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.

Protein Restriction Effects on Healthspan and Lifespan in Drosophila melanogaster Are Additive With a Longevity-Promoting Diet.

Aging of the organism is associated diminished response to external stimuli including weakened immune function, resulting in diseases that impair health and lifespan. Several dietary restriction modalities have been reported to improve health and lifespan in different animal models, but it is unknown whether any of the lifespan-extending dietary treatments could be combined to achieve an additive effect. Here, we investigated the effects of halving amino acids components in the HUNTaa diet, a synthetic medium known to extend lifespan in Drosophila. We found that dietary restriction by halving the entire amino acid components (DR group) could further extend lifespan and improve resistance to oxidative stress, desiccation stress, and starvation than flies on HUNTaa diet alone (wt group). Transcriptome analysis of Drosophila at 40, 60, and 80 days of age revealed that genes related to cell proliferation and metabolism decreased with age in the wt group, whereas background stimulus response and amino acid metabolism increased with age. However, these trends differed in the DR group, that is, the DR flies had downregulated stress response genes, including reduced background immune activation. Infection experiments demonstrated that these flies survived longer after feeding infection with Serratia marcescens and Enterococcus faecalis, suggesting that these flies had stronger immune function, and therefore reduced immune senescence. These results demonstrated that halving the entire amino acid components in the HUNTaa diet further extended health and lifespan and suggested that lifespan-extending diet and dietary restriction treatment could be combined to achieve additive beneficial results.

Atg4b Overexpression Extends Lifespan and Healthspan in Drosophila melanogaster.

Autophagy plays important but complex roles in aging, affecting health and longevity. We found that, in the general population, the levels of ATG4B and ATG4D decreased during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. We therefore analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila, and found that, indeed, Atg4b overexpression increased resistance to oxidative stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, our study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.

Persistent inflammation and neuronal loss in the mouse brain induced by a modified form of attenuated herpes simplex virus type I.

Herpes simplex virus-1 (HSV-1) is a widespread neurotropic virus that can reach the brain and cause a rare but acute herpes simplex encephalitis (HSE) with a high mortality rate. Most patients present with changes in neurological and behavioral status, and survivors suffer long-term neurological sequelae. To date, the pathogenesis leading to brain damage is still not well understood. HSV-1 induced encephalitis in the central nervous system (CNS) in animals are usually very diffuse and progressing rapidly, and mostly fatal, making the analysis difficult. Here, we established a mouse model of HSE via intracerebral inoculation of modified version of neural-attenuated strains of HSV-1 (deletion of ICP34.5 and inserting a strong promoter into the latency-associated transcript region), in which the LMR-αΔpA strain initiated moderate productive infection, leading to strong host immune and inflammatory response characterized by persistent microglia activation. This viral replication activity and prolonged inflammatory response activated signaling pathways in neuronal damage, amyloidosis, Alzheimer's disease, and neurodegeneration, eventually leading to neuronal loss and behavioral changes characterized by hypokinesia. Our study reveals detailed pathogenic processes and persistent inflammatory responses in the CNS and provides a controlled, mild and non-lethal HSE model for studying long-term neuronal injury and increased risk of neurodegenerative diseases due to HSV-1 infection.

dNAGLU Extends Life Span and Promotes Fitness and Stress Resistance in Drosophila.

To identify new factors that promote longevity and healthy aging, we studied Drosophila CG13397, an ortholog of the human NAGLU gene, a lysosomal enzyme overexpressed in centenarians. We found that the overexpression of CG13397 (dNAGLU) ubiquitously, or tissue specifically, in the nervous system or fat body could extend fly life span. It also extended the life span of flies overexpressing human Aß42, in a Drosophila Alzheimer's disease (AD) model. To investigate whether dNAGLU could influence health span, we analyzed the effect of its overexpression on AD flies and found that it improved the climbing ability and stress resistance, including desiccation and hunger, suggesting that dNAGLU improved fly health span. We found that the deposition of Aß42 in the mushroom body, which is the fly central nervous system, was reduced, and the lysosomal activity in the intestine was increased in dNAGLU over-expressing flies. When NAGLU was overexpressed in human U251-APP cells, which expresses a mutant form of the Aß-precursor protein (APP), APP-p.M671L, these cells exhibited stronger lysosomal activity and and enhanced expression of lysosomal pathway genes. The concentration of Aß42 in the cell supernatant was reduced, and the growth arrest caused by APP expression was reversed, suggesting that NAGLU could play a wider role beyond its catalytic activity to enhance lysosomal activity. These results also suggest that NAGLU overexpression could be explored to promote healthy aging and to prevent the onset of neurodegenerative diseases, including AD.

ETS1 acts as a regulator of human healthy aging via decreasing ribosomal activity.

