A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Multiomic characterization of RNA microenvironments by oligonucleotide-mediated proximity-interactome mapping.

RNA molecules form complex networks of molecular interactions that are central to their function and to cellular architecture. But these interaction networks are difficult to probe in situ. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a method for elucidating the biomolecules near an RNA of interest, within its native context. O-MAP uses RNA-fluorescence in situ hybridization-like oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA in situ, enabling nearby molecules to be enriched by streptavidin pulldown. This induces exceptionally precise biotinylation that can be easily optimized and ported to new targets or sample types. Using the noncoding RNAs 47S, 7SK and Xist as models, we develop O-MAP workflows for discovering RNA-proximal proteins, transcripts and genomic loci, yielding a multiomic characterization of these RNAs' subcellular compartments and new regulatory interactions. O-MAP requires no genetic manipulation, uses exclusively off-the-shelf parts and requires orders of magnitude fewer cells than established methods, making it accessible to most laboratories.

X-factors in human disease: impact of gene content and dosage regulation.

The gene content of the X and Y chromosomes has dramatically diverged during evolution. The ensuing dosage imbalance within the genome of males and females has led to unique chromosome-wide regulatory mechanisms with significant and sex-specific impacts on X-linked gene expression. X inactivation or silencing of most genes on one X chromosome chosen at random in females profoundly affects the manifestation of X-linked diseases, as males inherit a single maternal allele, while females express maternal and paternal alleles in a mosaic manner. An additional complication is the existence of genes that escape X inactivation and thus are ubiquitously expressed from both alleles in females. The mosaic nature of X-linked gene expression and the potential for escape can vary between individuals, tissues, cell types and stages of life. Our understanding of the specialized nature of X-linked genes and of the multilayer epigenetic regulation that influence their expression throughout the organism has been helped by molecular studies conducted by tissue-specific and single-cell-specific approaches. In turn, the definition of molecular events that control X silencing has helped develop new approaches for the treatment of some X-linked disorders. This review focuses on the peculiarities of the X chromosome genetic content and epigenetic regulation in shaping the manifestation of congenital and acquired X-linked disorders in a sex-specific manner.

Moderation of mitochondrial respiration mitigates metabolic syndrome of aging.

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously discovered mitochondrial complex IV moderate inhibitor in drinking water and orally administered to standard-diet-fed, wild-type C57BL/6J mice every day for up to 16 mo. No manifestation of any apparent toxicity or deleterious effect on studied mouse models was observed. The impacts of an added inhibitor on a variety of mitochondrial functions were analyzed, such as respiratory activity, mitochondrial bioenergetics, and biogenesis, and a few age-associated comorbidities, including reactive oxygen species (ROS) production, glucose abnormalities, and obesity in mice. It was found that mitochondrial quality, dynamics, and oxidative metabolism were greatly improved, resulting in lean mice with a specific reduction in visceral fat plus superb energy and glucose homeostasis during their aging period compared to the control group. These results strongly suggest that a mild interference in ATP synthesis through moderation of mitochondrial activity could effectively up-regulate mitogenesis, reduce ROS production, and preserve mitochondrial integrity, thereby impeding the onset of metabolic syndrome. We conclude that this inhibitory intervention in mitochondrial respiration rectified the age-related physiological breakdown in mice by protecting mitochondrial function and markedly mitigated certain undesired primary outcomes of metabolic syndrome, such as obesity and type 2 diabetes. This intervention warrants further research on the treatment of metabolic syndrome of aging in humans.

Sci-Hi-C: A single-cell Hi-C method for mapping 3D genome organization in large number of single cells.

The highly dynamic nature of chromosome conformation and three-dimensional (3D) genome organization leads to cell-to-cell variability in chromatin interactions within a cell population, even if the cells of the population appear to be functionally homogeneous. Hence, although Hi-C is a powerful tool for mapping 3D genome organization, this heterogeneity of chromosome higher order structure among individual cells limits the interpretive power of population based bulk Hi-C assays. Moreover, single-cell studies have the potential to enable the identification and characterization of rare cell populations or cell subtypes in a heterogeneous population. However, it may require surveying relatively large numbers of single cells to achieve statistically meaningful observations in single-cell studies. By applying combinatorial cellular indexing to chromosome conformation capture, we developed single-cell combinatorial indexed Hi-C (sci-Hi-C), a high throughput method that enables mapping chromatin interactomes in large number of single cells. We demonstrated the use of sci-Hi-C data to separate cells by karytoypic and cell-cycle state differences and to identify cellular variability in mammalian chromosomal conformation. Here, we provide a detailed description of method design and step-by-step working protocols for sci-Hi-C.

Massively multiplex single-cell Hi-C.

We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.

