Showing papers in "Protein & Cell in 2019"

Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes.

Abstract: Exosomes are cup-shaped small (30–150 nm) extracellular vesicles with the structure of lipid bilayer membrane (Tkach and Thery, 2016) containing proteins, mRNAs and microRNAs that mediate intercellular communication (Valadi et al., 2007). Unlike other extracellular vesicles, exosomes are released into the extracellular space when the multivesicular bodies (MVBs) fuse with the plasma membrane (Colombo et al., 2014). Almost all cell types can secret exosomes and exosomes exist in diverse biological fluids, such as blood, urine, saliva, hydrothorax and breast milk (Thery et al., 2006). Up to now, a number of studies have demonstrated the functions of exosomes in disease development and the potential clinical applications in diagnosis and therapy (Shao et al., 2016). To conduct reproducible studies on exosomal content and function, storage conditions need to have minimal impact on exosomes. There have been a few studies providing partial confirmation of the effect of different storage conditions on exosomes currently. Using exosomes from urine (Zhou et al., 2006) and conditioned medium (Lee et al., 2016) respectively to investigate the influence of storage temperature on exosomes as measured by Western blot, both groups have concluded that storage below −70 °C for a long time is the best temperature for the recovery of exosomes. On the other hand, Sokolova et al. (2011) applied nanoparticle tracking analysis (NTA) to measure the size changes of exosomes at different temperatures, revealing that storage at 37 °C led to more reduction in exosome sizes than that at 4 °C. However, in this study no information about changes in the particle concentration was reported. Some other studies revealed the effect of pH, storage temperature and cycles of freezing and thawing only on the yield of exosome isolation, but not on quantity changes during storage (Akers et al., 2016; Ban et al., 2015; Zhao et al., 2017). Therefore, the standard criterion of exosomal preservation condition is still undefined. Herein, we used HEK 293T cells and ExtraPEG method (Rider et al., 2016) to investigate the influence of multiple storage conditions (temperature, cycles of freezing and thawing, pH) on the quantity changes and cellular uptake of exosomes. ExtraPEG is a new polyethylene glycol (PEG) precipitation method for the purification exosomes without affecting their biological activity. Generally, ultracentrifugation (UC) (Mincheva-Nilsson et al., 2016) is most reliable but time-consuming; and precipitation methods such as ExoQuick (patent number: US20130337440 A1) and ExtraPEG can obtain higher yields of exosomes but with impurity of coprecipitated proteins. First, exosomes from the conditioned medium were extracted by ExtraPEG or UC method. After isolation, transmission electron microscope (TEM), NTA and Western blot were performed to analyze exosomes. Exosomes extracted by UC or ExtraPEG were similar in cupshaped structure (Fig. S1A and S1B), size distribution (Fig. S1C and S1D). And as representative exosome biomarkers, ALG-2-interacting protein X (ALIX), heat shock protein 70 (HSP70) and tumor susceptibility gene 101 (TSG101) were detected in exosomal protein while β-tubulin, widely used as an internal reference to analyze intracellular protein levels, was not detected in exosome samples (Fig. S1E and S1F). These data indicated exosomes were successfully isolated by ExtraPEG method and suitable for the following experiments. After isolation, the exosome pellets were divided equally into several portions and each portion was stored at different temperatures (−80 °C, −20 °C, 4 °C, 37 °C and 60 °C), or through 1–5 cycles of freezing to −80 °C and thawing, or at different pH levels (pH 4, pH 7 and pH 10). After 24 h, NTA and Western blot were performed to measure the remaining quantity of exosomes. Regarding temperatures, the exosomes stored at 4 °C had the highest concentration (Fig. 1A). Consistent with the NTA results, the exosomes stored at 4 °C showed higher levels of representative exosome markers ALIX, HSP70 and TSG101 (Fig. 1B). With the increasing cycles of freezing and thawing, the exosomal concentration and protein levels of ALIX, HSP70 and TSG101 all decreased (Fig. 1D and 1E). For different pH levels, the loss of exosomal concentration and three exosome markers ALIX, HSP70 and TSG101 at pH 4 and pH 10 was more than that at pH 7 (Fig. 1E and 1F). Interestingly, exosomes stored at pH 4 decreased more sharply than that at pH 10 (Fig. 1F and 1G), suggesting that acidic

Phage display screening of therapeutic peptide for cancer targeting and therapy.

Abstract: Recently, phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018. Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process; proving to be a powerful technique in the screening of peptide with high affinity and selectivity. In this review, we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ, in vitro, in vivo, and ex vivo screening methods. We will then discuss prominent examples of solid tumor targeting-peptides; namely peptide targeting tumor vasculature, tumor microenvironment (TME) and over-expressed receptors on cancer cells identified through phage display screening. We will also discuss the current challenges and future outlook for targeting peptide-based therapeutics in the clinics.

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation.

Abstract: A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2:MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.

Chemical screen identifies a geroprotective role of quercetin in premature aging

Abstract: Aging increases the risk of various diseases. The main goal of aging research is to find therapies that attenuate aging and alleviate aging-related diseases. In this study, we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established. Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects. Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs. RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C. Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs. Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

Sialylation is involved in cell fate decision during development, reprogramming and cancer progression.

