Towards the peak: The 10-year journey of the National Research Facility for Phenotypic and Genetic Analysis of Model Animals(Primate Facility) and a call for international collaboration in non-human primate research

Some 10 years ago, in early 2012, we started discussing the establishment of the National Research Facility for Phenotypic and Genetic Analysis of Model Animals(Primate Facility).Even though the Primate Facility is still under construction, the rapid passing of a decade is a good excuse to look back and reflect for a moment.

Harvesting the fruits of the first stage of the Primate Genome Project

Primates are highly successful mammals with significant morphological,behavioral,and physiological diversity.Studying the genomes of non-human primates,as the closest relative of humans,can provide insights into human evolution,genetic structure,and potential drug targets relevant to human health,thus making important contributions to medical research.Additionally,primate genome research can support ecological balance and resource conservation and promote sustainable development and human well-being.Despite the existence of more than 500 primate species belonging to 80 genera and 16 families worldwide,with new species still being discovered in recent years(Fan et al.,2017;Khanal et al.,2021;Roos et al.,2020),genome sequencing efforts have been limited to a relatively small number of species from only 22 genera(Ensembl v103).Notably,approximately 72%of primate genera remain unsequenced,leading to significant knowledge gaps in our understanding of their evolutionary history.This situation presents considerable challenges for the development,utilization,and protection of primate genetic resources.It is for these compelling reasons that we initiated the Primate Genome Project(PGP)(Wu et al.,2022).

Immunobiology of COVID-19: Mechanistic and therapeutic insights from animal models

The distribution of the immune system throughout the body complicates in vitro assessments of coronavirus disease 2019(COVID-19)immunobiology,often resulting in a lack of reproducibility when extrapolated to the whole organism.Consequently,developing animal models is imperative for a comprehensive understanding of the pathology and immunology of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.This review summarizes current progress related to COVID-19 animal models,including non-human primates(NHPs),mice,and hamsters,with a focus on their roles in exploring the mechanisms of immunopathology,immune protection,and long-term effects of SARS-CoV-2 infection,as well as their application in immunoprevention and immunotherapy of SARS-CoV-2 infection.Differences among these animal models and their specific applications are also highlighted,as no single model can fully encapsulate all aspects of COVID-19.To effectively address the challenges posed by COVID-19,it is essential to select appropriate animal models that can accurately replicate both fatal and non-fatal infections with varying courses and severities.Optimizing animal model libraries and associated research tools is key to resolving the global COVID-19 pandemic,serving as a robust resource for future emerging infectious diseases.

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.

Bat-derived oligopeptide LE6 inhibits the contact-kinin pathway and harbors anti-thromboinflammation and stroke potential

Thrombosis and inflammation are primary contributors to the onset and progression of ischemic stroke.The contact-kinin pathway,initiated by plasma kallikrein(PK)and activated factor XII(FXIIa),functions bidirectionally with the coagulation and inflammation cascades,providing a novel target for therapeutic drug development in ischemic stroke.In this study,we identified a bat-derived oligopeptide from Myotis myotis(Borkhausen,1797),designated LE6(Leu-Ser-Glu-Glu-Pro-Glu,702 Da),with considerable potential in stroke therapy due to its effects on the contact kinin pathway.Notably,LE6 demonstrated significant inhibitory effects on PK and FXIIa,with inhibition constants of 43.97μmol/L and 6.37μmol/L,respectively.In vitro analyses revealed that LE6 prolonged plasma recalcification time and activated partial thromboplastin time.In murine models,LE6 effectively inhibited carrageenan-induced mouse tail thrombosis,FeCl3-induced carotid artery thrombosis,and photochemically induced intracerebral thrombosis.Furthermore,LE6 significantly decreased inflammation and stroke injury in transient middle cerebral artery occlusion models.Notably,the low toxicity,hemolytic activity,and bleeding risk of LE6,along with its synthetic simplicity,underscore its clinical applicability.In conclusion,as an inhibitor of FXIIa and PK,LE6 offers potential therapeutic benefits in stroke treatment by mitigating inflammation and preventing thrombus formation.

Study of tree shrew biology and models: A booming and prosperous field for biomedical research

The tree shrew(Tupaia belangeri)has long been proposed as a suitable alternative to non-human primates(NHPs)in biomedical and laboratory research due to its close evolutionary relationship with primates.In recent years,significant advances have facilitated tree shrew studies,including the determination of the tree shrew genome,genetic manipulation using spermatogonial stem cells,viral vector-mediated gene delivery,and mapping of the tree shrew brain atlas.However,the limited availability of tree shrews globally remains a substantial challenge in the field.Additionally,determining the key questions best answered using tree shrews constitutes another difficulty.Tree shrew models have historically been used to study hepatitis B virus(HBV)and hepatitis C virus(HCV)infection,myopia,and psychosocial stress-induced depression,with more recent studies focusing on developing animal models for infectious and neurodegenerative diseases.Despite these efforts,the impact of tree shrew models has not yet matched that of rodent or NHP models in biomedical research.This review summarizes the prominent advancements in tree shrew research and reflects on the key biological questions addressed using this model.We emphasize that intensive dedication and robust international collaboration are essential for achieving breakthroughs in tree shrew studies.The use of tree shrews as a unique resource is expected to gain considerable attention with the application of advanced techniques and the development of viable animal models,meeting the increasing demands of life science and biomedical research.