X chromosome regulation: diverse patterns in development, tissues and disease.

Genes on the mammalian X chromosome are present in one copy in males and two copies in females. The complex mechanisms that regulate the X chromosome lead to evolutionary and physiological variability in gene expression between species, the sexes, individuals, developmental stages, tissues and cell types. In early development, delayed and incomplete X chromosome inactivation (XCI) in some species causes variability in gene expression. Additional diversity stems from escape from XCI and from mosaicism or XCI skewing in females. This causes sex-specific differences that manifest as differential gene expression and associated phenotypes. Furthermore, the complexity and diversity of X dosage regulation affect the severity of diseases caused by X-linked mutations.

Drug discovery approaches targeting the incretin pathway.

Incretin pathway plays an important role in the development of diabetes medications. Interventions in DPP-4 and GLP-1 receptor have shown remarkable efficacy in experimental and clinical studies and imperatively become one of the most promising therapeutic approaches in the T2DM drug discovery pipeline. Herein, we analyzed the actionmechanismsof DPP-4 and GLP-1 receptor targeting the incretin pathway in T2DM treatment. We gave an insight into the structural requirements for the potent DPP-4 inhibitors and revealed a classification of DPP-4 inhibitors by stressing on the binding modes of these ligands to the enzyme. We then reviewed the drug discovery strategies for the development of peptide and non-peptide GLP-1 receptor agonists (GLP-1 RAs). Furthermore, the drug design strategies for DPP-4 inhibitors and GLP-1R agonists were detailed accurately. This review might provide an efficient evidence for the highly potent and selective DPP-4 inhibitors and the GLP-1 RAs, as novel medicines for patients suffering from T2DM.

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.

Anti-diabetic potential of Pueraria lobata root extract through promoting insulin signaling by PTP1B inhibition.

Type 2 diabetes mellitus is a fast-growing epidemic affecting people globally. We initiated the project by searching the possible target of the Pueraria lobata root extract (P. lobata). We conducted the IC50 assays of P. lobata on the four diabetes-related proteins: PTP1B, TCPTP, SHP-2 and DPP-4. Results indicated that P. lobata exhibited high PTP1B inhibitory activity with IC50 of 0.043 mg/ml. Treated insulin-resistant HepG2 cells with 0.0115 mg/ml of P. lobata increased the glucose uptake by two times compared with the negative control. Further, we performed OGTT test on the diabetic C57BL/6 male mice. 20% decreased blood glucose (AUC) was obtained with a dose of 1 g/kg P. lobata compared with the negative control. Herein, we were able to demonstrate the antidiabetic effects of P. lobata might be related to the inhibition of PTP1B and therefore, bettering the insulin signaling pathway.

Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes.

High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type-specific 3D organization of lincRNA genes.

Surrogating and redirection of pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a novel class of potent and selective DPP-4 inhibitors.

The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in vitro DPP-4 inhibitory activity (IC50: 80 nM), while maintaining other key cellular parameters such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on docking analysis and structure-based drug design knowledge resulted in d1. Compound d1 has nearly 2-fold increase of inhibitory activity (IC50: 49 nM) and over 1000-fold selectivity against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose excursion by 34% at the dose of 10 mg/kg in diabetic mice. Herein we report the optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4 inhibitors.

Escape from X inactivation varies in mouse tissues.

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3-7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.

Synthesis, biological evaluation and molecular docking analysis of 2-phenyl-benzofuran-3-carboxamide derivatives as potential inhibitors of Staphylococcus aureus Sortase A.

In Gram-positive bacteria, Sortase A (Srt A) is a critical cysteine transpeptidase that is responsible for recognizing and assembling surface virulence proteins through the recognition of a LPXTG (leucine, proline, X, threonine, and glycine, where X is any amino acid) signal. Mutants lacking genes for Srt A attenuate infections without affecting microbial viability. Here a series of 2-phenyl-benzofuran-3-carboxamide derivatives were synthesized and identified as potent Srt A inhibitors. Activity assays revealed that multiple compounds exhibited excellent inhibitory activity against Srt A compared with known Sortase A inhibitor pHMB (IC50=130µM). Structural activity relationships (SARs) demonstrated that the amide group at 3-position was essential for inhibitory activity. Replacement of the hydroxyl group at the 2-phenyl position of benzofuran with other substitutions such as a methoxyl, halogen or nitro group reduced the enzyme inhibitory activity in most cases. The compound Ia-22 was found to be the most potent inhibitor against the enzyme with an IC50 value of 30.8µM. Molecular docking studies showed Ia-22 shared similar binding pattern with substrate LPXTG in the binding pocket of Srt A (PDB: 2KID) including i-butyl stretching, L-shape pattern kinking, and H-bond interaction with Srt A functional site residues Cys184, Trp194 and Arg197.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Synthesis, biological evaluation and molecular docking of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives as potential Staphylococcus aureus Sortase A inhibitors.