Abstract: Sialylation, or the covalent addition of sialic acid to the terminal end of glycoproteins, is a biologically important modification that is involved in embryonic development, neurodevelopment, reprogramming, oncogenesis and immune responses. In this review, we have given a comprehensive overview of the current literature on the involvement of sialylation in cell fate decision during development, reprogramming and cancer progression. Sialylation is essential for early embryonic development and the deletion of UDP-GlcNAc 2-epimerase, a rate-limiting enzyme in sialic acid biosynthesis, is embryonically lethal. Furthermore, the sialyltransferase ST6GAL1 is required for somatic cell reprogramming, and its downregulation is associated with decreased reprogramming efficiency. In addition, sialylation levels and patterns are altered during cancer progression, indicating the potential of sialylated molecules as cancer biomarkers. Taken together, the current evidences demonstrate that sialylation is involved in crucial cell fate decision.

Developing potent PROTACs tools for selective degradation of HDAC6 protein

Abstract: Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups on histone and non-histone proteins, thereby playing a vital role in the modulation of gene expression and protein activity. Eighteen HDACs have been identified in human and subdivided into four classes including I, II (IIa, IIb), III and IV (Seto et al., 2014). Among them, HDAC6 is a unique IIb HDAC with dominant cytoplasmic localization and two functional catalytic domains. Besides the functions for deacetylation of histone, and modulation of α-tubulin, HSP90 and cortactin, HDAC6 also participates in protein trafficking and degradation, cell shape and migration (Valenzuela-Fernandez et al., 2008). The deregulation of HDAC6 is related to various diseases, such as neurodegenerative diseases, cancer and pathological autoimmune response (Batchu et al., 2016). Hence, it is especially important for directly controlling cellular HDAC6 protein levels to achieve therapeutic purposes. The traditional approaches of reducing cellular protein levels mainly rely on genetic modifications, such as RNA interference, transcription activator-like effector nucleases, recombination-based gene knockout and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) (Boettcher et al., 2015). However, these approaches have failed to a certain degree to achieve acute and reversible changes of gene function. Furthermore, the complications of potential genetic compensation and/or spontaneous mutations arising in geneknockout models may lead to misinterpretations (Davisson et al., 2012; El-Brolosy et al., 2017). Therefore, it is urgent for developing a rapid, robust, and reversible approach to directly modulate HDAC6 protein levels. Known as a chemical based protein knockdown strategy, PROteolysis-TArgeting Chimera (PROTAC) has emerged as a novel and powerful method for the degradation of interested proteins. The PROTACs are heterobifunctional molecules, which consist of three parts: a ligand for binding target protein, a ligand for recruiting E3 ligase and a linker connecting the two ligands (Lai et al., 2017). Consequently, the PROTACs mediated interaction of the target protein and a E3 ligase caused ubiquitination and subsequent degradation of the target protein by the ubiquitin-proteasome system (UPS) (Fig. 1A). It has been proved that PROTAC technology can achieve efficient degradation of proteins with excellent selectivity in a quick and direct manner (Yang et al., 2018; Zhou et al., 2018). Moreover, the PROTAC also worked well for mutated proteins (Sun et al., 2018). Herein, we report the development of HDAC6-targeting degraders based on the PROTAC strategy. The newly designed PROTACs induced significant degradation of HDAC6 in a panel of cell lines, exhibited excellent selectivity against other HDACs, and demonstrated efficient inhibition of cell proliferation. Besides, the degradation process was well illustrated by fluorescence-based visualization. To design novel HDAC6-targeting PROTACs, we chose a selective HDAC6 inhibitor Nexturastat A (Nex A) as the HDAC6 binder (Bergman et al., 2012). According to the recently released co-crystal structure of HDAC6 in complex with Nex A (Miyake et al., 2016), the aliphatic chain was oriented outside of the ligand binding pocket. Based on the PROTACs design principles, Pomalidomide (Poma, a ligand for E3 ligase CRBN) was introduced onto the end of aliphatic chain of Nex A via different linkers (Lopez-Girona et al., 2012). As shown in the simulated diagram (Fig. 1B), the PROTACs should actively bind HDAC6 and CRBN simultaneously. The synthesis of the HDAC6 degraders was shown in Supplementary Materials (Scheme 1). Next, the resulting HDAC6-targeting PROTAC molecules were tested. To evaluate the degradation capability of our PROTACs for HDAC6 protein, we analyzed the cellular levels of HDAC6 in HeLa cells by Western blot after incubation with four different PROTACs. It was found that all PROTACs can effectively induce HDAC6 degradation after 24 h. Among them, NP8 was the most potent degrader which can significantly reduce the HDAC6 protein level at 100 nmol/L (Fig. 1C). We then went on to evaluate the degradation potential of NP8 in a panel of cell lines from different origins. NP8 consistently induced significant degradation of HDAC6 in all the cell lines we tested, while the multiple myeloma cell line MM.1S exhibited the best sensitivity to NP8 (Fig. S1). The NP8-induced degradation was specific for HDAC6 since the other representative HDAC family members were not affected by NP8 treatment (Figs. 1D and S2). Time-lapse experiment showed that NP8 induced fast and effective degradation of HDAC6 in just 2 h post drug treatment (Fig. 1E). The half degradation concentration (DC50) of NP8

Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase.

