Novel Tree Shrew Cytochrome P450 2Ds (CYP2D8a and CYP2D8b) Are Functional Drug-Metabolizing Enzymes that Metabolize Bufuralol and Dextromethorphan.

Tree shrews are a nonprimate species used in a range of biomedical studies. Recent genome analysis of tree shrews found that the sequence identities and the numbers of genes of cytochrome P450 (CYP or P450), an important family of drug-metabolizing enzymes, are similar to those of humans. However, tree shrew P450s have not yet been sufficiently identified and analyzed. In this study, novel CYP2D8a and CYP2D8b cDNAs were isolated from tree shrew liver and were characterized, along with human CYP2D6, dog CYP2D15, and pig CYP2D25. The amino acid sequences of these tree shrew CYP2Ds were 75%-78% identical to human CYP2D6, and phylogenetic analysis showed that they were more closely related to human CYP2D6 than rat CYP2Ds, similar to dog and pig CYP2Ds. For tree shrew CYP2D8b, two additional transcripts were isolated that contained different patterns of deletion. The gene and genome structures of CYP2Ds are generally similar in dogs, humans, pigs, and tree shrews. Tree shrew CYP2D8a mRNA was most abundantly expressed in liver, among the tissue types analyzed, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Tree shrew CYP2D8b mRNA was also expressed in liver, but at a level 7.3-fold lower than CYP2D8a mRNA. Liver microsomes and recombinant protein of both tree shrew CYP2Ds metabolized bufuralol and dextromethorphan, selective substrates of human CYP2D6, but the activity level of CYP2D8a greatly exceeded that of CYP2D8b. These results suggest that tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver. SIGNIFICANCE STATEMENT: Novel tree shrew CYP2D8a and CYP2D8b cDNAs were isolated from liver. Their amino acid sequences were 75%-78% identical to human CYP2D6. For CYP2D8b, two additional transcripts contained different patterns of deletion. Tree shrew CYP2D8a mRNA was abundantly expressed in liver, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Recombinant tree shrew CYP2Ds catalyzed the oxidation of bufuralol and dextromethorphan. Tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1-6) in tree shrews.

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1-5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1-6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85-90 % and 81-89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1-6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1-6, which are orthologous to human FMOs (FMO1-5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

RNA-Seq and miRNA-Seq data from Epstein-Barr virus-infected tree shrews reveal a ceRNA network contributing to immune microenvironment regulation.

Epstein-Barr virus (EBV) infection in humans is ubiquitous and associated with various diseases. Remodeling of the immune microenvironment is the primary cause of EBV infection and pathogenesis; however, the underlying mechanism has not been fully elucidated. In this study, we used whole-transcriptome RNA-Seq to detect mRNAs, long non-coding RNAs (lncRNA), and microRNA (miRNA) profiles in the control group, 3 days, and 28 days after EBV infection, based on the tree shrew model that we reported previously. First, we estimated the proportion of 22 cell types in each sample using CIBERSORT software and identified 18 high-confidence DElncRNAs related to immune microenvironment regulation after EBV infection. Functional enrichment analysis of these differentially expressed lncRNAs primarily focused on the autophagy, endocytosis, and ferroptosis signalling pathways. Moreover, EBV infection affects miRNA expression patterns, and many miRNAs are silenced. Finally, three competing endogenous RNA regulatory networks were built using lncRNAs that significantly correlated with immune cell types, miRNAs that responded to EBV infection, and potentially targeted the mRNA of the miRNAs. Among them, MRPL42-AS-5 might act as an hsa-miR-296-5p "sponge" and compete with target mRNAs, thus increasing mRNA expression level, which could induce immune cell infiltration through the cellular senescence signalling pathway against EBV infection. Overall, we conducted a complete transcriptomic analysis of EBV infection in vivo for the first time and provided a novel perspective for further investigation of EBV-host interactions.

Cognitive Deficits and Alzheimer's Disease-Like Pathologies in the Aged Chinese Tree Shrew.

Alzheimer's disease (AD) is the most common chronic progressive neurodegenerative disease in the elderly. It has an increasing prevalence and a growing health burden. One of the limitations in studying AD is the lack of animal models that show features of Alzheimer's pathogenesis. The tree shrew has a much closer genetic affinity to primates than to rodents and has great potential to be used for research into aging and AD. In this study, we aimed to investigate whether tree shrews naturally develop cognitive impairment and major AD-like pathologies with increasing age. Pole-board and novel object recognition tests were used to assess the cognitive performance of adult (about 1 year old) and aged (6 years old or older) tree shrews. The main AD-like pathologies were assessed by Western blotting, immunohistochemical staining, immunofluorescence staining, and Nissl staining. Our results showed that the aged tree shrews developed an impaired cognitive performance compared to the adult tree shrews. Moreover, the aged tree shrews exhibited several age-related phenotypes that are associated with AD, including increased levels of amyloid-ß (Aß) accumulation and phosphorylated tau protein, synaptic and neuronal loss, and reactive gliosis in the cortex and the hippocampal tissues. Our study provides further evidence that the tree shrew is a promising model for the study of aging and AD.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol.

