Identification of novel mammalian viruses in tree shrews(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer,infectious diseases,metabolic disorders,and mental health conditions.Herein,we used metatranscriptomic sequencing to analyze plasma,as well as oral and anal swab samples,from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses.In total,eight mammalian viruses with complete genomes were identified,belonging to six viral families,including Flaviviridae,Hepeviridae,Parvovirinae,Picornaviridae,Sedoreoviridae,and Spinareoviridae.Notably,the presence of rotavirus was recorded in tree shrews for the first time.Three viruses-hepacivirus 1,parvovirus,and picornavirus-exhibited low genetic similarity(<70%)with previously reported viruses at the whole-genome scale,indicating novelty.Conversely,three other viruses-hepacivirus 2,hepatovirus A and hepevirus-exhibited high similarity(>94%)to known viral strains.Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants.These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews,highlighting the necessity for further research into their potential for crossspecies transmission.

Using Tree Shrews (Tupaia belangeri)as a Novel Animal Model of Liver Transplantation

Liver transplantation (LT)is most effective and promising approach for end-stage liver disease.However,there remains room for further improvement and innovation,for example, to reduce ischemic reperfusion injury,transplant rejection and immune tolerance.A good animal model of LT is essential for such innovation in transplant research.Although rat LT model has been used since the last century,it has never been an ideal model because the results observed in rat may not be applied to human because these two species are genetically distinct from each other.In this study,we for the first time performed LT using the tree shrew (Tupaia belangeri),a species in the Order Scandentia which is closely related with primates,and evaluated the possibility to adopt this species as a new model of LT.We performed LT on 30 animals using the two-cuff technique, examining the success rate,the survival rate and the immunological reaction.The recipient operation time was 60 min averagely,and we limited the time of the anhepatic phase within 20 min.Twenty-seven (90%)of the animals survived for at least 3 days after the transplantation. Thirteen animals that did not receive any immunosuppressive drug died in 8 days mostly because of acute rejection effect (n=9),similar to the reaction in human but not in experimental rat.The rest 14 animals that were given rapamycin survived significantly longer (38 days)and half of them survived for 60 days until the end of the study.Our results suggest that performing LT in tree shrews can yield high success rate and high survival rate.More importantly,the tree shrews share similar immunological reaction with human.In addition,previous genomics study found that the tree shrews share more proteins with human.In sum,the tree shrews may outperform the experimental rats and could be used as a better and cost-effective animal model for LT.

The characterization of auditory brainstem response(ABR) waveforms: A study in tree shrews(Tupaia belangeri)

To characterize the patterns of ABR waves in tree shrews, we must understand the hearing sensitivity and auditory function of healthy adult tree shrews. Fifteen tree shrews(30 ears) were stimulated with clicks and tone-pips at 11 different frequencies from 1 to 60 kHz. The ABR waves were recorded and analyzed. The ABR consisted of five to seven positive waves in the first 10 ms after a click stimulus, and the average hearing threshold of component III was 27.86 ± 3.78 dB SPL. Wave III was the largest and most clear. The ABR threshold was related to the tone-pip sitmulus by a "U" shaped curve. The sensitive frequency was approximately 8 kHz in tree shrews. The latencies systematically decreased with increasing stimulus frequencies. The ABR amplitudes of wave III increased as the sound pressure level increased. All of these results provide an empirical basis for future studies of hearing diseases in tree shrews.

Basal physiological parameters in domesticated tree shrews(Tupaia belangeri chinensis)

Establishing non-human primate models of human diseases is an efficient way to narrow the large gap between basic studies and translational medicine.Multifold advantages such as simplicity of breeding,low cost of feeding and facility of operating make the tree shrew an ideal non-human primate model proxy.Additional features like vulnerability to stress and spontaneous diabetic characteristics also indicate that the tree shrew could be a potential new animal model of human diseases.However,basal physiological indexes of tree shrew,especially those related to human disease,have not been systematically reported.Accordingly,we established important basal physiological indexes of domesticated tree shrews including several factors:(1)body weight,(2)core body temperature and rhythm,(3)diet metabolism,(4)locomotor rhythm,(5)electroencephalogram,(6)glycometabolism and(7)serum and urinary hormone level and urinary cortisol rhythm.We compared the physiological parameters of domesticated tree shrew with that of rats and macaques.Results showed that(a)the core body temperature of the tree shrew was 39.59±0.05°C,which was higher than that of rats and macaques;(b)Compared with wild tree shrews,with two activity peaks,domesticated tree shrews had only one activity peak from 17:30 to 19:30;(c)Compared with rats,tree shrews had poor carbohydrate metabolism ability;and(d)Urinary cortisol rhythm indicated there were two peaks at 8:00 and 17:00 in domesticated tree shrews,which matched activity peaks in wild tree shrews.These results provided basal physiological indexes for domesticated tree shrews and laid an important foundation for diabetes and stress-related disease models established on tree shrews.

