genome. Compared with the previously published four genomes of the giant panda, our genome is characterized by the highest completeness and the correct sequence orientation. A gap-free and 850 kb length of immunoglobulin heavy-chain gene cluster was manually annotated in close proximity to the telomere of chromosome 14. Additionally, we developed an algorithm to predict the centromere position of each chromosome. We also constructed a complete chromatin structure for “Jing Jing”, which includes inter-chromosome interaction pattern, A/B compartment, topologically associated domain (TAD), TAD-clique and promoter-enhancer interaction (PEI).ConclusionsWe presented an improved chromosome-level genome and complete chromatin structure for the giant panda. This is a valuable resource for the future genetic and genomic studies on giant panda.     

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.     

大熊猫神经营养素-4基因在大肠杆菌中的表达

本通过PCR技术,直接从大熊猫基因组DNA上克隆得到其神经营养素—4的成熟肽编码序列,通过序列分析发现,该基因在进化上具有较高的保守性。将神经营养素—4成熟肽完整编码序列克隆至pGEX—4T—3表达载体,并经IPTG诱导在大肠杆菌中进行原核生物表达,获得了大熊猫重组蛋白神经营养素—4。重组表达蛋白经纯化后,进行大鼠肾上腺嗜铬瘤细胞神经营养因子的活性鉴定,发现其能够诱导神经细胞分化产生突触,具有预期的生物学活性。对大熊猫神经营养素—4的基因工程研究,为大熊猫癫痫的基因治疗奠定了基础。     

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.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.     

Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury.     

Preliminary investigation on iodine nutrition in captive giant pandas

Background/ObjectiveThe giant panda belongs to the family Ursidae and, as a species of bear, still retains the simple digestive system of a Carnivoran. However, under the pressure of a specific habitat they had to adapt to a plant mono-diet consisting of bamboo with different species and growth stages around the year. A plant-based diet has relatively low iodine content with risk of iodine deficiency. Furthermore, bamboo contains cyanogenic glycosides releasing cyanide whose detoxification metabolite the thiocyanate acts as antagonist against iodine uptake and storage in the thyroid. To date very little is known about the iodine nutritional status of the giant panda, thus this study was conducted to receive the first information about the iodine nutrition of captive giant panda.Subjects/MethodsHere we investigated the iodine content of bamboo with different plant parts/vegetation stage and species and further compounds of the captive giant panda diet. Next, the urinary iodine (UI) and urinary thiocyanate (UT) levels of infant, sub-adult, adult and geriatric captive giant pandas was measured during the periods when the pandas consume both bamboo leaves- and culm (bamboo leaf-culm stage). Afterwards, the UI of 19 adult giant pandas was measured again for the different iodine intake during bamboo shoot stage. Finally, in this study part also the fecal iodine concentration was analyzed for calculation of total iodine excretion in relation to the iodine intake.ResultsBamboo leaves had the highest iodine content (453 μg/kg dry matter (DM)), followed by the shoots (84 μg/kg DM, p < 0.05), while bamboo culm had the lowest value (12 μg/kg DM, p < 0.05). During bamboo leaf-culm stage, giant pandas of different age groups had different UI and UT levels (p < 0.05). Furthermore, UI and UT were positively correlated among sub-adult, adult and geriatric giant pandas (p < 0.05). In adult giant pandas during bamboo shoot stage, the iodine excretion in feces was not different from that in urine while their total iodine excretion was less than their iodine intake (p < 0.05). Moreover, during bamboo shoot stage, the UI level of adult giant pandas was much lower than noted during bamboo leaf-culm stage (p < 0.05).ConclusionsOur results indicate that UI of captive giant pandas was related to their age as well as to the vegetation stage/part of bamboo they consumed reflecting a different periodic iodine supply. Thiocyanate and fecal excretion should be emphasized when considering the iodine nutrition of giant pandas.     

cDNA cloning and expression of ghrelin in giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein (prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide. The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids (Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart.     

马边大风顶自然保护区大熊猫种群生存力模拟分析

采用漩涡模型Vortex mondel 713 , 模拟了马边大风顶自然保护区大熊猫种群在未来100年内的变动趋势。结果显示: 在无交配限制、无密度制约、无近亲交配衰退等条件下, 马边大风顶自然保护区大熊猫种群数量呈下降趋势; 在考虑近交衰退的影响后, 遗传多样性水平降低, 灭绝率提高; 竹子开花虽能加速大熊猫种群的绝灭, 但由于保护区分布有多个竹种,因此并不会对大熊猫种群产生灾难性影响; 但是人为捕杀可迅速减少大熊猫种群数量, 加速其灭绝过程。因此, 对该保护区大熊猫进行保护的最重要措施就是严格控制人为捕杀, 并保护栖息地及走廊带。     

Prosurvival Bcl-2 proteins stabilize pancreatic mitochondria and protect against necrosis in experimental pancreatitis