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species.In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree.Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs.These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

Expression of Testis-specific Serine/Threonine Kinases during the Reproductive and Nonreproductive Seasons and Their Localization in Mature Spermatozoa of Tree Shrews (Tupaia belangeri).

Tree shrews display obvious reproductive cycles, and sexually mature male tree shrews produce little or no sperm with extremely low motility during the nonreproductive season; the mechanism underlying this phenomenon remains unknown. Because testis-specific serine/threonine kinases (TSSK) are specifically expressed in the testis and male germ cells of mammals, we hypothesized that they may have an important role in spermatogenesis or sperm function regulation in tree shrews. In addition, the expression, distribution, subcellular localization, and dynamic changes of TSSK in tree shrew sperm are unclear. Here we show that during the reproductive season, the seminiferous tubules were significantly larger as compared with the nonreproductive season and contained mature sperm and other germ cells. The mRNA expression of Tssk genes in testis was significantly higher than that in other tissues, and the mRNA level in the testis during the reproductive season was significantly higher than that in nonreproductive season. In addition, the mRNA level of Tssk3 in the testis and sperm was significantly higher than that of other members. Specifically, Tssk1 mRNA was distributed in the acrosome and throughout the flagellum of tree shrew sperm, Tssk2 was present in the acrosome, Tssk3 was localized to postacrosomal region and relocated to the main part of the flagellum after capacitation, and Tssk6 was distributed in the acrosome and postacrosomal region. These results indicate that the TSSK are important regulating reproductive function in tree shrews.

The regulation of expression and splicing of transcription factors are related to the muscle damage caused by radiation in tree shrews.

Radiotherapy-induced muscle injury (RIMI) is a major complication of radiotherapy for nasopharyngeal carcinoma. Transcription factor (TF) expression and alternative splicing are crucial events in transcriptional and posttranscriptional regulation, respectively, and are known to be involved in key signaling pathways contributing to a variety of human disorders, including radiation injury. To investigate the TFs and alternative splicing events involved in RIMI, we constructed a tree shrew model as described previously in which the RIMI group received 20 Gy of irradiation on the tensor veli palatini (TVP) muscles. The irradiated muscles were evaluated by RNA sequencing (RNA-seq) 6 months later, and the results compared with those for normal TVP muscles. The alt5p and alt3p events were the two main types of differentially regulated alternative splicing events (RASEs) identified via the Splice sites Usage Variation Analysis (SUVA) software, and these RASEs were highly conserved in RIMI. According to functional enrichment analysis, the differentially RASEs were primarily enriched in pathways related to transcriptional regulation. Furthermore, we identified 16 alternative splicing TFs (ASTFs) in ASTF-differentially expressed gene (DEG) networks based on co-expression analysis, and the regulatory networks were chiefly enriched in pathways linked to cell proliferation and differentiation. This study revealed that RASEs and ASTF-DEG networks may both play important regulatory roles in gene expression network alteration in RIMI. Future studies on the targeting mechanisms and early interventions directed at RASEs and ASTF-DEG networks may aid in the treatment of RIMI.

Mechanism of cognitive impairment induced by d-galactose and l-glutamate through gut-brain interaction in tree shrews.

d-Galactose (d-gal) and l-glutamate (l-glu) impair learning and memory. The mechanism of interaction between the gut microbiome and brain remains unclear. In this study, a model of cognitive impairment was induced in tree shrews by intraperitoneal (ip) injection of d-gal (600 mg/kg/day), intragastric (ig) administration with l-glu (2000 mg/kg/day), and the combination of d-gal (ip, 600 mg/kg/day) and l-glu (ig, 2000 mg/kg/day). The cognitive function of tree shrews was tested by the Morris water maze method. The expression of Aß1-42 proteins, the intestinal barrier function proteins occludin and P-glycoprotein (P-gp), and the inflammatory factors NF-κB, TLR2, and IL-18 was determined by immunohistochemistry. The gut microbiome was analyzed by 16SrRNA high-throughput sequencing. After administering d-gal and l-glu, the escape latency increased (p < .01), and the times of crossing the platform decreased (p < .01). These changes were greater in the combined administration of d-gal and l-glu (p < .01). The expression of Aß1-42 was higher in the perinuclear region of the cerebral cortex (p < .01) and intestinal cell (p < .05). There was a positive correlation between the cerebral cortex and intestinal tissue. Moreover, the expression of NF-κB, TLR2, IL-18, and P-gp was higher in the intestine (p < .05), while the expression of occludin and the diversity of gut microbes were lower, which altered the biological barrier of intestinal mucosal cells. This study indicated that d-gal and l-glu could induce cognitive impairment, increase the expression of Aß1-42 in the cerebral cortex and intestinal tissue, decrease the gut microbial diversity, and alter the expression of inflammatory factors in the mucosal intestines. The dysbacteriosis may produce inflammatory cytokines to modulate neurotransmission, causing the pathogenesis of cognitive impairment. This study provides a theoretical basis to explore the mechanism of learning and memory impairment through the interaction of microbes in the gut and the brain.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes.

Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72-78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes.

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82-87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.

Characteristics of Angiotensin I-converting enzyme 2, type II transmembrane serine protease 2 and 4 in tree shrew indicate it as a potential animal model for SARS-CoV-2 infection.

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.

Mapping of c-Fos expression in male tree shrew forebrain.

The tree shrew is susceptible to stimuli. However, mapping of c-Fos expression in male tree shrew forebrain has not been explored. The present results provided the first detailed mapping of c-Fos expression in the forebrain of the tree shrew (Tupaia belangeri chinensis). Acute restraint stress rapidly increased the density of c-Fos-immunoreactive (-ir) neurons in the medial orbital cortex (MO), infralimbic cortex, intermediate part of the lateral septal nucleus (LSi), ventral part of the lateral septal nucleus (LSv), anterior part of the bed nucleus of the stria terminalis, posterior part of the bed nucleus of the stria terminalis (STP), paraventricular nucleus of the hypothalamus, supraoptic nucleus, lateral hypothalamic area, ventromedial hypothalamic nucleus (VMH), and medial amygdaloid nucleus (MeA). Furthermore, a significant increase in c-Fos expression was observed in the MO, LSi, LSv, STP, VMH, arcuate hypothalamic nucleus, anterior amygdaloid area, MeA, and cortical amygdaloid nucleus immediately after acute footshock stress. In addition, the distinct patterns of c-Fos expression in the forebrain were shown in context-, restraint-, or footshock-treated tree shrews. In general, the present study provides the first detailed maps of c-Fos expression in male tree shrew forebrain immediately after various stimuli.

The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein.

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.

Lipidomic analysis of meibomian gland secretions from the tree shrew: Identification of candidate tear lipids critical for reducing evaporation.

Lipids secreted from the meibomian glands form the outermost layer of the tear film and reduce its evaporation. Abnormal changes in the quantities or compositions of lipids present in meibomian gland secretions (meibum) are known to lead to dry eye disease, although the underlying mechanism is not yet well understood. The tree shrew is the non-primate mammal most closely related to humans. To assess the utility of the tree shrew as a model for the study of dry eye disease, we analyzed the lipid profile of tree shrew meibum using an untargeted ESI-MS and MS/MSall shotgun approach. The resulting lipidome shared many similarities with human meibum, while displaying some interesting differences. For example, several classes of lipids, including wax esters, cholesteryl esters, diesters, and (O-acyl)-ω-hydroxy fatty acids, had relatively longer chain lengths in tree shrew meibum. These increases in length may promote more effective reduction of tear evaporation in the tree shrew, which likely underlies the much longer blinking interval of this mammal. Our results suggest that the tree shrew could be an effective model for the study of dry eye.

Upregulation of chemokine CXCL10 enhances chronic pulmonary inflammation in tree shrew collagen-induced arthritis.

Chronic pulmonary inflammation (CPI) gives rise to serious lung injuries in rheumatoid arthritis (RA) patients. However, the molecular mechanism underlying the pathogenesis of RA-associated CPI remains little understood. Here we established a novel tree shrew-based collagen-induced arthritis (TsCIA) model to study RA-associated CPI. Our results showed that typical CPI but not fibrosis developed pathologically in the TsCIA model. Furthermore, abnormal up-regulation of pulmonary chemokine CXCL10 was directly associated with lung damage. Specific blockage of CXCR3 (a CXCL10 receptor) significantly decreased the severity of CPI by decreasing the recruitment of inflammatory cells. Therefore, CXCL10 is proposed as a key player responsible for the development of TsCIA-associated CPI. Our findings also suggest that CXCR3 could be developed as a potential diagnosis biomarker for RA-associated CPI.

Molecular cloning and characterization of APOBEC3 family in tree shrew.

