Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gßγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, ß-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Hippocampal delivery of neurotrophic factor-α1/carboxypeptidase E gene prevents neurodegeneration, amyloidosis, memory loss in Alzheimer's Disease male mice.

Alzheimer's Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aß1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aß1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aß1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation.

The effectiveness of using giant panda as a surrogate for protecting sympatric species.

The use of umbrella species to promote biodiversity conservation is practiced worldwide. The giant panda (Ailuropoda melanoleuca) an iconic species for world wildlife conservation, that inhabits regions with significant biodiversity. Given that the functions at wildlife of different trophic levels and in different body size groups are different within the ecosystem, it is unknown whether those groups of wildlife co-occurring with giant pandas are each likewise protected. To examine the umbrella effect of giant pandas on sympatric species, we used an extensive dataset of wildlife from more than 78% of giant panda habitats. We analysed the changes in distribution for four wildlife categories (large carnivores, large herbivores, medium carnivores and medium herbivores) using a generalized linear mixed model, and the underlying driving factors using binomial logistic regression models. Changes in forests in giant panda habitats were evaluated using Fragstats. The results have shown that the counts of herbivores and medium carnivores increased significantly during the decade. However, those of large carnivores significantly declined. Forest cover and nature reserves showed significant and positive effects on wildlife in 2001 and 2011, while the human population had significant and negative impacts on the herbivores and carnivores. Our results have also suggested that there has been a slight alleviation in forest fragmentation in areas unaffected by earthquakes. We concluded that the umbrella strategy of using the giant panda as an umbrella species achieved partial success by promoting the recovery of herbivores and medium carnivores. Meanwhile, this has indicated that the strategy was not sufficient for large carnivores, and therefore not enough for local ecosystems, given the critical role of large carnivores. We have suggested integrating habitat patches, controlling human disturbance, and preparing for potential human-wildlife conflict management in the Giant Panda National Park to restore large carnivore populations and maintain ecosystem functioning.

Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via ß-arrestin/ERK signaling.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in ß-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was ß-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited ß-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of ß-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the ß-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells.

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Climate change and landscape-use patterns influence recent past distribution of giant pandas.

Climate change is one of the most pervasive threats to biodiversity globally, yet the influence of climate relative to other drivers of species depletion and range contraction remain difficult to disentangle. Here, we examine climatic and non-climatic correlates of giant panda (Ailuropoda melanoleuca) distribution using a large-scale 30 year dataset to evaluate whether a changing climate has already influenced panda distribution. We document several climatic patterns, including increasing temperatures, and alterations to seasonal temperature and precipitation. We found that while climatic factors were the most influential predictors of panda distribution, their importance diminished over time, while landscape variables have become relatively more influential. We conclude that the panda's distribution has been influenced by changing climate, but conservation intervention to manage habitat is working to increasingly offset these negative consequences.

Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E-∆N Promotes Proliferation and Invasion of Pancreatic Cancer Cells via Upregulation of CXCR2 Gene Expression.

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.

Carboxypeptidase E is a prediction marker for tumor recurrence in early-stage hepatocellular carcinoma.

Tumor recurrence and metastasis are the major causes of death for hepatocellular carcinoma (HCC) patients who are able to receive curative resection. Identifying the predicting biomarkers for tumor recurrence would improve their survival. RNA extracted from fresh frozen tumors and adjacent non-tumor liver tissues of 120 HCC patients were obtained from Taiwan Liver Cancer Network (TLCN) in year 2010 for determination of the carboxypeptidase E (CPE) expression level (including its splicing mutant CPE-ΔN) in the tumor tissue (T) and paired non-tumor liver tissue (N) by real-time quantitative polymerase chain reaction. All patients were male, had chronic hepatitis B virus infection, were in the early pathology stage, and received curative resection. The T/N ratio of the CPE expression level was correlated with the updated survival data from TLCN in 2015. The CPE expression level in the 120 HCC patients was divided into three groups according to the T/N ratio: <1, ≥1 and ≤2, and >2, respectively. By multivariate analyses, the recurrence-free survival (RFS) was only significantly associated with the pathology stage and the CPE expression level. For overall survival (OS), only the CPE expression level was the significant prognostic factor. The CPE expression level was also significantly correlated with the tumor recurrence for both stage I (p = 0.0106) and stage II patients (p = 0.0006). The CPE mRNA expression level in HCC can be a useful biomarker for predicting tumor recurrence in HCC patients who are in the early pathology stage and able to receive curative resection.

A mechanistic study of the proapoptotic effect of tolfenamic acid: involvement of NF-κB activation.