Abstract: N6-methyladenosine (m6A), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5'-GGACU-3' consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel β-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNA-binding interface comprises basic residues from zinc finger 1 and hydrophobic residues from β-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA m6A modification.

Release and uptake mechanisms of vesicular Ca 2+ stores

Abstract: Cells utilize calcium ions (Ca2+) to signal almost all aspects of cellular life, ranging from cell proliferation to cell death, in a spatially and temporally regulated manner. A key aspect of this regulation is the compartmentalization of Ca2+ in various cytoplasmic organelles that act as intracellular Ca2+ stores. Whereas Ca2+ release from the large-volume Ca2+ stores, such as the endoplasmic reticulum (ER) and Golgi apparatus, are preferred for signal transduction, Ca2+ release from the small-volume individual vesicular stores that are dispersed throughout the cell, such as lysosomes, may be more useful in local regulation, such as membrane fusion and individualized vesicular movements. Conceivably, these two types of Ca2+ stores may be established, maintained or refilled via distinct mechanisms. ER stores are refilled through sustained Ca2+ influx at ER-plasma membrane (PM) membrane contact sites (MCSs). In this review, we discuss the release and refilling mechanisms of intracellular small vesicular Ca2+ stores, with a special focus on lysosomes. Recent imaging studies of Ca2+ release and organelle MCSs suggest that Ca2+ exchange may occur between two types of stores, such that the small stores acquire Ca2+ from the large stores via ER-vesicle MCSs. Hence vesicular stores like lysosomes may be viewed as secondary Ca2+ stores in the cell.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Abstract: Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 A resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Tongue coating microbiome as a potential biomarker for gastritis including precancerous cascade

Abstract: The development of gastritis is associated with an increased risk of gastric cancer. Current invasive gastritis diagnostic methods are not suitable for monitoring progress. In this work based on 78 gastritis patients and 50 healthy individuals, we observed that the variation of tongue-coating microbiota was associated with the occurrence and development of gastritis. Twenty-one microbial species were identified for differentiating tongue-coating microbiomes of gastritis and healthy individuals. Pathways such as microbial metabolism in diverse environments, biosynthesis of antibiotics and bacterial chemotaxis were up-regulated in gastritis patients. The abundance of Campylobacter concisus was found associated with the gastric precancerous cascade. Furthermore, Campylobacter concisus could be detected in tongue coating and gastric fluid in a validation cohort containing 38 gastritis patients. These observations provided biological evidence of tongue diagnosis in traditional Chinese medicine, and indicated that tongue-coating microbiome could be a potential non-invasive biomarker, which might be suitable for long-term monitoring of gastritis.

Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects.

Abstract: Genetic engineering of T cells to express chimeric antigen receptors (CARs) is an efficient approach for clinical therapy of hematological malignancies (Kuwana et al., 1987; Eshhar et al., 1993; Barrett et al., 2014). The CARs endow T cells with the ability to recognize specific antigens and bind them in an MHC-independent manner, thereby overcoming some of the mechanisms that mediate tumor immune escape. In addition, by providing co-stimulatory signals, CARs endow T cells with enhanced cytotoxicity and persistence compared with primary T cells. A typical CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb) for antigen recognition and signaling domains for coactivation (Eshhar et al., 1993; Sadelain et al., 2013). To date, CAR-T cell therapy has been most effective in immunotherapy of CD19 B cell acute lymphoblastic leukemia, with a complete response in more than 75% of cases (Sadelain et al., 2013). However, there are still some challenges for CAR-T-mediated treatments. Side effects like offtargeting, cytokine release syndrome (CRS) and neuronal toxicities have been reported, and these may induce lethal responses (Morgan et al., 2010; Park et al., 2011). In addition, no response, incomplete tumor regression, and tumor recurrence were also observed after CAR-T treatment. For example, 10%–20% of patients were non-responsive to CD19 CAR-T clinical therapy (Lee et al., 2015; Park et al., 2018). Even in cases with a complete response, about 50% of them suffered tumor recurrence in one year, and one third of them had a CD19 relapse (Maude et al., 2018; Orlando et al., 2018). These disappointing results are associated with early CAR-T cell disappearance or poor cell function, which leads to incomplete tumor regression or loss of long-term antitumor effects. Cytokines are important factors for T cell development and homeostasis. In addition to the TCR and costimulatory receptors, cytokines provide stimulatory signals for full T cell activation, and have pleiotropic effects on T cell proliferation, differentiation and function. Currently, IL-2 is the main cytokine used to culture cells for adoptive cell therapy, as it plays an important role in the proliferation and functional effect of T cells. However, T cells cultured with IL-2 are phenotypically heterogeneous, being predominantly composed of effector memory cells which have sufficient functional effect but are sensitive to death. IL-7 has a critical role in the development and maturation of T cells. It promotes the generation of naïve and central memory T cell subsets and regulates their homeostasis. IL15 mediates the formation and homeostasis of CD8 memory T cells. It has been reported that IL-7 and IL-15 are able to instruct T cells toward memory stem-like phenotypes, which are less differentiated and have a superior capacity for expansion and survival (Cieri et al., 2013). Here, we systematically compared the effects of IL-7/IL-15 and IL-2 on the expansion, apoptosis and anti-tumor responses of CAR-T cells. We first constructed the anti-CD19 CAR (19BB-CAR) using an anti-CD19 mAb (clone FMC63)-derived scFv linked to the CD8α hinge and transmembrane regions, followed by a 4-1BB intracellular signaling domain and the CD3ζ signaling moiety. The 19BB-CAR and enhanced green fluorescent protein (eGFP) sequences were ligated and subcloned into the lentiviral vector FUW with a substitutive EF1α promoter (Fig. S1A). The cultured primary T cells were stimulated with anti-CD3/anti-CD28 Dynabeads and cytokine IL-2 before transduction with 19BB-CAR lentiviral particles. Using Protein L binding to the variable immunoglobulin light chains of the CAR, we found that CAR expression is directly correlated to eGFP expression (Fig. S1B). The CAR was highly expressed in IL-2-cultured T cells three days after infection (Fig. S1C). The CAR-T cells were expanded 100-fold in 2 weeks under IL-2 stimulation (Fig. S1D). To test the specificity of 19BB-CAR-T cells, we co-incubated them with two human leukemia cell lines, Raji (CD19) and K562 (CD19). The secretion of IL-2, IFN-γ and TNF-α by 19BB-CAR-T cells was significantly increased upon coincubation with CD19 Raji but not CD19 K562 cells (Fig. S1E). Accordingly, cytotoxicity assays showed that 19BB-CAR-T cells specifically lysed CD19 Raji but not CD19 K562 cells (Fig. S1F). These data suggest that 19BBCAR-T cells specifically recognize the CD19 molecule.