Adaptation to reduced energy production during aging is a fundamental issue for maintaining healthspan or prolonging life span. Currently, however, the underlying mechanism in long-lived people remains poorly understood. Here, we analyzed transcriptomes of 185 long-lived individuals (LLIs) and 86 spouses of their children from two independent Chinese longevity cohorts and found that the ribosome pathway was significantly down-regulated in LLIs. We found that the down-regulation is likely controlled by ETS1 (ETS proto-oncogene 1), a transcription factor down-regulated in LLIs and positively coexpressed with most ribosomal protein genes (RPGs). Functional assays showed that ETS1 can bind to RPG promoters, while ETS1 knockdown reduces RPG expression and alleviates cellular senescence in human dermal fibroblast (HDF) and embryonic lung fibroblast (IMR-90) cells. As protein synthesis/turnover in ribosomes is an energy-intensive cellular process, the decline in ribosomal biogenesis governed by ETS1 in certain female LLIs may serve as an alternative mechanism to achieve energy-saving and healthy aging.

An Aerolysin-like Pore-Forming Protein Complex Targets Viral Envelope to Inactivate Herpes Simplex Virus Type 1.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.

Cohesin promotes HSV-1 lytic transcription by facilitating the binding of RNA Pol II on viral genes.

BACKGROUND: Herpes Simplex Virus type I (HSV-1) is a large double-stranded DNA virus that enters productive infection in epithelial cells and reorganizes the host nucleus. Cohesin, a major constituent of interphase and mitotic chromosomes comprised of SMC1, SMC3, and SCC1 (Mcd1/Rad21), SCC3 (SA1/SA2), have diverse functions, including sister chromatid cohesion, DNA double-stranded breaks repair, and transcriptional control. Little is known about the role of cohesin in HSV-1 lytic infection. METHODS: We measured the effect on HSV-1 transcription, genome copy number, and viral titer by depleting cohesin components SMC1 or Rad21 using RNAi, followed by immunofluorescence, qPCR, and ChIP experiments to gain insight into cohesin's function in HSV-1 transcription and replication. RESULTS: Here, we report that cohesion subunits SMC1 and Rad21 are recruited to the lytic HSV-1 replication compartment. The knockdown results in decreased viral transcription, protein expression, and maturation of viral replication compartments. SMC1 and Rad21 knockdown leads to the reduced overall RNA pol II occupancy level but increased RNA pol II ser5 phosphorylation binding on viral genes. Consistent with this, the knockdown increased H3K27me3 modification on these genes. CONCLUSIONS: These results suggest that cohesin facilitates HSV-1 lytic transcription by promoting RNA Pol II transcription activity and preventing chromatin's silencing on the viral genome.

Correction to: Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

An amendment to this paper has been published and can be accessed via the original article.

Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

BACKGROUND: Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS: We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58 days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS: Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS: These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.

Hypoxia and cancer related pathology.

Hypoxic environments occur normally at high altitude, or in underground burrows and in deep sea habitats. They also occur pathologically in human ischemia and in hypoxic solid tumors. Hypoxia in various cancer types and its related molecular mechanisms are associated with a poor clinical outcome. This review will discuss how hypoxia can influence two aspects of tumorigenesis, namely the direct, cell-intrinsic oncogenic effects, as well as the indirect effects on tumor progression mediated by an altered tumor microenvironment. We will also discuss recent progress in identifying the functional roles of hypoxia-related factors (HIFs), along with their regulators and downstream target genes, in cancer stem cells and therapy. Importantly, we propose, using convergent evolution schemes to identify novel biomarkers for both hypoxia adaptation and hypoxic solid tumors as an important strategy in the future.

HSV-1 infection and pathogenesis in the tree shrew eye following corneal inoculation.

Herpes simplex virus type I (HSV-1) infection causes inflammation in the cornea known as herpes simplex virus keratitis (HSK), a common but serious corneal disease. It is not entirely clear whether the virus during recurring infection comes from the trigeminal ganglia or the eye tissue, including the retina and ciliary ganglion. Because the tree shrew is closely related to primates and tree shrew eye anatomic structures are similar to humans, we studied HSV-1 corneal infection in the tree shrew. We found that HSK symptoms closely mimic those found in human HSK showing typical punctiform and dendritic viral keratitis during the acute infection period. Following the HSV-specific lesions, complications such as stromal scarring, corneal thickening (primary infection), opacity, and neovascularization were observed. In the tree shrew model, following ocular inoculation, the cornea becomes infected, and viral protein can be detected using anti-HSV-1 antibodies in the epithelial layer and retina neuronal ganglion cells. The HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 3 days post-infection (dpi), peaking at 5 dpi. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the Latency Associated Transcripts (LATs) continue to accumulate. Interestingly, after the acute infection, we still detected abundant active HSV-1 in tree shrew eyes. Further, we found HSV-1 persistent in the ciliary ganglion and cornea. These findings are discussed in support of the tree shrew as a non-human primate HSK model, which could be useful for mechanistic studies of HSK.

YTHDF1 links hypoxia adaptation and non-small cell lung cancer progression.