The APOBEC3 family is a series antiviral factors that inhibit the replication of many viruses, such as HIV-1 and HBV. Tree shrews (Tupaia belangeri) possess great potential as an animal model for human diseases and therapeutic responses. However, the APOBEC3 family is unknown in tree shrews. Recent work has showed the presence of the APOBEC3 family in tree shrews. In this work, the cDNA sequences of five APOBEC3 members were identified in tree shrews, namely, tsAPOBEC3A, -3C, -3F, -3G and -3H. The results showed that their sequences encoded a zinc (Z)-coordinating-domain as a characteristic of APOBEC3 proteins. Phylogenetic analysis revealed that the tree shrew APOBEC3 (tsAPOBEC3) genes have occurred independently and that they are clustered with other mammalian APOBEC3 members. Transcript expression analysis indicated that tsAPOBEC3 genes are constitutively expressed, and high in immune-related tissues. tsAPOBEC3 gene expression was up-regulated in hepatocytes and PBMCs by IFN-α stimulation. Finally, tsAPOBEC3 proteins could edit both sides of DNA by inserting G→A and C→T hypermutations. Overall, the results suggest that the tsAPOBEC3 family could play a key role in defense immunity through distinct editing mechanisms. Our results provided insights into the genetic basis for the development of a tree shrew model for studying viral infection. Future studies will focus on deepening our understanding on the antiviral functions of these editing enzymes in tree shrew.

Auditory brainstem responses after electrolytic lesions in bilateral subdivisions of the medial geniculate body of tree shrews.

This study aimed to establish a tree shrew model of bilateral electrolytic lesions in the medial geniculate body (MGB) to determine the advantages of using a tree shrew model and to assess the pattern of sound processing in tree shrews after bilateral electrolytic damage in different parts of the MGB. The auditory brainstem responses (ABRs) of a normal control group (n = 30) and an electrical damage group (n = 30) were tested at 0 h, 24 h, 48 h, 72 h, 7 days, 15 days, and 30 days after surgery. (1) The bilateral ablations group exhibited a significant increase in the ABR threshold of the electrolytic damage group between pre- and post-operation. (2) There were significant increases in the I-VI latencies at 0 h after MGBd and MGBm lesions and at 24 h after MGBv lesion. (3) The amplitudes of wave VI were significantly decreased at 24 h and 48 h after MGBd lesion, at 72 h and 7 days after MGBm lesion, and at 24 h, 48 h, 72 h, and 7 days after MGBv lesion. (1) The electrolytic damage group suffered hearing loss that did not recover and appeared to be difficult to fully repair after bilateral ablation. (2) The latencies and amplitudes of responses in the MGB following bilateral electrolytic lesion were restored to pre-operation levels after 15-30 days, suggesting that a portion of the central nuclei lesion was reversible. (3) The tree shrew auditory animal model has many advantages compared to other animal models, such as greater complexity of brain structure and auditory nuclei fiber connections, which make the results of this experiment more useful for clinical diagnoses compared with studies using rats and guinea pigs.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging.

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews (Tupaia belangeri chinensis). The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

Transcriptome Profiles Using Next-Generation Sequencing Reveal Liver Changes in the Early Stage of Diabetes in Tree Shrew (Tupaia belangeri chinensis).

Determining the liver changes during the early stages of diabetes is critical to understand the nature of the disease and development of novel treatments for it. Advances in the use of animal models and next-generation sequencing technologies offer a powerful tool in connection between liver changes and the diabetes. Here, we created a tree shrew diabetes model akin to type 1 diabetes by using streptozotocin to induce hyperglycemia and hyperlipidemia. Using RNA-seq, we compiled liver transcriptome profiles to determine the differentially expressed genes and to explore the role of hyperglycemia in liver changes. Our results, respectively, identified 14,060 and 14,335 genes in healthy tree shrews and those with diabetes, with 70 genes differentially expressed between the two groups. Gene orthology and KEGG annotation revealed that several of the main biological processes of these genes were related to translational processes, steroid metabolic processes, oxidative stress, inflammation, and hypertension, all of which are highly associated with diabetes and its complications. These results collectively suggest that STZ induces hyperglycemia in tree shrew and that hyperglycemia induced oxidative stress led to high expression of aldose reductase, inflammation, and even cell death in liver tissues during the early stage of diabetes.

Identification of the full-length ß-actin sequence and expression profiles in the tree shrew (Tupaia belangeri).

The tree shrew (Tupaia belangeri) diverges from the primate order (Primates) and is classified as a separate taxonomic group of mammals - Scandentia. It has been suggested that the tree shrew can be used as an animal model for studying human diseases; however, the genomic sequence of the tree shrew is largely unidentified. In the present study, we reported the full-length cDNA sequence of the housekeeping gene, ß-actin, in the tree shrew. The amino acid sequence of ß-actin in the tree shrew was compared to that of humans and other species; a simple phylogenetic relationship was discovered. Quantitative polymerase chain reaction (qPCR) and western blot analysis further demonstrated that the expression profiles of ß-actin, as a general conservative housekeeping gene, in the tree shrew were similar to those in humans, although the expression levels varied among different types of tissue in the tree shrew. Our data provide evidence that the tree shrew has a close phylogenetic association with humans. These findings further enhance the potential that the tree shrew, as a species, may be used as an animal model for studying human disorders.