JMJD3 in the regulation of human diseases

Abstract: In recent years, many studies have shown that histone methylation plays an important role in maintaining the active and silent state of gene expression in human diseases. The Jumonji domain-containing protein D3 (JMJD3), specifically demethylate di- and trimethyl-lysine 27 on histone H3 (H3K27me2/3), has been widely studied in immune diseases, infectious diseases, cancer, developmental diseases, and aging related diseases. We will focus on the recent advances of JMJD3 function in human diseases, and looks ahead to the future of JMJD3 gene research in this review.

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Abstract: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation. However, the underlying cellular and molecular mechanisms remain unidentified. Here, we generated non-integrative induced pluripotent stem cells (iPSCs) from fibroblasts of a CADASIL patient harboring a heterozygous NOTCH3 mutation (c.3226C>T, p.R1076C). Vascular smooth muscle cells (VSMCs) differentiated from CADASIL-specific iPSCs showed gene expression changes associated with disease phenotypes, including activation of the NOTCH and NF-κB signaling pathway, cytoskeleton disorganization, and excessive cell proliferation. In comparison, these abnormalities were not observed in vascular endothelial cells (VECs) derived from the patient’s iPSCs. Importantly, the abnormal upregulation of NF-κB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor, providing a potential therapeutic strategy for CADASIL. Overall, using this iPSC-based disease model, our study identified clues for studying the pathogenic mechanisms of CADASIL and developing treatment strategies for this disease.

Down-regulation of the let-7i facilitates gastric cancer invasion and metastasis by targeting COL1A1.