Regeneration of islet β-cells in tree shrews and rats

Backgroud: Current understanding of injury and regeneration of islet β-cells in diabetes is mainly based on rodent studies. The tree shrew is now generally accepted as being among the closest living relatives of primates, and has been widely used in animal experimentation. However, there are few reports on islet cell composition and regeneration of β-cells in tree shrews.Methods: In this study, we examined the changes in islet cell composition and regeneration of β-cells after streptozotocin(STZ) treatment in tree shrews compared with Sprague-Dawley rats. Injury and regeneration of islet β-cells were observed using hematoxylin and eosin(HE) staining and immunohistochemical staining for insulin, glucagon, somatostatin and PDX-1.Results: Our data showed that in rats islet injury was most obvious on day 3 after injection, and islet morphologies were significantly restored by day 21. Regeneration of islet β-cells was very pronounced in rats, and mainly involved regeneration of centro-acinar cells and transformation of extra-islet ductal cells. In tree shrews, the regeneration of islet β-cells was not as significant. On days 3 and 7, only scattered regenerated cells were observed in the remaining islets. Further, no regeneration of centro-acinar cells was observed.Conclusion: The results suggest that the repair mechanism of islet β-cells in tree shrews is similar to that of humans.

Alteration of the p53 gene during tree shrews' hepatocarcinogenesis

OBJECTIVE: To detect the expression and variation of the p53 gene in hepatocarcinogenesis of treeshrews induced by hepatitis B virus （HBV） and aflatoxin B1（AFB1）.METHODS: Tree shrews were divided into four groups: group A, infected with HBV and fed withAFB1; group B, only infected with HBV; group C, fed with AFB1 alone; and group D normal control.The tree shrews underwent liver biopsy every. 15 weeks. Liver and tumor tissues were detected byimmunohistochemistry and molecular biotechnologies.RESULTS: The incidence of hepatocellular carcinoma （HCC） was higher in group A （66.7%） than ingroups B （0） and C （30%）. HCC occurrence was earlier in group A than in group C （120.0±16.6 wkvs 153.3±5.8 wk, t=3.336, P【0.01）. Mutated p53 protein was not found in all groups before 75weeks of experiment. At the 105th week, the expression rates of mutated p53 protein were 78.6%,60.0% and 71.4% in groups A, B and C respectively, which were significantly higher than that in groupD （10%） （X2≥5.03, P【0.05）. An abnormal band of the p53 gene was detected in groups A and C.The mutational points of the p53 gene in liver cancer of tree shrews were at codon 275, 78 and 13.Nucleotide sequence and amino acids sequence of tree shrew’s wild-type p53 were 91.7% and 93.4% inhomology, compared with those of human p53, respectively.CONCLUSIONS: Remarkable synergistic effect on HCC exists between HBV and AFB1. Mutated p53protein expressed before occurrence of HCC promotes the development of HCC. HBV and AFB1 maysynergistically induce p53 gene mutation.

Human-like adrenal features in Chinese tree shrews revealed by multi-omics analysis of adrenal cell populations and steroid synthesis

The Chinese tree shrew(Tupaia belangeri chinensis)has emerged as a promising model for investigating adrenal steroid synthesis,but it is unclear whether the same cells produce steroid hormones and whether their production is regulated in the same way as in humans.Here,we comprehensively mapped the cell types and pathways of steroid metabolism in the adrenal gland of Chinese tree shrews using single-cell RNA sequencing,spatial transcriptome analysis,mass spectrometry,and immunohistochemistry.We compared the transcriptomes of various adrenal cell types across tree shrews,humans,macaques,and mice.Results showed that tree shrew adrenal glands expressed many of the same key enzymes for steroid synthesis as humans,including CYP11B2,CYP11B1,CYB5A,and CHGA.Biochemical analysis confirmed the production of aldosterone,cortisol,and dehydroepiandrosterone but not dehydroepiandrosterone sulfate in the tree shrew adrenal glands.Furthermore,genes in adrenal cell types in tree shrews were correlated with genetic risk factors for polycystic ovary syndrome,primary aldosteronism,hypertension,and related disorders in humans based on genome-wide association studies.Overall,this study suggests that the adrenal glands of Chinese tree shrews may consist of closely related cell populations with functional similarity to those of the human adrenal gland.Our comprehensive results(publicly available at http://gxmujyzmolab.cn:16245/scAGMap/)should facilitate the advancement of this animal model for the investigation of adrenal gland disorders.