A series of novel 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed, synthesized and evaluated for their in vitro inhibitory activities against Staphylococcus aureus Sortase A with known Sortase A inhibitor pHMB as positive compound (IC50=130µM). Most compounds exhibited excellent inhibitory activity (IC50=19.8-184.2µM). Structure-activity relationship studies demonstrated that substitution at 7-position and 2-position of benzoxazole had great influence on the activities. Specifically, the substituent at 7-position is indispensable for inhibitory activity. The molecular docking studies revealed the i-butyl amide group went towards the ß6/ß7 loop-ß8 substructure of the protein and the benzoxazole core lied in a hydrophobic pocket composed of Ala118, Val166, Val168, Val169 and Ile182, shaping the whole molecule into a L-shape mode to be recognized by Sortase A.

Female bias in Rhox6 and 9 regulation by the histone demethylase KDM6A.

The Rhox cluster on the mouse X chromosome contains reproduction-related homeobox genes expressed in a sexually dimorphic manner. We report that two members of the Rhox cluster, Rhox6 and 9, are regulated by de-methylation of histone H3 at lysine 27 by KDM6A, a histone demethylase with female-biased expression. Consistent with other homeobox genes, Rhox6 and 9 are in bivalent domains prior to embryonic stem cell differentiation and thus poised for activation. In female mouse ES cells, KDM6A is specifically recruited to Rhox6 and 9 for gene activation, a process inhibited by Kdm6a knockdown in a dose-dependent manner. In contrast, KDM6A occupancy at Rhox6 and 9 is low in male ES cells and knockdown has no effect on expression. In mouse ovary where Rhox6 and 9 remain highly expressed, KDM6A occupancy strongly correlates with expression. Our study implicates Kdm6a, a gene that escapes X inactivation, in the regulation of genes important in reproduction, suggesting that KDM6A may play a role in the etiology of developmental and reproduction-related effects of X chromosome anomalies.

Discovery of novel potent and selective ligands for 5-HT2A receptor with quinazoline scaffold.

A series of compounds with quinazoline scaffold were designed, synthesized and evaluated as novel potent 5-HT2A receptor ligands. N-(4-Chlorophenyl)-2-(piperazin-1-yl)quinazolin-4-amine (5o) has a Ki value of 14.04 ± 0.21 nM, with a selectivity more than 10,000 fold over 5-HT1A receptors (D1 and D2-like receptors). The functional assay showed that this compound is an antagonist to 5-HT2A receptor with an IC50 value of 1.66 µM.

Design, synthesis, and biological evaluation of 2-substituted ethenesulfonic acid ester derivatives as selective PTP1B inhibitors.

Fifteen 2-substituted ethenesulfonic acid ester derivatives were designed, synthesized, and evaluated for the inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) and T-Cell protein tyrosine phosphatase (TCPTP). The structural activity relationship (SAR) of these compounds are discussed to clarify the impact of the linker and the optimized tail on the inhibitory activity of PTP1B and selectivity over TCPTP. Most of the compounds exhibit excellent inhibitory activities against PTP1B with IC50 values of 1.5-8.9 µM. SAR analysis reveal that the substituents at the hydrophobic tail significantly alter the inhibitory activity against PTP1 B and selectivity over TCPTP, e.g. compound 5d showed excellent inhibitory activity to PTP1B with IC50 = 7.8 µM, and -6-fold selectivity over TCPTP. Combined with our previous findings, we confirm that the linker length and the substituted hydrophobic tail have decisive influence on the PTP1B inhibitory activity and selectivity.

Identification of 2-substituted ethenesulfonic acid ester derivatives as novel, potent and selective inhibitors of protein tyrosine phosphatase 1B.

Sixteen 2-substituted ethenesulfonic acid ester derivatives were designed, synthesized and evaluated for the inhibitory activity against tyrosine phosphatase 1B (PTP1B) and T-Cell protein tyrosine phosphatase (TCPTP). The structural activity relationship (SAR) of these compounds demonstrated that the hydrophilic head, aromatic center and the hydrophobic tail affected the inhibitory activities against PTP1B and the selectivity over TCPTP. Most of the compounds exhibited excellent inhibitory activity against PTP1B with IC50 value of 1.0 µM - 31.2 µM. SAR analysis revealed that the hydrophilic head was indispensable in the maintain of inhibitory activity against PTP1B, the aromatic center significantly altered the selectivity of PTP1B over TCPTP, and the hydrophobic tail significantly altered the inhibitory activity against PTP1B.