Hypoxia occurs naturally at high-altitudes and pathologically in hypoxic solid tumors. Here, we report that genes involved in various human cancers evolved rapidly in Tibetans and six Tibetan domestic mammals compared to reciprocal lowlanders. Furthermore, m6A modified mRNA binding protein YTHDF1, one of evolutionary positively selected genes for high-altitude adaptation is amplified in various cancers, including non-small cell lung cancer (NSCLC). We show that YTHDF1 deficiency inhibits NSCLC cell proliferation and xenograft tumor formation through regulating the translational efficiency of CDK2, CDK4, and cyclin D1, and that YTHDF1 depletion restrains de novo lung adenocarcinomas (ADC) progression. However, we observe that YTHDF1 high expression correlates with better clinical outcome, with its depletion rendering cancerous cells resistant to cisplatin (DDP) treatment. Mechanistic studies identified the Keap1-Nrf2-AKR1C1 axis as the downstream mediator of YTHDF1. Together, these findings highlight the critical role of YTHDF1 in both hypoxia adaptation and pathogenesis of NSCLC.

Correction to: ZIKV infection effects changes in gene splicing, isoform composition and lncRNA expression in human neural progenitor cells.

In the original publication of this article [1], two grants from National Science Foundation of China and Yunnan Provincial Government (U1602226) and by National Science Foundation of China (2016YFC1200404) were omitted in the 'Funding' section. The correct 'Funding' section is below.

Transcriptome evidence reveals enhanced autophagy-lysosomal function in centenarians.

Centenarians (CENs) are excellent subjects to study the mechanisms of human longevity and healthy aging. Here, we analyzed the transcriptomes of 76 centenarians, 54 centenarian-children, and 41 spouses of centenarian-children by RNA sequencing and found that, among the significantly differentially expressed genes (SDEGs) exhibited by CENs, the autophagy-lysosomal pathway is significantly up-regulated. Overexpression of several genes from this pathway, CTSB, ATP6V0C, ATG4D, and WIPI1, could promote autophagy and delay senescence in cultured IMR-90 cells, while overexpression of the Drosophila homolog of WIPI1, Atg18a, extended the life span in transgenic flies. Interestingly, the enhanced autophagy-lysosomal activity could be partially passed on to their offspring, as manifested by their higher levels of both autophagy-encoding genes and serum beclin 1 (BECN1). In light of the normal age-related decline of autophagy-lysosomal functions, these findings provide a compelling explanation for achieving longevity in, at least, female CENs, given the gender bias in our collected samples, and suggest that the enhanced waste-cleaning activity via autophagy may serve as a conserved mechanism to prolong the life span from Drosophila to humans.

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

ZIKV infection effects changes in gene splicing, isoform composition and lncRNA expression in human neural progenitor cells.

BACKGROUND: The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes microcephaly and Guillain-Barré syndrome in infected individuals. To obtain insights into the mechanism of ZIKV infection and pathogenesis, we analyzed the transcriptome of ZIKV infected human neural progenitor cells (hNPCs) for changes in alternative splicing (AS), gene isoform (ISO) composition and long noncoding RNAs (lncRNAs) expression. METHODS: We analyzed differentially expressed lncRNAs, AS, ISO from RNA-seq data in ZIKV infected hNPCs. RESULTS: We obtained 149 differentially expressed lncRNAs, including potential viral targets to modulate cellular processes such as cell cycle, apoptosis and immune response. The infection induced 262 cases of AS occurring in 229 genes, which were enriched in cell death, RNA processing, transport, and neuron development. Among 691 differentially expressed ISOs, upregulated ISOs were enriched in signaling, regulation of transcription, and amino acid biosynthesis, while downregulated ISOs were mostly enriched in cell cycle. Importantly, these analyses revealed specific links between ZIKV induced changes in cellular pathways and the type of changes in the host transcriptome, suggesting important regulatory mechanisms. CONCLUSIONS: Our analyses revealed candidate lncRNAs, AS events and ISOs which may function in ZIKV infection induced cell cycle disruption, apoptosis and attenuation of neurogenesis, and shed light on the roles of lncRNAs, AS and ISOs in virus-host interactions, and would facilitate future studies of ZIKV infection and pathogenesis.

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair.

CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs). CTCF depletion increased chromosomal instability, marked by chromosome breakage and end fusions, elevated genotoxic stress-induced genomic DNA fragmentation, and activated the ataxia telangiectasia mutated (ATM) kinase. We show that CTCF could be recruited to drug-induced 53BP1 foci and known fragile sites, as well as to I-SceI endonuclease-induced DSBs. Laser irradiation analysis revealed that this recruitment depends on ATM, Nijmegen breakage syndrome (NBS), and the zinc finger DNA-binding domain of CTCF. We demonstrate that CTCF knockdown impaired homologous recombination (HR) repair of DSBs. Consistent with this, CTCF knockdown reduced the formation of γ-radiation-induced Rad51 foci, as well as the recruitment of Rad51 to laser-irradiated sites of DNA lesions and to I-SceI-induced DSBs. We further show that CTCF is associated with DNA HR repair factors MDC1 and AGO2, and directly interacts with Rad51 via its C terminus. These analyses establish a direct, functional role of CTCF in DNA repair and provide a potential link between genome organization and genome stability.