The position of tree shrews in the mammalian tree:Comparing multi-gene analyses with phylogenomic results leaves monophyly of Euarchonta doubtful

The well-accepted Euarchonta grandorder is a pruned version of Archonta nested within the Euarchontoglires(or Supraprimates)clade.At present,it includes tree shrews(Scandentia),flying lemurs(Dermoptera)and primates(Primates).Here,a phylogenomic dataset containing 1912 exons from 22 representative mammals was compiled to investigate the phylogenetic relationships within this group.Phylogenetic analyses and hypothesis testing suggested that tree shrews can be classified as a sister group to Primates or to Glires or even as a basal clade within Euarchontoglires.Further analyses of both modified and original previously published datasets found that the phylogenetic position of tree shrews is unstable.We also found that two of three exonic indels reported as synapomorphies of Euarchonta in a previous study do not unambiguously support the monophyly of such a clade.Therefore,the monophyly of both Euarchonta and Sundatheria(Dermoptera+Scandentia)are suspect.Molecular dating and divergence rate analyses suggested that the ancestor of Euarchontoglires experienced a rapid divergence,which may cause the unresolved position of tree shrews even using the whole genomic data.

Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells

Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to generate transgenic tree shrews.However,the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear.Here,we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages.We found that SSCs lost spermatogenesis ability after long-term expansion(>50 passages),as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia(SPG)-derivedspermatocytesor spermatids marking spermatogenesis.RNA sequencing(RNA-seq)analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers.Specifically,DNA damage response and repair genes(e.g.,MRE11,SMC3,BLM,and GEN1)were down-regulated,whereas genes associated with mitochondrial function(e.g.,NDUFA9,NDUFA8,NDUFA13,and NDUFB8)were up-regulated after expansion.The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells.Supplementation with nicotinamide adenine dinucleotide(NAD+)precursor nicotinamide riboside(NR)exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture.Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.

Shrew攻击对拥塞控制协议的影响及仿真分析

Shrew攻击是一种典型的低速率拒绝服务攻击,其以隐蔽、高效的攻击使大量面向连接的服务性能显著降低甚至瘫痪。针对不同层面Internet拥塞控制之间的内在联系和相互影响,揭示了自适塞控制机制的安全漏洞的根源。在仿真实验中采用多种TCP、IP拥塞控制算法,模拟了有无Shrew攻击的情况下各种算法产生的性能和参数的变化。试验结果表明:持续的Shrew攻击能使服务功能近于瘫痪,同时,SFQ和DRR等公平性算法则能够有效抑制此类攻击。

Bartonella species in small mammals and their potential vectors in Asia

In this article,authors review the current knowledge of Bartonella infection in small mammals including rodents,insectivores,bats and exotic small mammal pets and their vectors in Asia.Species of Bartonella are Gram-negative intracellular bacteria that infect erythrocytes of various mammalian and non-mammalian animals and mainly transmitted by blood sucking arthropod vectors.The genus Bartonella includes several species of important human diseases with severe clinical signs.Several new Bartonella species were isolated from rodents and other small mammals,and from human patients in Asia.Bartonella species are identified using standard polymerase chain reaction amplification and a sequencing targeting two housekeeping genes(glt.A and rpoB) and the internal transcribed spacer fragment.Authors also discuss the implications in term of potential emerging zoonotic diseases.

An annotated checklist of mammals of Kenya

Kenya has a rich mammalian fauna. We reviewed recently published books and papers including the six volumes of Mammals of Africa to develop an up-to-date annotated checklist of all mammals recorded from Kenya. A total of 390 species have been identified in the country, including 106 species of rodents,104 species of bats, 63 species of even-toed ungulates(including whales and dolphins), 36 species of insectivores and carnivores, 19 species of primates,five species of elephant shrews, four species of hyraxes and odd-toed ungulates, three species of afrosoricids, pangolins, and hares, and one species of aardvark, elephant, sirenian and hedgehog. The number of species in this checklist is expected to increase with additional surveys and as the taxonomic status of small mammals(e.g., bats, shrews and rodents) becomes better understood.

清醒活动树鼩Tupaia(Tree Shrew)神经元单位放电的记录

鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报道,本文报告一种记录清醒活动状态树鼩的单位放电技术考虑到树鼩形体甚小、体重仅100多克。要分离单位放电,必须拥有一种特殊规格的微型微推进器,以适应特殊实验之需。本研究设计一种采用差动结构原理实现微推进的装置,整个装置由微推进器与基座组成。微推进器的参数如下:重量8.8克;微调范围:2.0毫米;微调读数5微米;微调可控范围:小于2微米;外形尺寸:13×13×51毫米。微推进器相对于基座有直径为1.8毫米的径向偏心移动范围。基座重0.65克,实验在局麻下,在立体定向仪控制下埋植基座。术后第二天即可实验观察。

Tree shrew (Tupaia belangeri)as a novel laboratory disease animal model

The tree shrew(Tupaia belangeri) is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.

