Comparative proteomics in captive giant pandas to identify proteins involved in age-related cataract formation.

Approximately 20% of aged captive giant pandas (Ailuropoda melanoleuca) have cataracts that impair their quality of life. To identify potential biomarkers of cataract formation, we carried out a quantitative proteomics analysis of 10 giant pandas to find proteins differing in abundance between healthy and cataract-bearing animals. We identified almost 150 proteins exceeding our threshold for differential abundance, most of which were associated with GO categories related to extracellular localization. The most significant differential abundance was associated with components of the proteasome and other proteins with a role in proteolysis or its regulation, most of which were depleted in pandas with cataracts. Other modulated proteins included components of the extracellular matrix or cytoskeleton, as well as associated signaling proteins and regulators, but we did not find any differentially expressed transcription factors. These results indicate that the formation of cataracts involves a complex post-transcriptional network of signaling inside and outside lens cells to drive stress responses as a means to address the accumulation of protein aggregates triggered by oxidative damage. The modulated proteins also indicate that it should be possible to predict the onset of cataracts in captive pandas by taking blood samples and testing them for the presence or absence of specific protein markers.

Increased Non-MAIT CD161+CD8+ T Cells Display Pathogenic Potential in Chronic HBV Infection.

BACKGROUND & AIMS: CD161-expressing CD8+ T cells consist of mucosal-associated invariant T cells with semi-invariant T-cell receptor (TCR) use and non-mucosal-associated invariant T CD161+CD8+ T cells with polyclonal TCR repertoire. Although CD161+CD8+ T cells are enriched in liver and embrace hepatitis B virus (HBV)-specific T cells in chronic hepatitis B (CHB) patients, their roles in disease progression remain poorly understood. This study aimed to decipher their profiling and dynamic changes during chronic HBV infection. METHODS: Blood samples from 257 CHB patients and nontumor liver specimens from 73 HBV-positive patients were analyzed for CD161+CD8+ T-cell characterization by flow cytometry, TCR repertoire determination, transcriptomic analyses, and cell experiments. RESULTS: CD161+CD8+ T cells were increased and hyperactivated in patients, while positive correlation between the CD161+CD8+ T-cell ratio and HBV-DNA level suggested this was insufficient to control HBV replication. The overlap of complementarity determining region 3 sequences supported the switch between CD161-CD8+ and CD161+CD8+ populations. Although CD161+CD8+ T cells were endowed with innateness phenotype and enhanced antiviral capacity, the population from patients had impaired type I cytokine production, and increased interleukin 17 and granzyme B secretion. The increased CD161+CD8+ T cells and their increased granzyme B secretion correlated positively with inflammation-associated liver injury. Hepatic CD161+CD8+ T cells showed neutrophil-related pathogenic potential because they had increased transcript signatures and proinflammatory cytokine production in neutrophil recruitment- and response-related pathways that changed consistently in the injured liver. CONCLUSIONS: Our results highlight the reduced antiviral potency but increased pathogenic potential of CD161+CD8+ T cells in CHB patients, supporting CD161 expression as a marker of pathogenic CD8+ T subset and the intervention target for liver injury.

RNA-Seq analysis in giant pandas reveals the differential expression of multiple genes involved in cataract formation.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is an endangered mammalian species native to China. Fewer than 2500 giant pandas are known to exist, many of which are bred in captivity as a means to preserve and repopulate the species. Like other captive mammals, giant pandas acquire age-related cataracts, reducing their quality of life. Recent comparative genome-wide methylation analysis revealed 110 differentially methylated genes associated with cataract formation including six also associated with the formation of age-related cataracts in humans. RESULTS: To investigate the pathological pathway in greater detail, here we used RNA-Seq analysis to investigate the differential expression profiles of genes in three giant pandas with cataracts and three healthy controls. We identified more than 700 differentially expressed genes, 29 of which were selected for further analysis based on their low q-value. We found that many of the genes encoded regulatory and signaling proteins associated with the control of cell growth, migration, differentiation and apoptosis, supporting previous research indicating a key role for apoptosis in cataract formation. CONCLUSION: The identification of genes involved in the formation of age-related cataracts could facilitate the development of predictive markers, preventative measures and even new therapies to improve the life of captive animals.

