Ling Xu

Evaluating the phylogenetic position of Chinese tree shrew (Tupaia belangeri chinensis) based on complete mitochondrial genome: implication for using tree shrew as an alternative experimental animal to primates in biomedical research.

Tree shrew (Tupaia belangeri) is currently placed in Order Scandentia and has a wide distribution in Southeast Asia and Southwest China. Due to its unique characteristics, such as small body size, high brain-to-body mass ratio, short reproductive cycle and life span, and low-cost of maintenance, tree shrew has been proposed to be an alternative experimental animal to primates in biomedical research. However, there are some debates regarding the exact phylogenetic affinity of tree shrew to primates. In this study, we determined the mtDNA entire genomes of three Chinese tree shrews (T. belangeri chinensis) and one Malayan flying lemur (Galeopterus variegatus). Combined with the published data for species in Euarchonta, we intended to discern the phylogenetic relationship among representative species of Dermoptera, Scandentia and Primates. The mtDNA genomes of Chinese tree shrews and Malayan flying lemur shared similar gene organization and structure with those of other mammals. Phylogenetic analysis based on 12 concatenated mitochondrial protein-encoding genes revealed a closer relationship between species of Scandentia and Glires, whereas species of Dermoptera were clustered with Primates. This pattern was consistent with previously reported phylogeny based on mtDNA data, but differed from the one reconstructed on the basis of nuclear genes. Our result suggested that the matrilineal affinity of tree shrew to primates may not be as close as we had thought. The ongoing project for sequencing the entire genome of Chinese tree shrew will provide more information to clarify this important issue.

Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

Significance RIG-I-like receptors (RLRs) belong to a family of DExD/H box RNA helicases and comprise three members: RIG-I, MDA5, and LGP2. The function of RLRs as key cytoplasmic sensors of pathogen-associated molecular patterns has been subjected to numerous pathogenic challenges and undergone dynamic evolution, making it an excellent example for studying innate immunity evolution. We characterized the loss of RIG-I and found that tMDA5 acted as the pattern-recognition receptor responsible for sensing Sendai virus infection in the tree shrew, with an involvement of tLGP2 and tMITA. The positively selective sites in tMDA5 were crucial for the tMDA5-mediated antiviral signaling pathway. Our results provided insights into the evolutionary adaptation and functional diversity of antiviral activity in vertebrates. The function of the RIG-I-like receptors (RLRs; including RIG-I, MDA5, and LGP2) as key cytoplasmic sensors of viral pathogen-associated molecular patterns (PAMPs) has been subjected to numerous pathogenic challenges and has undergone a dynamic evolution. We found evolutionary evidence that RIG-I was lost in the Chinese tree shrew lineage. Along with the loss of RIG-I, both MDA5 (tMDA5) and LGP2 (tLGP2) have undergone strong positive selection in the tree shrew. tMDA5 or tMDA5/tLGP2 could sense Sendai virus (an RNA virus posed as a RIG-I agonist) for inducing type I IFN, although conventional RIG-I and MDA5 were thought to recognize distinct RNA structures and viruses. tMDA5 interacted with adaptor tMITA (STINGTMEM173/ERIS), which was reported to bind only with RIG-I. The positively selected sites in tMDA5 endowed the substitute function for the lost RIG-I. These findings provided insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Promoter variant rs2301228 on the neural cell adhesion molecule 1 gene confers risk of schizophrenia in Han Chinese.

BACKGROUND Schizophrenia is recognized as a disorder of the brain and neuronal connectivity. The neural cell adhesion molecule 1 (NCAM1) gene plays a crucial role in regulating neuronal connectivity. METHODS We conducted a two-stage association analysis on 17 NCAM1 SNPs in two independent Han Chinese schizophrenia case-control cohorts (discovery sample from Hunan Province: 986 patients and 1040 normal controls; replication sample from Yunnan Province: 564 cases and 547 healthy controls). Allele, genotype and haplotype frequencies were compared between case and control samples. Transcription factor binding site prediction and luciferase reporter assays were employed to assess the potential function of promoter SNPs. We detected developmental changes at the transcriptional level of NCAM1 during neuron differentiation in Macaca mulatta neural progenitor cells (NPC). Serum levels of NCAM1 were measured in 72 cases and 88 controls. RESULTS A promoter variant, rs2301228, was found to be associated with schizophrenia at the allelic level and was validated in a replication cohort. Luciferase reporter assays demonstrated that risk allele rs2301228-A significantly down-regulated NCAM1 gene transcription compared to the G-allele. Concordantly, schizophrenia patients had a significantly lower level of serum NCAM1 compared to healthy donors. During the NPC neuronal differentiation, NCAM1 mRNA was significantly increased, suggesting a critical role of this gene in neural development. CONCLUSIONS Our results provide direct evidence for NCAM1 as a susceptibility gene for schizophrenia, which offers support to a neurodevelopmental model and neuronal connectivity hypothesis in the onset of schizophrenia.

