Biomarkers of aging for the identification and evaluation of longevity interventions.

With the rapid expansion of aging biology research, the identification and evaluation of longevity interventions in humans have become key goals of this field. Biomarkers of aging are critically important tools in achieving these objectives over realistic time frames. However, the current lack of standards and consensus on the properties of a reliable aging biomarker hinders their further development and validation for clinical applications. Here, we advance a framework for the terminology and characterization of biomarkers of aging, including classification and potential clinical use cases. We discuss validation steps and highlight ongoing challenges as potential areas in need of future research. This framework sets the stage for the development of valid biomarkers of aging and their ultimate utilization in clinical trials and practice.

A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association.

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.

Accelerated evolution of an Lhx2 enhancer shapes mammalian social hierarchies.

Social hierarchies emerged during evolution, and social rank influences behavior and health of individuals. However, the evolutionary mechanisms of social hierarchy are still unknown in amniotes. Here we developed a new method and performed a genome-wide screening for identifying regions with accelerated evolution in the ancestral lineage of placental mammals, where mammalian social hierarchies might have initially evolved. Then functional analyses were conducted for the most accelerated region designated as placental-accelerated sequence 1 (PAS1, P = 3.15 × 10-18). Multiple pieces of evidence show that PAS1 is an enhancer of the transcription factor gene Lhx2 involved in brain development. PAS1s isolated from various amniotes showed different cis-regulatory activity in vitro, and affected the expression of Lhx2 differently in the nervous system of mouse embryos. PAS1 knock-out mice lack social stratification. PAS1 knock-in mouse models demonstrate that PAS1s determine the social dominance and subordinate of adult mice, and that social ranks could even be turned over by mutated PAS1. All homozygous mutant mice had normal huddled sleeping behavior, motor coordination and strength. Therefore, PAS1-Lhx2 modulates social hierarchies and is essential for establishing social stratification in amniotes, and positive Darwinian selection on PAS1 plays pivotal roles in the occurrence of mammalian social hierarchies.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

Accurate Drug Repositioning through Non-tissue-Specific Core Signatures from Cancer Transcriptomes.

Experimental large-scale screens for drug repositioning are limited by restriction to in vitro conditions and lack of applicability to real human conditions. Here, we developed an in silico screen in human in vivo conditions using a reference of single gene mutations' non-tissue-specific "core transcriptome signatures" (CSs) of 8,476 genes generated from the TCGA database. We developed the core-signature drug-to-gene (csD2G) software to scan 3,546 drug treatment profiles against the reference signatures. csD2G significantly outperformed conventional cell line-based gene perturbation signatures and existing drug-repositioning methods in both coverage and specificity. We highlight this with 3 demonstrated applications: (1) repositioned category of psychiatric drugs to inhibit the TGF-ß pathway; (2) antihypertensive calcium channel blockers predicted to activate AMPK and inhibit AKT pathways, and validated by clinical electronic medical records; and (3) 7 drugs predicted and validated to selectively target the AKT-FOXO and AMPK pathways and thus regulate worm lifespan.

Immune mechanisms induced by an HSV-1 mutant strain: Discrepancy analysis of the immune system gene profile in comparison with a wild-type strain.

Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7, ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy.

Molecular and phenotypic biomarkers of aging.

Individuals of the same age may not age at the same rate. Quantitative biomarkers of aging are valuable tools to measure physiological age, assess the extent of 'healthy aging', and potentially predict health span and life span for an individual. Given the complex nature of the aging process, the biomarkers of aging are multilayered and multifaceted. Here, we review the phenotypic and molecular biomarkers of aging. Identifying and using biomarkers of aging to improve human health, prevent age-associated diseases, and extend healthy life span are now facilitated by the fast-growing capacity of multilevel cross-sectional and longitudinal data acquisition, storage, and analysis, particularly for data related to general human populations. Combined with artificial intelligence and machine learning techniques, reliable panels of biomarkers of aging will have tremendous potential to improve human health in aging societies.

Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta.

Novel regenerative therapies may stem from deeper understanding of the mechanisms governing cardiovascular lineage diversification. Using enhancer mapping and live imaging in avian embryos, and genetic lineage tracing in mice, we investigated the spatio-temporal dynamics of cardiovascular progenitor populations. We show that expression of the cardiac transcription factor Nkx2.5 marks a mesodermal population outside of the cardiac crescent in the extraembryonic and lateral plate mesoderm, with characteristics of hemogenic angioblasts. Extra-cardiac Nkx2.5 lineage progenitors migrate into the embryo and contribute to clusters of CD41+/CD45+ and RUNX1+ cells in the endocardium, the aorta-gonad-mesonephros region of the dorsal aorta and liver. We also demonstrated that ectopic expression of Nkx2.5 in chick embryos activates the hemoangiogenic gene expression program. Taken together, we identified a hemogenic angioblast cell lineage characterized by transient Nkx2.5 expression that contributes to hemogenic endothelium and endocardium, suggesting a novel role for Nkx2.5 in hemoangiogenic lineage specification and diversification.