Abstract: Globally, gastric cancer is the most common malignant tumor and the second highest contributor to cancer deaths after lung cancer (Murray et al., 2012). Despite improved success with treatment of early stage gastric cancer (Fuse et al., 2016), the five-year survival rate of advanced staged gastric cancer patients is still low. The aggressive growth characteristics of the tumor and metastasis are key factors responsible for poor overall survival in these patients (Ozkan et al., 2005). Therefore, investigation of the molecular mechanisms that underlie the aggressive behavior of gastric cancers, and identification of potential target genes for therapeutic interventions, is a key imperative. Aberrant miRNA expression is a key contributor to tumorigenesis in humans (Croce and Calin, 2005; Iorio et al., 2005; Wu et al., 2009; Acunzo and Croce, 2016). Our previous study showed that as a crucial hub gene in gastric cancer, COL1A1 is directly regulated by let-7i miRNA and its high expression levels in gastric cancer have been linked to increased tumor invasiveness (Shi et al., 2015). Downregulation of Let-7i in several cancers was shown to be associated with unfavorable prognosis (Yang et al., 2008; Yang et al., 2013). However, whether let-7i influences progression of gastric cancers is not known. In the present study, we assessed the let-7i expression level and its effects in gastric cancer samples and cell lines. The binding sites of COL1A1 and let-7i were predicted using bioinformatics software and their regulatory mechanism verified. Further, we also analyzed expression levels of COL1A1 in gastric cancer tissues and cell lines. The results suggest that increased let-7i expression may lead to decrease in proliferative, metastatic and invasive properties of cancer cells. Expression levels of let-7i were assessed in 40 pairs of gastric cancer tissue specimens and their corresponding adjacent normal tissue samples by qRT-PCR. The result showed that let-7i expression was significantly low in gastric cancer than in normal tissues (Fig. 1A, P < 0.001). Further, expression levels of let-7i were lower in gastric cancer cell lines (SGC-7901, MGC-803, AGS, N87) as compared to that in normal gastric epithelial cells GES-1, while no significant difference in this respect was observed in the MKN-45 cell lines (Fig. 1B). A statistically significant association was observed between low expression level of let-7i and T stage (P < 0.05; Fig. S1A), and lymph node metastasis (P < 0.05; Fig. S1B). The effects of let-7i restoration on regulation of gastric cancer cell vitality and cell proliferation were assessed by transfecting let-7i mimic or miRNA negative control into two human gastric cancer cell lines, SGC-7901 and MGC-803, which have relatively lower levels of let-7i expression. As expected, ectopic let-7i expression markedly suppressed viability of SGC-7901 (P < 0.05; Fig. 1C) and MGC-803 cell lines (P < 0.05; Fig. S2A) as assessed by use of cell counting kits. Furthermore, over expression of let-7i also reduced proliferation of both SGC-7901 (P < 0.05, Fig. 1D) and MGC-803 cells (P < 0.05, Fig. S2B), 48 h after transfection, as revealed on colony formation assay. Overexpression of let-7i also reduced invasive and migratory ability of both SGC-7901 (P < 0.05, Fig. 1E and 1F) and MGC-803 cells (P < 0.05, Fig. S2C and S2D). These findings suggest that let-7i reduced cell viability and proliferative ability and inhibited invasive and migratory properties of gastric cancer cells in vitro. SGC-7901 cell lines which stably expressing let-7i and miRNA-control were subcutaneously injected into the dorsal flank of nude mice to evaluate the in vivo effects of let-7i on gastric cancer tumor growth. The result showed reduced tumor volume and tumor weight in nude mice with let-7i mimic injection (Fig. 1G–I), which suggests a role of let-7i in modulating in vivo gastric cancer progression. In addition, SGC-7901 cells stably expressing let-7i and miRNA-control cells were transplanted through the lateral tail vein to evaluate the effects of let-7i expression on tumor metastasis. Macroscopic observation and histological analysis of the livers showed that the ectopic expression of let-7i significantly inhibited metastasis in organs (Fig. 1J). We already assessed the expression of COL1A1 in gastric cancer tissues and hypothesized COL1A1 to be one of the target genes of let-7i in gastric cancer cells which mediates its effect through a TF-miRNA co-regulated network identified in our previous study. We reassessed COL1A1 expression in gastric cancer tissue samples and

Inhibition of Rac1-dependent forgetting alleviates memory deficits in animal models of Alzheimer's disease

Abstract: Accelerated forgetting has been identified as a feature of Alzheimer's disease (AD), but the therapeutic efficacy of the manipulation of biological mechanisms of forgetting has not been assessed in AD animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small GTPase, has been shown to regulate active forgetting in Drosophila and mice. Here, we showed that Rac1 activity is aberrantly elevated in the hippocampal tissues of AD patients and AD animal models. Moreover, amyloid-beta 42 could induce Rac1 activation in cultured cells. The elevation of Rac1 activity not only accelerated 6-hour spatial memory decay in 3-month-old APP/PS1 mice, but also significantly contributed to severe memory loss in aged APP/PS1 mice. A similar age-dependent Rac1 activity-based memory loss was also observed in an AD fly model. Moreover, inhibition of Rac1 activity could ameliorate cognitive defects and synaptic plasticity in AD animal models. Finally, two novel compounds, identified through behavioral screening of a randomly selected pool of brain permeable small molecules for their positive effect in rescuing memory loss in both fly and mouse models, were found to be capable of inhibiting Rac1 activity. Thus, multiple lines of evidence corroborate in supporting the idea that inhibition of Rac1 activity is effective for treating AD-related memory loss.

RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy.

Abstract: RNA splicing contributes to a broad spectrum of post-transcriptional gene regulation during normal development, as well as pathological manifestation of heart diseases. However, the functional role and regulation of splicing in heart failure remain poorly understood. RNA binding protein (RBP), a major component of the splicing machinery, is a critical factor in this process. RNA binding motif protein 24 (RBM24) is a tissue-specific RBP which is highly expressed in human and mouse heart. Previous studies demonstrated the functional role of RBM24 in the embryonic heart development. However, the role of RBM24 in postnatal heart development and heart disease has not been investigated. In this paper, using conditional RBM24 knockout mice, we demonstrated that ablation of RBM24 in postnatal heart led to rapidly progressive dilated cardiomyopathy (DCM), heart failure, and postnatal lethality. Global splicing profiling revealed that RBM24 regulated a network of genes related to cardiac function and diseases. Knockout of RBM24 resulted in misregulation of these splicing transitions which contributed to the subsequent development of cardiomyopathy. Notably, our analysis identified RBM24 as a splice factor that determined the splicing switch of a subset of genes in the sacomeric Z-disc complex, including Titin, the major disease gene of DCM and heart failure. Together, this study identifies regulation of RNA splicing by RBM24 as a potent player in remodeling of heart during postnatal development, and provides novel mechanistic insights to the pathogenesis of DCM.

Transcriptional mechanism of IRF8 and PU.1 governs microglial activation in neurodegenerative condition.