Initiation of the Primate Genome Project

A crucial step for understanding human evolution is to identify the genomic changes that occurred during primate evolution,thus allowing investigators to reconstruct the ancestral states preceding the human condition.In the past several decades,the primate clade has been a research focus in genome sequencing due to its unique phylogenetic position and key importance.

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.

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1,TRF2,and RAD50

Telomeres are nucleoprotein structures located at the end of each chromosome,which function in terminal protection and genomic stability.Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo.As relatively long-lived mammals based on body size,bats display unique telomeric patterns,including the upregulation of genes involved in alternative lengthening of telomeres(ALT),DNA repair,and DNA replication.At present,however,the relevant molecular mechanisms remain unclear.In this study,we performed cross-species comparison and identified EPAS1,a well-defined oxygen response gene,as a key telomeric protector in bat fibroblasts.Bat fibroblasts showed high expression of EPAS1,which enhanced the transcription of shelterin components TRF1 and TRF2,as well as DNA repair factor RAD50,conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion.Based on a human single-cell transcriptome atlas,we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation.Using in vitro-cultured human pulmonary endothelial cells,we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans.In addition,the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence.In conclusion,we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging,drawing insights from the longevity of bats.

Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques

As of June 2020, Coronavirus Disease 2019(COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years,making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection. To examine the effect of age on death, we established a SARSCoV-2 infection model in Chinese rhesus macaques(Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b^+ and CD8^+ cells in lungs at one-week post infection(wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b^+ cells, and persistent infiltration of CD8^+ cells in the lungs at 2 wpi. In addition,peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

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.

Biological implications and limitations of a cynomolgus monkey with naturally occurring Parkinson's disease

We recently identified a cynomolgus monkey with naturally occurring Parkinson's disease(PD), indicating that PD may not be a uniquely human disease(Li et al., 2020). In our previous study, four lines of evidence, including typical PD clinical symptoms, pharmacological responses, pathological hallmarks, and genetic mutations, strongly supported the identification of a monkey with spontaneous PD(Figure 1).

Single-nucleus transcriptomic profiling of multiple organs in a rhesus macaque model of SARS-CoV-2 infection

Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000single-nucleus transcriptomes of the lung, liver,kidney, and cerebral cortex in rhesus macaques(Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multiorgan dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019(COVID-19).Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway,which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy(an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection,which may facilitate the development of therapeutic interventions for COVID-19.

Low color temperature artificial lighting can slow myopia development: Long-term study using juvenile monkeys

The incidence of myopia has increased rapidly in recent decades, suggesting that environmental factors, such as light,may be an important cause. Correlated color temperature(CCT) is a commonly used index to quantify the spectral composition of light. Here, we used 32 juvenile monkeys(16 females and 16 males) and selected four kinds of light with typical but different CCTs to study the relationship between CCT and ocular axial elongation.

Northern pig-tailed macaques(Macaca leonina)infected with SARS-CoV-2 show rapid viral clearance and persistent immune response

Coronavirus disease 2019(COVID-19),which is caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2),has become an unprecedented global health emergency.At present,SARS-CoV-2-infected nonhuman primates are considered the gold standard animal model for COVID-19 research.Here,we showed that northern pig-tailed macaques(Macaca leonina,NPMs)supported SARS-CoV-2 replication.Furthermore,compared with rhesus macaques,NPMs showed rapid viral clearance in lung tissues,nose swabs,throat swabs,and rectal swabs,which may be due to higher expression of interferon(IFN)-αin lung tissue.However,the rapid viral clearance was not associated with good outcome.In the second week post infection,NPMs developed persistent or even more severe inflammation and body injury compared with rhesus macaques.These results suggest that viral clearance may have no relationship with COVID-19 progression and SARS-CoV-2-infected NPMs could be considered as a critically ill animal model in COVID-19 research.