BMP restricts stemness of intestinal Lgr5+ stem cells by directly suppressing their signature genes.

The intestinal epithelium possesses a remarkable self-renewal ability, which is mediated by actively proliferating Lgr5+ stem cells. Bone morphogenetic protein (BMP) signalling represents one major counterforce that limits the hyperproliferation of intestinal epithelium, but the exact mechanism remains elusive. Here we demonstrate that epithelial BMP signalling plays an indispensable role in restricting Lgr5+ stem cell expansion to maintain intestinal homeostasis and prevent premalignant hyperproliferation on damage. Mechanistically, BMP inhibits stemness of Lgr5+ stem cells through Smad-mediated transcriptional repression of a large number of stem cell signature genes, including Lgr5, and this effect is independent of Wnt/ß-catenin signalling. Smad1/Smad4 recruits histone deacetylase HDAC1 to the promoters to repress transcription, and knockout of Smad4 abolishes the negative effects of BMP on stem cells. Our findings therefore demonstrate that epithelial BMP constrains the Lgr5+ stem cell self-renewal via Smad-mediated repression of stem cell signature genes to ensure proper homeostatic renewal of intestinal epithelium.

Genome-wide analysis of histone acetylation dynamics during mouse embryonic stem cell neural differentiation.

Epigenetic modification as an intrinsic fine-tune program cooperates with key transcription factors to regulate the cell fate determination. The histone acetylation participating in neural differentiation of pluripotent stem cells is expected but not well studied. Here, using acetylated histone H3 ChIP-sequencing (ChIP-seq), we demonstrate that the histone H3 acetylation level is gradually increased on the neural gene loci while decreased on the neural-inhibitory gene loci during mouse embryonic stem cell (mESC) neural differentiation. We further show that histone deacetylase 1 (HDAC1) is essential for neural commitment by targeting Nodal signaling. Thus, our study reveals a mechanism by which the epigenetic modification of histone acetylation/deacetylation interacts with extracellular signaling in mESC neural fate determination. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference number GSE66025.

Genome-wide ChIP-seq and RNA-seq analyses of Pou3f1 during mouse pluripotent stem cell neural fate commitment.

Appropriate neural initiation of the pluripotent stem cells in the early embryos is critical for the development of the central nervous system. This process is regulated by the coordination of extrinsic signals and intrinsic programs. However, how the coordination is achieved to ensure proper neural fate commitment is largely unknown. Here, taking advantage of genome-wide ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses, we demonstrate that the transcriptional factor Pou3f1 is an upstream activator of neural-promoting genes, and it is able to repress neural-inhibitory signals as well. Further studies revealed that Pou3f1 could directly bind neural lineage genes like Sox2 and downstream targets of neural inhibition signaling such as BMP and Wnt. Our results thus identify Pou3f1 as a critical dual-regulator of the intrinsic transcription factors and the extrinsic cellular signals during neural fate commitment. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference number GSE69865.

The effects of graded levels of calorie restriction: IV. Non-linear change in behavioural phenotype of mice in response to short-term calorie restriction.

Animals have to adjust their activities when faced with caloric restriction (CR) to deal with reduced energy intake. If CR is pronounced, allostasis can push individuals into alternate physiological states which can result in important health benefits across a wide range of taxa. Here we developed a new approach to determine the changes in behavioural phenotype associated with different levels of CR. We exposed C57BL/6 male mice to graded CR (from 0 to 40%) for three months and defined their behavioural phenotype using hidden Markov models of their movement and body temperature. All 40% CR mice exhibited a state-shift in behavioural phenotype and only some exposed to 30% CR did. We show for the first time that mice changed their activity characteristics rather than changed their activities. This new phenotyping approach provides an avenue to determine the mechanisms linking CR to healthspan.

Improved nucleosome-positioning algorithm iNPS for accurate nucleosome positioning from sequencing data.

Accurate determination of genome-wide nucleosome positioning can provide important insights into global gene regulation. Here, we describe the development of an improved nucleosome-positioning algorithm-iNPS-which achieves significantly better performance than the widely used NPS package. By determining nucleosome boundaries more precisely and merging or separating shoulder peaks based on local MNase-seq signals, iNPS can unambiguously detect 60% more nucleosomes. The detected nucleosomes display better nucleosome 'widths' and neighbouring centre-centre distance distributions, giving rise to sharper patterns and better phasing of average nucleosome profiles and higher consistency between independent data subsets. In addition to its unique advantage in classifying nucleosomes by shape to reveal their different biological properties, iNPS also achieves higher significance and lower false positive rates than previously published methods. The application of iNPS to T-cell activation data demonstrates a greater ability to facilitate detection of nucleosome repositioning, uncovering additional biological features underlying the activation process.

The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways.

The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positive transcription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. In this study, we showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. Our results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals.

Integrating genomic, epigenomic, and transcriptomic features reveals modular signatures underlying poor prognosis in ovarian cancer.