Characterization of a MAVS ortholog from the Chinese tree shrew (Tupaia belangeri chinensis)

Human mitochondrial antiviral signaling protein (hMAVS, also known as IPS-1, VISA, or Cardif) is essential for antiviral innate immunity. The Chinese tree shrew (Tupaia belangeri chinenses), a close relative of primates, is emerging as a potential animal model for investigating viral infection. However, there is a lack of biological knowledge about the antiviral innate immunity of the tree shrew. In this study, we identified and characterized the function of the Chinese tree shrew MAVS gene (tMAVS). The cDNA of tMAVS was 2771 bp in length and encoded a polypeptide of 501 amino acids. Phylogenetic analyses based on the amino acid sequences revealed a closer affinity of tMAVS with those of primates. Quantitative real-time PCR analysis indicated that tMAVS mRNA was constitutively expressed in all seven tissues analyzed in this study. The tMAVS mRNA expression was rapidly and significantly increased after RNA virus infections. Ectopic-expression of tMAVS significantly potentiated the virus-triggered activation of IRF3, NF-κB and interferon-β (IFN-β), whereas knockdown of tMAVS displayed the opposite effect. Furthermore, tMAVS mutants lacking the caspase activation and recruitment (CARD) domains or the transmembrane (TM) domain were unable to induce IFN-β. Similar with hMAVS, mitochondrial localization of tMAVS was dependent on its domain. Collectively, this study revealed evolutionary conservation of the MAVS antiviral signaling pathway in the Chinese tree shrew.

Diverse interleukin-7 mRNA transcripts in Chinese tree shrew (Tupaia belangeri chinensis).

Interleukin-7 (IL7) is a pleiotropic cytokine that is actively involved in the immune system. The Chinese tree shrew (Tupaia belangeri chinensis) has been proposed as an alternative experimental animal to primates in biomedical research. However, there is a lack of biological knowledge about the immune system of the tree shrew. In this study, we cloned the IL7 gene (tIL7) in the Chinese tree shrew and quantified the expression of mRNA transcripts in eight tissues (heart, liver, spleen, lung, kidney, intestine, skeletal muscle and brain) from 20 individuals. Eleven tIL7 mRNA transcripts were identified in different tissues. The canonical form (tIL7c) had a length of 1817 bp and encoded a predicted gene product with 177 amino acids. Phylogenetic analyses based on the amino acid sequences revealed a considerably large genetic difference between tree shrew and human. Quantification of mRNA expression of transcripts tIL7c, tIL7-sv1, tIL7-sv2 and tIL7-sv3 showed that these transcripts were expressed in all tissues, albeit the expression levels varied in different tissues. Transcripts tIL7c, tIL7-sv1, and tIL7-sv2 had the lowest expression in brain, and tIL7-sv3 had a dramatically high mRNA expression in skeletal muscle and heart. The mRNA expression levels of tIL7c and tIL7-sv1 were significantly increased upon ploy(I:C) stimulation in tree shrew primary renal cells. As with human full-length IL7, tIL7c, tIL7-sv1, tIL7-sv2 and tIL7-sv3 showed similar a subcellular localization pattern. Our results identified diverse tIL7 transcripts in the Chinese tree shrew, which may play a potential role in modulating IL7-regulated biological effects.

IDH1 p.R132 mutations may not be actively involved in the carcinogenesis of hepatocellular carcinoma

Background Recent studies have identified prevalent isocitrate dehydrogenase 1 (IDH1) codon 132 mutations (p.R132) in gliomas and acute myeloid leukemia (AML). The IDH1 mutations lead to a loss of its normal enzymatic activity and acquisition of neomorphic activity in production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), which finally cause alterations of multiple gene expression of tumorigenesis-associated α-KG-dependent enzymes. The aim of this study was to determine whether IDH1 p.R132 mutations are involved in the carcinogenesis of hepatocellular carcinoma. Material/Methods A total of 87 Han Chinese patients with primary hepatocellular carcinoma (HCC) were analyzed by direct DNA sequencing for IDH1 p.R132 mutations. The expression levels of multiple α-KG-dependent enzymes and associated genes were quantified in HepG2 cells overexpressing IDH1 p.R132 mutants by Western blotting and real-time PCR. Results None of 87 Han Chinese patients with HCC harbored any IDH1 p.R132 mutations. The protein levels of HIF-1α and histone methylation marker (H3K4me3 and H3K79me2) were determined in HepG2 cells overexpressing IDH1 p.R132 mutants, but we discerned no difference. Measurement of mRNA expression levels of VEGF, GLUT1, and HOXA genes also showed no significant difference between cells overexpressing IDH1 wild-type and p.R132 mutants. Conclusions Our negative results, together with some previous reports of the absence of IDH1 p.R132 mutations in HCC tissues, suggests that IDH1 p.R132 mutations are not actively involved in the development of HCC.