A novel missense mutation in the HSF4 gene of giant pandas with senile congenital cataracts.

Cataracts are a common cause of visual impairment and blindness in mammals. They are usually associated with aging, but approximately one third of cases have a significant genetic component. Cataracts are increasingly prevalent among aging populations of captive giant pandas (Ailuropoda melanoleuca) and it is therefore important to identify genetic determinants that influence the likelihood of cataract development in order to distinguish between congenital and age-related disease. Here we screened for cataract-related genetic effects using a functional candidate gene approach combined with bioinformatics to identify the underlying genetic defect in a giant panda with congenital cataracts. We identified a missense mutation in exon 10 of the HSF4 gene encoding heat shock transcription factor 4. The mutation causes the amino acid substitution R377W in a highly conserved segment of the protein between the isoform-specific and downstream hydrophobic regions. Predictive modeling revealed that the substitution is likely to increase the hydrophobicity of the protein and disrupt interactions with spatially adjacent amino acid side chains. The mutation was not found in 13 unaffected unrelated animals but was found in an unrelated animal also diagnosed with senile congenital cataract. The novel missense mutation in the HSF4 gene therefore provides a potential new genetic determinant that could help to predict the risk of cataracts in giant pandas.

Genome-wide analysis of methylation in giant pandas with cataract by methylation-dependent restriction-site associated DNA sequencing (MethylRAD).

The giant panda (Ailuropoda melanoleuca) is a native species to China. They are rare and endangered and are regarded as the 'national treasure' and 'living fossil' in China. For the time being, there are only about 2500 giant pandas in the world. Therefore, we still have to do much more efforts to protect the giant pandas. In captive wildlife, the cataract incidence of mammalian always increases with age. Currently, in China, the proportion of elderly giant pandas who suffering from cataract has reached 20%. The eye disorder thus has a strong influence on the physical health and life quality of the elderly giant pandas. To discover the genes associated with the pathogenesis of cataract in the elderly giant panda and achieve the goal of early assessment and diagnosis of cataract in giant pandas during aging, we performed whole genome methylation sequencing in 3 giant pandas with cataract and 3 healthy giant pandas using methylation-dependent restriction-site associated DNA sequencing (MethylRAD). In the present study, we obtained 3.62M reads, on average, for each sample, and identified 116 and 242 differentially methylated genes (DMGs) between the two groups under the context of CCGG and CCWGG on genome, respectively. Further KEGG and GO enrichment analyses determined a total of 110 DMGs that are involved in the biological functions associated with pathogenesis of cataract. Among them, 6 DMGs including EEA1, GARS, SLITRK4, GSTM3, CASP3, and EGLN3 have been linked with cataract in old age.

The relation between mental health, homosexual stigma, childhood abuse, community engagement, and unprotected anal intercourse among MSM in China.

The aim of the study was to explore the relation of various factors with unprotected anal intercourse (UAI) and provide some insight for HIV intervention on Chinese men who have sex with men (MSM). The current cross-sectional study recruited 365 MSM in Dalian, China. More than half of the respondents (117 respondents, 51.8% of the sample) had engaged in UAI. The multivariable logistic regression model suggested that poorer mental health (AOR: 7.16; 95% CI: 3.14-16.31), self-stigma (AOR: 1.53; 95% CI: 1.00-2.34), and experience(s) of physical abuse in childhood (AOR: 5.85; 95% CI: 1.77-19.30) were significantly and positively related to UAI. Community engagement was negatively associated with UAI (p < 0.05). It appears it is necessary to incorporate mental health services, eliminate the stigma against homosexuality, and facilitate MSM-related community engagement into intervention strategies to prevent UAI among Chinese MSM. Targeted UAI interventions in the subgroup with a history of childhood physical abuse should also be of great concern.

Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos.

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43-48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.