Ovarian cancer has a poor prognosis, with different outcomes for different patients. The mechanism underlying this poor prognosis and heterogeneity is not well understood. We have developed an unbiased, adaptive clustering approach to integratively analyze ovarian cancer genome-wide gene expression, DNA methylation, microRNA expression, and copy number alteration profiles. We uncovered seven previously uncategorized subtypes of ovarian cancer that differ significantly in median survival time. We then developed an algorithm to uncover molecular signatures that distinguish cancer subtypes. Surprisingly, although the good-prognosis subtypes seem to have not been functionally selected, the poor-prognosis ones clearly have been. One subtype has an epithelial-mesenchymal transition signature and a cancer hallmark network, whereas the other two subtypes are enriched for a network centered on SRC and KRAS. Our results suggest molecular signatures that are highly predictive of clinical outcomes and spotlight "driver" genes that could be targeted by subtype-specific treatments.

Prognostic value of combined serum biomarkers in predicting outcomes in cervical cancer patients.

BACKGROUND: We evaluated the prognostic value of pretreatment serum biomarkers in predicting outcomes in cervical cancer patients subjected to treatment. METHODS: Serum samples collected from 60 cervical cancer patients and 60 age-matched healthy individuals were used for the detection of 22 biomarkers, prior to therapy. Cox multivariate analysis and classification and regression tree analysis (CART) were performed to evaluate the prognostic factors. RESULTS: Cox multivariate analysis disclosed that carbohydrate antigen 153 (CA153), squamous cell carcinoma antigen (SCC) and tumor necrosis factor-α (TNF-α) are associated with prognosis in cervical cancer. CART analysis led to the stratification of patients into 3 groups: (1) serum concentrations of CA153 ≥17.60 µg/l, (2) serum concentrations of CA153 <17.60 µg/l and TNF-α ≥10.60 pg/ml, and (3) serum concentrations of CA153 <17.60 µg/l and TNF-α <10.60 pg/ml. The 2-y overall survival rates for Groups 1, 2 and 3 were 33.3%, 60.0% and 93.9%, respectively. CONCLUSIONS: Higher serum concentrations of TNF-α, SCC and CA153 before therapy are independently associated with poor prognosis in patients with stage I and II disease. Combined usage of these three biomarkers allows efficient evaluation of outcomes in cervical cancer patients.

An aging program at the systems level?

Many genes and pathways are known to modulate lifespan in various organisms, but it remains unclear whether there exists a common aging program, and how individual variations of lifespan can occur in an isogenic population. Recent studies on aging regulation at the systems and epigenetic levels point to the possibility of regulating and potentially reversing the aging epigenome and transcriptome, resulting in differential aging status and aging rate in different individuals. Here, the author summarize some of these findings and discuss the possibility of integrating multiple layers of aging regulation at the systems level, to identify an aging program that can explain lifespan variations introduced by environmental and developmental history.

Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis.

Although microarray and expressed sequence tag (EST)-based approaches have been used to profile gene expression during baculovirus infection, the response of host genes to baculovirus infection and the interaction between baculovirus and its host remain largely unknown. To determine the host response to Bombyx mori nucleopolyhedrovirus infection and the dynamic interaction between the virus and its host, eight digital gene expression libraries were examined in a Bm5 cell line before infection and at 1.5, 3, 6, 12, 24, 48, and 96 h postinfection. Gene set enrichment analysis of differentially expressed genes at each time point following infection showed that gene sets including cytoskeleton, transcription, translation, energy metabolism, iron ion metabolism, and the ubiquitin-proteasome pathway were altered after viral infection. In addition, a time course depicting protein-protein interaction networks between the baculovirus and the host were constructed and revealed that viral proteins interact with a multitude of cellular machineries, such as the proteasome, cytoskeleton, and spliceosome. Several viral proteins, including IE2, CG30, PE38, and PK-1/2, were predicted to play key roles in mediating virus-host interactions. Based on these results, we tested the role of the ubiquitin-proteasome pathway and iron ion metabolism in the viral infection cycle. Treatment with a proteasome inhibitor and deferoxamine mesylate in vitro and in vivo confirmed that these pathways regulate viral infection. Taken together, these findings provide new insights into the interaction between the baculovirus and its host and identify molecular mechanisms that can be used to block viral infection and improve baculovirus expression systems.

Understanding biological functions through molecular networks.

The completion of genome sequences and subsequent high-throughput mapping of molecular networks have allowed us to study biology from the network perspective. Experimental, statistical and mathematical modeling approaches have been employed to study the structure, function and dynamics of molecular networks, and begin to reveal important links of various network properties to the functions of the biological systems. In agreement with these functional links, evolutionary selection of a network is apparently based on the function, rather than directly on the structure of the network. Dynamic modularity is one of the prominent features of molecular networks. Taking advantage of such a feature may simplify network-based biological studies through construction of process-specific modular networks and provide functional and mechanistic insights linking genotypic variations to complex traits or diseases, which is likely to be a key approach in the next wave of understanding complex human diseases. With the development of ready-to-use network analysis and modeling tools the networks approaches will be infused into everyday biological research in the near future.