Abstract: Microglial activation occurs in divergent neuropathological conditions. Such microglial event has the key involvement in the progression of CNS diseases. However, the transcriptional mechanism governing microglial activation remains poorly understood. Here, we investigate the microglial response to traumatic injury-induced neurodegeneration by the 3D fluorescence imaging technique. We show that transcription factors IRF8 and PU.1 are both indispensible for microglial activation, as their specific post-developmental deletion in microglia abolishes the process. Mechanistically, we reveal that IRF8 and PU.1 directly target the gene transcription of each other in a positive feedback to sustain their highly enhanced expression during microglial activation. Moreover, IRF8 and PU.1 dictate the microglial response by cooperatively acting through the composite IRF-ETS motifs that are specifically enriched on microglial activation-related genes. This action of cooperative transcription can be further verified biochemically by the synergetic binding of IRF8 and PU.1 proteins to the composite-motif DNA. Our study has therefore elucidated the central transcriptional mechanism of microglial activation in response to neurodegenerative condition.

Mitochondrion-processed TERC regulates senescence without affecting telomerase activities

Abstract: Mitochondrial dysfunctions play major roles in ageing. How mitochondrial stresses invoke downstream responses and how specificity of the signaling is achieved, however, remains unclear. We have previously discovered that the RNA component of Telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. Cytosolic TERC-53 levels respond to mitochondrial functions, but have no direct effect on these functions, suggesting that cytosolic TERC-53 functions downstream of mitochondria as a signal of mitochondrial functions. Here, we show that cytosolic TERC-53 plays a regulatory role on cellular senescence and is involved in cognition decline in 10 months old mice, independent of its telomerase function. Manipulation of cytosolic TERC-53 levels affects cellular senescence and cognition decline in 10 months old mouse hippocampi without affecting telomerase activity, and most importantly, affects cellular senescence in terc-/- cells. These findings uncover a senescence-related regulatory pathway with a non-coding RNA as the signal in mammals.

Hepatitis B virus is degraded by autophagosome-lysosome fusion mediated by Rab7 and related components

Abstract: With an estimated 240 million chronically infected people worldwide, hepatitis B virus (HBV) infection is a major public health problem (Schweitzer et al., 2015). Despite more than 30 years of intense research, many aspects of the HBV life cycle still remain unknown. Recent progress demonstrated that efficient HBV replication is dependent on autophagy (Liu et al., 2014; Xie et al., 2016; Lin et al., 2017). Autophagy is a conserved catabolic process by which long-lived proteins and damaged organelles are sequestered in the cytoplasm and removed for recycling and important for maintaining cellular homeostasis (Mizushima and Komatsu, 2011), and involves the formation of autophagosomes, known as early autophagy, and their fusion with lysosomes and lysosomal cargo degradation, known as late autophagy. HBV induces partial autophagy to facilitate its own replication. Several reports indicated that HBV induces partial autophagy to facilitate its own replication through the actions of hepatitis B x (HBx) protein and the small HBV surface protein (HBsAg) (Sir et al., 2010; Li et al., 2011; Liu et al., 2014). According to these findings, the HBV life cycle is closely bound to autophagy. Yet, the exact mechanism of how autophagic flux affects HBV replication remains unclear. Thus, we investigated how HBV gene expression, replication and assembly are associated with autophagy. To examine the effect of different autophagic phases on HBV production, human hepatoma cells HepG2.2.15 (Fig. S1A) were treated with the PI3KC3 inhibitor 3-methyladenine (3-MA), the Rab7 inhibitor CID1067700 (CID) or the lysosome inhibitor chloroquine (CQ). Immunofluorescence microscopy showed that the number of LC3 puncta was decreased by 3-MA treatment and increased following treatment with CID and CQ. Moreover, the ratios of LC3II/Actin were applied for assessing the autophagic activity, with betaactin used as the internal reference for normalization (Xie et al., 2016). Western blot analysis of cellular lysates revealed that 3-MA elevated the autophagic cargo p62 expression level but decreased the levels of LC3-II and hepatitis B core antigen (HBcAg) (Fig. S1B). However, inhibitors of late autophagyCID and CQ elevated the levels of p62 and HBcAg in HepG2.2.15 cells (Fig. S1B). Moreover, 3-MA reduced the amount of secreted and intracellular HBsAg, the levels of HBV DNA in culture supernatants and intracellular HBV RIs, while the CID and CQ significantly increased their production (Fig. S1C). The effect of different autophagy inhibitors was also confirmed in primary human hepatocytes (PHHs) (Fig. S1D and S1E). Consistently, blocking the initiation of autophagy reduced HBV replication but interference with its late phase resulted in increased HBV production. As the autophagy is a process mediating the degradation of cargos within the autophagosomes, it should be assumed that a significant part of HBV proteins and other components may be eliminated by the autophagy. Rab7 belongs to a family of small GTPases and plays a central role in regulating endo-lysosomal membrane traffic (Wang et al., 2011; Inoue et al., 2015). Rab7 is also required for the maturation of late endosomes (LEs)/MVBs as well as autophagosomes by recruiting its effectors Pleckstrin homology domain containing protein family member 1 (PLEKHM1) and Rab7-interacting lysosomal protein (RILP), directing the trafficking of cargo along microtubules and participating in the fusion step with lysosomes (McEwan et al., 2015a). Moreover, Rab7 silencing prevents the fusion of autophagosomes and lysosomes (Liu et al., 2014). Accordingly, we chose Rab7 as the target to modulate the cellular autophagic process and to determine its role in the HBV life cycle. As shown in Figure 1A and 1B, Rab7 expression is decreased in hepatoma cells in the presence of HBV. Using specific siRNA, Rab7 silencing increased the number of LC3 puncta (Figs. 1C and S2A) and the levels of the autophagic cargo, LC3-II and p62, strongly increased after Rab7 silencing (Fig. 1D). Rab7 silencing significantly increased the amounts of HBcAg, HBV capsid and capsidassociated HBV DNA (Fig. 1D), and the levels of secreted