Creating animal models, why not use the Chinese tree shrew ( Tupaia belangeri chinensis)？

The Chinese tree shrew(Tupaia belangeri chinensis), a squirrel-like and rat-sized mammal, has a wide distribution in Southeast Asia, South and Southwest China and has many unique characteristics that make it suitable for use as an experimental animal. There have been many studies using the tree shrew(Tupaia belangeri) aimed at increasing our understanding of fundamental biological mechanisms and for the modeling of human diseases and therapeutic responses. The recent release of a publicly available annotated genome sequence of the Chinese tree shrew and its genome database(www.treeshrewdb.org) has offered a solid base from which it is possible to elucidate the basic biological properties and create animal models using this species. The extensive characterization of key factors and signaling pathways in the immune and nervous systems has shown that tree shrews possess both conserved and unique features relative to primates. Hitherto, the tree shrew has been successfully used to create animal models for myopia, depression, breast cancer, alcohol-induced or non-alcoholic fatty liver diseases, herpes simplex virus type 1(HSV-1) and hepatitis C virus(HCV) infections, to name a few. The recent successful genetic manipulation of the tree shrew has opened a new avenue for the wider usage of this animal in biomedical research. In this opinion paper, I attempt to summarize the recent research advances that have used the Chinese tree shrew, with a focus on the new knowledge obtained by using the biological properties identified using the tree shrew genome, a proposal for the genome-based approach for creating animal models, and the genetic manipulation of the tree shrew. With more studies using this species and the application of cutting-edge gene editing techniques, the tree shrew will continue to be under the spot light as a viable animal model for investigating the basis of many different human diseases.

基于数字信号处理的一种Shrew DDoS攻击检测方法

首先简要介绍了传统DDoS的一般检测方法,进而介绍了一种新型的DDoS攻击———Shrew DDoS攻击,这是一种不同于传统DDoS的低频攻击。针对这种攻击提出了一种基于数字信号处理的检测方法。

Comprehensive annotation of the Chinese tree shrew genome by large-scale RNA sequencing and long-read isoform sequencing

The Chinese tree shrew(Tupaia belangeri chinensis)is emerging as an important experimental animal in multiple fields of biomedical research.Comprehensive reference genome annotation for both mRNA and long non-coding RNA(lncRNA)is crucial for developing animal models using this species.In the current study,we collected a total of 234 high-quality RNA sequencing(RNA-seq)datasets and two long-read isoform sequencing(ISO-seq)datasets and improved the annotation of our previously assembled high-quality chromosomelevel tree shrew genome.We obtained a total of 3514 newly annotated coding genes and 50576 lncRNA genes.We also characterized the tissuespecific expression patterns and alternative splicing patterns of mRNAs and lncRNAs and mapped the orthologous relationships among 11 mammalian species using the current annotated genome.We identified 144 tree shrew-specific gene families,including interleukin 6(IL6)and STT3 oligosaccharyltransferase complex catalytic subunit B(STT3B),which underwent significant changes in size.Comparison of the overall expression patterns in tissues and pathways across four species(human,rhesus monkey,tree shrew,and mouse)indicated that tree shrews are more similar to primates than to mice at the tissue-transcriptome level.Notably,the newly annotated purine rich element binding protein A(PURA)gene and the STT3B gene family showed dysregulation upon viral infection.The updated version of the tree shrew genome annotation(KIZ version 3:TS_3.0)is available at http://www.treeshrewdb.org and provides an essential reference for basic and biomedical studies using tree shrew animal models.

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis)is a small experimental animal with a close affinity to primates.This species has long been proposed to be an alternative experimental animal to primates in biomedical research.Despite decades of study,there is no pure breed for this animal,and the overall genetic diversity of wild tree shrews remains largely unknown.In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations,we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew.An analysis of a wild population of 117 individuals collected from the suburb of Kunming,China,showed that these loci exhibited a highly expected heterozygosity(0.616).These 12 microsatellites were sufficient for individual identification and parentage analysis.The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.

A bright future for the tree shrew in neuroscience research:Summary from the inaugural Tree Shrew Users Meeting

Tree shrews(Tupaia spp.)have been used in neuroscience research since the 1960s due to their evolutionary proximity to primates.The use of and interest in this animal model have recently increased,in part due to the adaptation of modern neuroscience tools in this species.These tools include quantitative behavioral assays,calcium imaging,optogenetics and transgenics.To facilitate the exchange and development of these new technologies and associated research findings,we organized the inaugural“Tree Shrew Users Meeting”which was held online due to the COVID-19 pandemic.Here,we review this meeting and discuss the history of tree shrews as an animal model in neuroscience research and summarize the current themes being investigated using this animal,as well as future directions.

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 of5hm C 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 mR NA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels.Our findings provide new insight into the dynamic changes in 5hm C levels in tree shrew brains during postnatal development and aging processes.