Monkeypox,wild animals,and potential big problem

A new outbreak of monkeypox virus(MPV)is currently occurring in several regions of the world.Since the beginning of May 2022,3040 cases have been reported to the World Health Organization(WHO)globally.Outside of its usual“endemic”base in Africa,the virus has so far been detected in 47 countries,including in Europe,America,Oceania,and Asia,where monkeypox is uncommon or previously unreported.On 25 June 2022,the WHO Emergency Committee declared that the outbreak does not currently constitute a Public Health Emergency of International Concern,but unanimously acknowledged the emergency nature of the event and that controlling further spread will require a vigorous response.Recently,new cases of MPV infection have been confirmed in Korea,Singapore,and Taiwan,China.Thus,a better understanding of MPV and enhanced surveillance are essential for preventing disease recurrence.

Exploring the primate genome:Unraveling the mysteries of evolution and human disease

Nonhuman primates(NHPs),our closest relatives,represent one of the most successful lineages of adaptive radiation in mammals.The primate order contains several hundred species living in varied ecological niches with phenotypic diversity.However,what we know about NHPs is still limited because of genome diversity.To address this gap,the Primate Genome Project(PGP)consortium recently made a comprehensive sequencing effort.

GSNOR negatively regulates the NLRP3 inflammasome via S-nitrosation of MAPK14

Hyperactivation of the NLRP3 inflammasome has been implicated in the pathogenesis of numerous diseases.However,the precise molecular mechanisms that modulate the transcriptional regulation of NLRP3 remain largely unknown.In this study,we demonstrated that S-nitrosoglutathione reductase(GSNOR)deficiency in macrophages leads to significant increases in the Nlrp3 and Il-1βexpression levels and interleukin-1β(IL-1β)secretion in response to NLRP3 inflammasome stimulation.Furthermore,in vivo experiments utilizing Gsnor^(−/−)mice revealed increased disease severity in both lipopolysaccharide(LPS)-induced septic shock and dextran sodium sulfate(DSS)-induced colitis models.Additionally,we showed that both LPS-induced septic shock and DSS-induced colitis were ameliorated in Gsnor^(−/−)Nlrp3^(−/−)double-knockout(DKO)mice.Mechanistically,GSNOR deficiency increases the S-nitrosation of mitogen-activated protein kinase 14(MAPK14)at the Cys211 residue and augments MAPK14 kinase activity,thereby promoting Nlrp3 and Il-1βtranscription and stimulating NLRP3 inflammasome activity.Our findings suggested that GSNOR is a regulator of the NLRP3 inflammasome and that reducing the level of S-nitrosylated MAPK14 may constitute an effective strategy for alleviating diseases associated with NLRP3-mediated inflammation.

Transferrin Is Up-Regulated by Microbes and Acts as a Negative Regulator of Immunity to Induce Intestinal Immunotolerance

Cross-talks (e.g., host-driven iron withdrawal and microbial iron uptake between host gastrointestinal tract and commensal microbes) regulate immunotolerance and intestinal homeostasis. However, underlying mechanisms that regulate the cross-talks remain poorly understood. Here, we show that bacterial products up-regulate iron-transporter transferrin and transferrin acts as an immunosuppressor by interacting with cluster of differentiation 14 (CD14) to inhibit pattern recognition receptor (PRR) signaling and induce host immunotolerance. Decreased intestinal transferrin is found in germ-free mice and human patients with ulcerative colitis, which are characterized by impaired intestinal immunotolerance. Intestinal transferrin and host immunotolerance are returned to normal when germ-free mice get normal microbial commensalism, suggesting an association between microbial commensalism, transferrin, and host immunotolerance. Mouse colitis models show that transferrin shortage impairs host’s tolerogenic responses, while its supplementation promotes immunotolerance. Designed peptide blocking transferrin–CD14 interaction inhibits immunosuppressive effects of transferrin. In monkeys with idiopathic chronic diarrhea, transferrin shows comparable or even better therapeutic effects than hydrocortisone. Our findings reveal that by up-regulating host transferrin to silence PRR signaling, commensal bacteria counteract immune activation induced by themselves to shape host immunity and contribute for intestinal tolerance.

Bioactive peptides from scorpion venoms:therapeutic scaffolds and pharmacological tools

Evolution and natural selection have endowed animal venoms,including scorpion venoms,with a wide range of pharmacological properties.Consequently,scorpions,their venoms,and/or their body parts have been used since time immemorial in traditional medicines,especially in Africa and Asia.With respect to their pharmacological potential,bioactive peptides from scorpion venoms have become an important source of scientific research.With the rapid increase in the characterization of various components from scorpion venoms,a large number of peptides are identified with an aim of combating a myriad of emerging global health problems.Moreover,some scorpion venom-derived peptides have been established as potential scaffolds helpful for drug development.In this review,we summarize the promising scorpion venoms-derived peptides as drug candidates.Accordingly,we highlight the data and knowledge needed for continuous characterization and development of additional natural peptides from scorpion venoms,as potential drugs that can treat related diseases.