Basic and translational aging research in China: present and future

Abstract: The percentage of elderly people in the world is increasing at an unprecedented pace; so it is in China, which has the world's largest population and a high ratio of the seniors (aged 60 and above) to working-age adults. The growing elderly population is presenting a major social challenge. Accordingly, it is not only imperative as a national strategic demand but also promises great scientific values to understand the biological process of aging, explore the mystery of healthy aging, delay the aging process, and treat the age-related diseases. This Perspective summarizes past and present advances of the basic and translational aging research in China and offers perspectives on future endeavors in this area.

Low-dose quercetin positively regulates mouse healthspan.

Abstract: Aging is the leading risk factor for many chronic diseases, accounting for almost 60% of all deaths worldwide. How to achieve healthy aging, alleviate aging-related diseases, and extend healthspan has become a main topic of biomedical research (He et al., 2019). Geroprotective compounds, such as metformin and rapamycin, have been shown to improve both healthspan and lifespan in mice (Martin-Montalvo et al., 2013; Bitto et al., 2016), whereas nicotinamide partially improves healthspan in mice (Mitchell et al., 2018). In addition, senolytics, compounds that eliminate senescent cells, have been proven to improve physical function and increase lifespan in mice (Xu et al., 2018). Although none have proven to be clinically reliable in delaying aging or treating frailty in humans, these compounds have already provoked enthusiasm for identifying a potential “elixir”. Therefore, the exploration of more geroprotective compounds, especially natural active compounds, holds great potential for the development of geriatric medicines. Quercetin (Que) is a natural bioflavonoid found in fruits and vegetables such as apples and onions. Que (50 mg/kg) in combination with dasatinib (5 mg/kg) (abbreviated as D + Q) has been shown to effectively eliminate senescent cells via induction of apoptosis, thus alleviating senescence-related phenotypes and improving physical function and lifespan in mice (Zhu et al., 2015; Xu et al., 2018). In addition, Que (10 mg/kg) in combination with dasatinib (5 mg/kg) has been reported to reduce hepatic steatosis (Ogrodnik et al., 2017). In each of these in vivo studies, however, Que was used at high doses ranging from 10 to 50 mg/kg body weight, which raises concerns about dose-dependent side effects such as headaches and limb tingling (Shoskes et al., 1999). As a selective tyrosine kinase receptor inhibitor, dasatinib is associated with warnings and precautions including pulmonary arterial hypertension and low blood cell counts. Therefore, high-dose Que and extra side effects of dasatinib would hamper potential clinical applications of Que in geriatric medicines. Through natural products screening using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), we recently identified Que as a geroprotective agent that counteracts accelerated and natural aging of hMSCs at a concentration of as low as 100 nmol/L, which is 100 times lower than the concentration of Que (10 μmol/L) previously used in combination with dasatinib as senolytic drugs to eliminate senescent cells in human umbilical vein cells (HUVECs) (Zhu et al., 2015; Geng et al., 2018). To explore the geroprotective effect of low-dose Que monotherapy in rodents, we evaluated the in vivo effect of long-term low-dose Que administration under physiologicalaging condition. Que was given to 14-month-old C57BL/6J male mice by weekly oral gavage at a concentration of 0.125 mg/kg body weight, which is 80–400 times lower than that of the previously tested D + Q (10–50 mg/kg body weight) regimens (Fig. 1A), with vehicle (10% PEG400 in PBS)treated mice as controls (Zhu et al., 2015; Xu et al., 2018). After eight months of treatment, Que-treated mice showed decreased hair loss with normal food intake, body weight, blood glucose and bone mineral density (Figs. 1B and S1A– D). Compared to vehicle-treated mice, mice subjected to Que treatment showed markedly improved exercise endurance in the RotaRod and treadmill tests, but normal grip strength by grip strength meter assay (Figs. 1C, 1D, and S1E–G). Accordingly, the cardiac function of these mice was examined by Doppler tissue imaging. Although ejection fraction (EF) and fractional shortening (FS) were unaffected, a higher frequency of the mitral ratio of peak early to late diastolic filling velocity (E/A) within the normal range was observed in Que-treated mice than in the age-matched controls (Figs. 1E and S1H). However, the lifespan was not prolonged by low-dose Que treatment observed up to the age of 31 months (Fig. S1I). Taken together, these data indicate that long-term low-dose Que administration alone sufficiently improves multiple aspects of healthspan, but not lifespan, in mice. To investigate how Que improved healthspan in mice, we collected 11 different kinds of tissues from 10-week young male mice (Y-Ctrl) and vehicle (O-Veh)and low-dose Quetreated 22-month old male mice (O-Que). No significant difference was observed in organ weights between O-Veh and O-Que (Fig. S2A). Given that exercise endurance and diastolic function were improved by Que, we particularly examined the changes in skeletal muscles (SKM), white adipose tissues (WAT), brown adipose tissues (BAT) and hearts. Upon Que treatment, the arrangement of muscle fibers became more regular and compact with less fibrosis

Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis

Abstract: RAP1 is a well-known telomere-binding protein, but its functions in human stem cells have remained unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter. RAP1 deficiency enhanced self-renewal and delayed senescence in hMSCs, but not in hNSCs, suggesting complicated lineage-specific effects of RAP1 in adult stem cells. Altogether, these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.