Genetic variants of the MAVS, MITA and MFN2 genes are not associated with leprosy in Han Chinese from Southwest China

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae), which has massive genomic decay and dependence on host metabolism. Accumulating evidence showed a crucial role of mitochondria in metabolism and innate immunity. We hypothesized that the mitochondrial-related antimicrobial/antiviral immune genes MAVS (mitochondrial antiviral signaling protein), MITA (mediator of IRF3 activation) and MFN2 (mitofusin 2) would confer a risk to leprosy. In this study, we performed a case-control study to analyze 11 tag and/or non-synonymous SNPs of the MAVS, MITA and MFN2 genes in 527 leprosy patients and 583 healthy individuals, and directly sequenced the three genes in 80 leprosy patients with a family history from Yunnan, Southwest China. We found no association between these SNPs and leprosy (including its subtypes) based on the frequencies of alleles, genotypes and haplotypes between the cases and controls. There was also no enrichment of potential pathogenic variants of the three genes in leprosy patients. Our results suggested that genetic variants of the MAVS, MITA and MFN2 genes might not affect the susceptibility to leprosy.

Identification and characterization of toll-like receptors (TLRs) in the Chinese tree shrew (Tupaia belangeri chinensis).

In mammals, the toll-like receptors (TLRs) play a major role in initiating innate immune responses against pathogens. Comparison of the TLRs in different mammals may help in understanding the TLR-mediated responses and developing of animal models and efficient therapeutic measures for infectious diseases. The Chinese tree shrew (Tupaia belangeri chinensis), a small mammal with a close relationship to primates, is a viable experimental animal for studying viral and bacterial infections. In this study, we characterized the TLRs genes (tTLRs) in the Chinese tree shrew and identified 13 putative TLRs, which are orthologs of mammalian TLR1-TLR9 and TLR11-TLR13, and TLR10 was a pseudogene in tree shrew. Positive selection analyses using the Maximum likelihood (ML) method showed that tTLR8 and tTLR9 were under positive selection, which might be associated with the adaptation to the pathogen challenge. The mRNA expression levels of tTLRs presented an overall low and tissue-specific pattern, and were significantly upregulated upon Hepatitis C virus (HCV) infection. tTLR4 and tTLR9 underwent alternative splicing, which leads to different transcripts. Phylogenetic analysis and TLR structure prediction indicated that tTLRs were evolutionarily conserved, which might reflect an ancient mechanism and structure in the innate immune response system. Taken together, TLRs had both conserved and unique features in the Chinese tree shrew.

Analysis of the complete mitochondrial genome and characterization of diverse NUMTs of Macaca leonina

As a non-human primate, the pig-tailed macaque has received wide attention because it can be infected by HIV-1. In this study, we determined the complete mtDNA sequence of the northern pig-tailed macaque (Macaca leonina). Unexpectedly, during the amplification of the mtDNA control region (D-loop region) we observed several D-loop-like sequences, which were NUMTs (nuclear mitochondrial sequences) and a total of 14 D-loop-like NUMT haplotypes were later identified in five individuals. The neighbor-joining tree and estimated divergence time based on these D-loop-like NUMT sequences of M. leonina provide some insights into the understanding of the evolutionary history of NUMTs. D-loop-like haplotypes G and H, which also exist in the nuclear genome of mulatta, appear to have been translocated into the nuclear genome before the divergence of M. mulatta and M. leonina. The other D-loop-like NUMT haplotypes were translocated into the nuclear genome of M. leonina after the divergence of the two species. Later sequence conversion was predicted to occur among these 14 D-loop-like NUMT haplotypes. The overall structure of the mtDNA of M. leonina was found to be similar to that seen in other mammalian mitochondrial genomes. Phylogenetic analysis based on the maximum likelihood method shows M. leonina clustered with Macaca silenus among the analyzed mammalian species.

Does the Genetic Feature of the Chinese Tree Shrew ( Tupaia belangeri chinensis) Support Its Potential as a Viable Model for Alzheimer’s Disease Research?

Alzheimers disease (AD) is a neurodegenerative disease with increasing incidence across the world and no cure at the present time. An ideal animal model would facilitate the understanding of the pathogenesis of AD and discovery of potential therapeutic targets. The Chinese tree shrew (Tupaia belangeri chinensis) has a closer genetic affinity to primates relative to rodents, and can attain ages of 8 years or older, which represents another advantage for the study of neurodegenerative diseases such as AD compared to primates. Here, we analyzed 131 AD-related genes in the Chinese tree shrew brain tissues based on protein sequence identity, positive selection, mRNA, and protein expression by comparing with those of human, rhesus monkey, and mouse. In particular, we focused on the Aβ and neurofibrillary tangles formation pathways, which are crucial to AD pathogenesis. The Chinese tree shrew had a generally higher sequence identity with human than that of mouse versus human for the AD pathway genes. There was no apparent selection on the tree shrew lineage for the AD-related genes. Moreover, expression pattern of the Aβ and neurofibrillary tangle formation pathway genes in tree shrew brain tissues resembled that of human brain tissues, with a similar aging-dependent effect. Our results provided an essential genetic basis for future AD research using the tree shrew as a viable model.