Recent studies demonstrate that tolfenamic acid (TA) induces apoptosis and suppresses the development and progression of several types of cancers. However, the underlying mechanisms are complex and remain to be fully elucidated. Nuclear factor-kappaB (NF-κB) plays a critical role in inflammation, cancer development and progression. Although non-steroidal anti-inflammatory drugs modulate NF-κB signaling pathway in different ways, the link between NF-κB and TA-induced apoptosis of colorectal cancer cells has yet to be thoroughly investigated. In this study, we examined the effects of TA on the NF-κB pathway and apoptosis. TA activated NF-κB transcriptional activity and binding affinity of NF-κB to DNA. TA-induced NF-κB activation was mediated by an increased phosphorylation and proteosomal degradation of IκB-α and subsequent p65 nuclear translocation. We also observed that TA stabilized p65 and increased nuclear accumulation via an increase of p65 phosphorylation at Ser276 residue, which was mediated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. The knockout of p53 blocked TA-induced transcriptional activation of NF-κB, but not the p65 nuclear accumulation. TA increased transcriptional activity of p53 and the binding affinity of p53 with p65, which are mediated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase-stimulated Ser276 phosphorylation. TA-induced apoptosis was ameliorated by the knockout of p65 and p53 and the point mutation of p65 at Ser276 residue. We demonstrate a novel molecular mechanism by which TA induced the NF-κB and apoptosis in human colorectal cancer cells.

Genetic consequences of historical anthropogenic and ecological events on giant pandas.

The giant panda (Ailuropoda melanoleuca) was taken to the brink of extinction in the 1980s through a combination of deforestation, large-scale loss of bamboo in the core of its range, poaching, and zoo collection, causing over 1000 deaths from the 1950s. It was thought that the drastic population decline was likely to impose a severe impact on population viability. Here, based on temporal genotyping of individuals, we show that this rapid decline did not significantly reduce the overall effective population size and genetic variation of this species, or of the two focal populations (Minshan and Qionglai) that declined the most. These results are contrary to previously assumptions, probably because the population decline has not produced the expected negative impact due to the short time scale involved (at most 10 generations), or because previous surveys underestimated the population size at the time of decline. However, if present-day habitat fragmentation and limited migration of giant pandas remains, we predict a loss of genetic diversity across the giant pandas' range in the near future. Thus, our findings highlight the substantial resilience of this species when facing demographic and environmental stochasticity, but key conservation strategies, such as enhancing habitat connectivity and habitat restoration should be immediately implemented to retain the extant genetic variation and maintain long-term evolutionary potential of this endangered species.

Graphene oxide as a multi-functional additive for compatilizer, enhancer, and barrier in ethylene vinyl alcohol copolymer/aramid pulp composites.

To improve the thermal, mechanical, and barrier properties of ethylene vinyl alcohol copolymer (EVOH)/aramid pulp (AP), graphene oxide (GO) was used as a compatilizer, enhancer, and barrier to fabricate EVOH-based composites. The results showed that graphene oxide serves as an ideal compatilizer to reinforce the interfacial action between the EVOH matrix and aramid pulp. The EVOH/AP/GO composite presented the best combination of thermal stability, tensile strength, oxygen barrier, and heat deformation temperature by adding only 1 wt% graphene oxide, compared to those of pure EVOH. Moreover, both scanning electron microscopy (SEM) and polarized optical microscopy (POM) photographs demonstrated that the aramid pulp dispersed homogeneously into the EVOH resin with the addition of 1 wt% graphene oxide. Our work provides a novel and facile way for producing a prominent EVOH-based composite, which can be potentially used in packaging fields in the future.

Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxy tryptamine receptor 1E (5-HTR1E) is reported to activate cAMP and ERK pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gßγ and Gq were not associated with 5-HTR1E activation, while blocking PKA inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, ß-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockout studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 ( RAMP1 ), Nuclear receptor 1 ( NR4A1 ) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Preclinical evaluation of combined antineoplastic effect of DLC1 tumor suppressor protein and suberoylanilide hydroxamic acid on prostate cancer cells.

Deleted in liver cancer (DLC1), a tumor suppressor gene in multiple cancers, is recurrently down regulated or inactivated by epigenetic mechanisms in primary prostate carcinomas (PCAs). In this study the methylation and acetylation profile of the DLC1 promoter region was examined in three PCA cell lines with low or undetectable DLC1 expression: LNCaP, its derivative C4-2B-2, and 22Rv1. Two histone deacetylase inhibitors (HDAC), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) induced histone acetylation of the DLC1 promoter in all three lines. DLC1 promoter methylation and deacetylation were detected in LNCaP and C4-2B-2 cells while in 22Rv1 cells DLC1 is silenced by deacetylation. Treatment with SAHA or TSA efficiently increased DLC1 expression in all lines, particularly in 22Rv1 cells, and activated the DLC1 promoter through the same Sp1 sites. The 22Rv1 cell line was selected to evaluate the efficacy of combined DLC1 transduction and SAHA treatment on tumor growth in athymic mice. Individually, DLC1 transduction and SAHA exposure reduced the tumor size by 75-80% compared to controls and in combination almost completely inhibited tumor growth. The antitumor effect was associated with the induction of apoptosis and inhibition of RhoA activity. SAHA alone significantly reduced RhoA activity, showing that this RhoGTPase is a target for SAHA. These results, obtained with a reliable preclinical in vivo test, predict that combined therapeutic agents targeting the pathways governing DLC1 function and HDAC inhibitors may be beneficial in management of prostate cancer.