Acinar cells in pancreatitis die through apoptosis and necrosis, the roles of which are different. The severity of experimental pancreatitis correlates directly with the extent of necrosis and inversely, with apoptosis. Apoptosis is mediated by the release of cytochrome c into the cytosol followed by caspase activation, whereas necrosis is associated with the mitochondrial membrane potential (ΔΨm) loss leading to ATP depletion. Here, we investigate the role of Bcl-2 proteins in apoptosis and necrosis in pancreatitis. We found up-regulation of prosurvival Bcl-2 proteins in pancreas in various experimental models of acute pancreatitis, most pronounced for Bcl-xL. This up-regulation translated into increased levels of Bcl-xL and Bcl-2 in pancreatic mitochondria. Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss and cytochrome c release in isolated mitochondria. Corroborating the results on mitochondria, Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss, ATP depletion and necrosis in pancreatic acinar cells, both untreated and hyperstimulated with CCK-8 (in vitro pancreatitis model). Together Bcl-xL/Bcl-2 inhibitors and CCK induced more necrosis than either treatment alone. Bcl-xL/Bcl-2 inhibitors also stimulated cytochrome c release in acinar cells leading to caspase-3 activation and apoptosis. However, different from their effect on pronecrotic signals, the stimulation by Bcl-xL/Bcl-2 inhibitors of apoptotic responses was less in CCK-treated than control cells. Therefore, Bcl-xL/Bcl-2 inhibitors potentiated CCK-induced necrosis but not apoptosis. Correspondingly, transfection with Bcl-xL siRNA stimulated necrosis but not apoptosis in the in vitro pancreatitis model. Further, in animal models of pancreatitis Bcl-xL up-regulation inversely correlated with necrosis, but not apoptosis. Results indicate that Bcl-xL and Bcl-2 protect acinar cells from necrosis in pancreatitis by stabilizing mitochondria against death signals. We conclude that Bcl-xL/Bcl-2 inhibition would aggravate acute pancreatitis, whereas Bcl-xL/Bcl-2 up-regulation presents a strategy to prevent or attenuate necrosis in pancreatitis.     

Polymorphism of follicle stimulating hormone beta (FSHβ) subunit gene and its association with litter traits in giant panda

The different SSCP patterns of the follicle stimulating hormone beta (FSHβ) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHβ gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHβ primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHβ primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.     

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.     

Genome of the Giant Panda Roundworm Illuminates Its Host Shift and Parasitic Adaptation

Baylisascaris schroederi, a roundworm (ascaridoid) parasite specific to the bamboo-feeding giant panda (Ailuropoda melanoleuca), represents a leading cause of mortality in wild giant panda populations. Here, we present a 293-megabase chromosome-level genome assembly of B. schroederi to infer its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential for transmission and rapid adaptation to new hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to the cuticle and basal metabolisms, indicate that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo-enriched gut of giant pandas by structurally increasing its cuticle thickness and efficiently utilizing host nutrients through gut parasitism. Additionally, we characterized the secretome of B. schroederi and predicted potential drug and vaccine targets for new control strategies. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to the giant panda as well as the host-shift events in ascaridoid parasite lineages. Our findings on the unique biology of B. schroederi will also aid in the development of prevention and treatment measures to protect giant panda populations from roundworm parasitism.     

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.     

Kretzoiarctos gen. nov., the Oldest Member of the Giant Panda Clade

The phylogenetic position of the giant panda, Ailuropoda melanoleuca (Carnivora: Ursidae: Ailuropodinae), has been one of the most hotly debated topics by mammalian biologists and paleontologists during the last century. Based on molecular data, it is currently recognized as a true ursid, sister-taxon of the remaining extant bears, from which it would have diverged by the Early Miocene. However, from a paleobiogeographic and chronological perspective, the origin of the giant panda lineage has remained elusive due to the scarcity of the available Miocene fossil record. Until recently, the genus Ailurarctos from the Late Miocene of China (ca. 8–7 mya) was recognized as the oldest undoubted member of the Ailuropodinae, suggesting that the panda lineage might have originated from an Ursavus ancestor. The role of the purported ailuropodine Agriarctos, from the Miocene of Europe, in the origins of this clade has been generally dismissed due to the paucity of the available material. Here, we describe a new ailuropodine genus, Kretzoiarctos gen. nov., based on remains from two Middle Miocene (ca. 12–11 Ma) Spanish localities. A cladistic analysis of fossil and extant members of the Ursoidea confirms the inclusion of the new genus into the Ailuropodinae. Moreover, Kretzoiarctos precedes in time the previously-known, Late Miocene members of the giant panda clade from Eurasia (Agriarctos and Ailurarctos). The former can be therefore considered the oldest recorded member of the giant panda lineage, which has significant implications for understanding the origins of this clade from a paleobiogeographic viewpoint.