Carboxypeptidase E and its splice variants: Key regulators of growth and metastasis in multiple cancer types.

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis.

Landscape-scale giant panda conservation based on metapopulations within China's national park system.

Historically, giant panda conservation in China has been compromised by disparate management of protected areas. It is thus crucial to address how giant panda populations can be managed cohesively on a landscape scale, an opportunity offered by China's newly established Giant Panda National Park. Here, we evaluated giant panda populations in a metapopulation context, based on range-wide data from the Fourth National Giant Panda Survey. We delineated metapopulations by geographic range, relative abundance, and relative density and assessed the extent of human disturbance each metapopulation faced. We found density-dependent and disturbance-influenced effects on habitat selection across metapopulations. We determined the main effects faced by each metapopulation regarding area sensitivity, population size, intraspecific competition, and disturbance. To enhance the landscape-scale conservation of giant pandas and various other wildlife across China's national park system, we propose that metapopulation management incorporates population status along with density-dependent and disturbance-related effects on habitat selection.

Neurotrophic factor-α1, a novel tropin is critical for the prevention of stress-induced hippocampal CA3 cell death and cognitive dysfunction in mice: comparison to BDNF.

Stress leads to brain pathology including hippocampal degeneration, cognitive dysfunction, and potential mood disorders. Hippocampal CA3, a most stress-vulnerable region, consists of pyramidal neurons that regulate cognitive functions e.g. learning and memory. These CA3 neurons express high levels of the neuroprotective protein, neurotrophic factor-α1 (NF-α1), also known as carboxypeptidase E (CPE), and receive contacts from granule cell projections that release BDNF which has neuroprotective activity. Whether NF-α1-CPE and/or BDNF are critical in protecting these CA3 neurons against severe stress-induced cell death is unknown. Here we show that social combined with the physical stress of maternal separation, ear tagging, and tail snipping at weaning in 3-week-old mice lacking NF-α1-CPE, led to complete hippocampal CA3 degeneration, despite having BDNF and active phosphorylated TrkB receptor levels similar to WT animals. Mice administered TrkB inhibitor, ANA12 which blocked TrkB phosphorylation showed no degeneration of the CA3 neurons after the weaning stress paradigm. Furthermore, transgenic knock-in mice expressing CPE-E342Q, an enzymatically inactive form, replacing NF-α1-CPE, showed no CA3 degeneration and exhibited normal learning and memory after the weaning stress, unlike NF-α1-CPE-KO mice. Mechanistically, we showed that radio-labeled NF-α1-CPE bound HT22 hippocampal cells in a saturable manner and with high affinity (Kd = 4.37 nM). Subsequently, treatment of the HT22cpe-/- cells with NF-α1-CPE or CPE-E342Q equivalently activated ERK signaling and increased BCL2 expression to protect these neurons against H2O2-or glutamate-induced cytotoxicity. Our findings show that NF-α1-CPE is more critical compared to BDNF in protecting CA3 pyramidal neurons against stress-induced cell death and cognitive dysfunction, independent of its enzymatic activity.

Geographic pattern of antibiotic resistance genes in the metagenomes of the giant panda.

The rise in infections by antibiotic-resistant bacteria poses a serious public health problem worldwide. The gut microbiome of animals is a reservoir for antibiotic resistance genes (ARGs). However, the correlation between the gut microbiome of wild animals and ARGs remains controversial. Here, based on the metagenomes of giant pandas (including three wild populations from the Qinling, Qionglai and Xiaoxiangling Mountains, and two major captive populations from Yaan and Chengdu), we investigated the potential correlation between the constitution of the gut microbiome and the composition of ARGs across the different geographic locations and living environments. We found that the types of ARGs were correlated with gut microbiome composition. The NMDS cluster analysis using Jaccard distance of the ARGs composition of the gut microbiome of wild giant pandas displayed a difference based on geographic location. Captivity also had an effect on the differences in ARGs composition. Furthermore, we found that the Qinling population exhibited profound dissimilarities of both gut microbiome composition and ARGs (the highest proportion of Clostridium and vancomycin resistance genes) when compared to the other wild and captive populations studies, which was supported by previous giant panda whole-genome sequencing analysis. In this study, we provide an example of a potential consensus pattern regarding host population genetics, symbiotic gut microbiome and ARGs. We revealed that habitat isolation impacts the ARG structure in the gut microbiome of mammals. Therefore, the difference in ARG composition between giant panda populations will provide some basic information for their conservation and management, especially for captive populations.