Analysis of VISTA expression and function in renal cell carcinoma highlights VISTA as a potential target for immunotherapy

Abstract: Tumors evade immune surveillances, in part via negative regulatory pathways (also called checkpoints) that also regulate immune tolerance to autoimmunity (Thommen et al., 2018). Checkpoint inhibitor therapy, i.e., anti-CTLA-4 and anti-PD-1, has been approved to be an effective therapeutic approach in a variety of cancers (Mariathasan et al., 2018). However, only a subset of cancer patients shows durable responses (Callahan et al., 2016), urging for a broader investigation beyond PD-1 and CTLA-4. Renal cell carcinoma (RCC), the most common kidney cancer, was often considered as an immunogenic tumor based on high levels of T cell infiltration (Finke et al., 1992). However, the infiltrating T cells in RCC were reported to be characterized by a low amount of expanded T cell clonotypes (Sittig et al., 2013). Unexpectedly, the objective response rates to antiPD-1 antibody were 18%–31% in PD-L1 RCC patients vs. 9%–18% in PD-L1 patients (Motzer et al., 2015; McDermott et al., 2016). Thus, there is an urgent need for investigation on immune evasion mechanisms in RCC, especially PD-1independent ones. We thus hypothesized that the low response rate to PD-1 blockade may be caused by co-expression of other checkpoint molecules in the immunosuppressive tumor microenvironment (TME). First, we analyzed the mRNA expression level of several checkpoint molecules in the B7 superfamily through GEPIA using data from TCGA and Oncoprint. We found there was no significant difference in CD274 (encoding PD-L1) expression between RCC tumors and adjacent non-tumoral tissues (Fig. S1A), regardless of RCC types, clear cell RCC (ccRCC), chromophobe RCC (chRCC) or papillary RCC (pRCC). Notably, C10orf54 (encoding VISTA) was significantly upregulated in tumors from patients with ccRCC and downregulated in chRCC tumors compared to adjacent non-tumoral tissues. CD276 (encoding B7-H3) was highly expressed in tumors from patients with ccRCC as well as pRCC, whereas VTCN1 (encoding B7S1) expression was significantly reduced in all RCC types compared to adjacent non-tumoral tissues. In addition, the expression levels of CD276 and C10orf54 were especially higher than CD274 in ccRCC tumors (Fig. S1B). These data might underscore the low response rates to PD-1/PD-L1 inhibitors in ccRCC. To evaluate the expression of the above checkpoint molecules at the protein level in ccRCC accounting for 75% of RCC, paired tumor and para-tumor tissues (2 cm away from tumors) were analyzed by immunofluorescence. The clinical and pathological characteristics of the patients were summarized in Table 1. Figures 1A and S2 show that VISTA was mostly expressed on CD45 cells in para-tumors and tumors, consistent with published data that human VISTA is predominantly expressed in hematopoietic tissues and highly expressed within myeloid compartment (Lines et al., 2014; Ni and Dong, 2017b, a). Moreover, the expression level of VISTA in para-tumors was significantly lower than that in tumor sections (Fig. 1B), in line with the expression pattern of VISTA mRNA. In contrast, the expression levels of B7-H3 and B7S1 proteins were low in both para-tumors and tumors with no significant difference between the two samples, inconsistent with its mRNA expression pattern (Fig. 1A and 1B). PD-L1 was predominantly expressed by CD45 cells (Figs. 1A and S2), and there was no significantly difference in PD-L1 expression between para-tumors and tumor tissues (Fig. 1B). To investigate whether ccRCC tumor cells express VISTA, sequential tumor sections were stained by anti-pan-cytokeratin and anti-VISTA, respectively. As shown in Figure 1C, pan-cytokeratin-expressing cells also showed VISTA expression, indicating that ccRCC tumor cells expressed VISTA, but at a relatively lower level. Since VISTA is mainly expressed on tumor-infiltrated CD45 cells, we next sought to identify which subsets of myeloid cells express VISTA. TILs from ccRCC patients were isolated following enzymatic digestion as our lab previously described (Xie et al., 2018). In addition, peripheral blood mononuclear cells (PBMCs) from the same patients were also used as controls. Different surface markers were employed to distinguish myeloid dendritic cells (mDCs, LinHLA-DRCD11CCD123), monocytes/macrophages (CD14HLA-DR) and monocytic myeloid-derived suppressor cells (mMDSCs, CD14HLA-DR). As shown in

TMEM43-S358L mutation enhances NF-κB-TGFβ signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

Abstract: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a genetic cardiac muscle disease that accounts for approximately 30% sudden cardiac death in young adults The Ser358Leu mutation of transmembrane protein 43 (TMEM43) was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype, ARVD5 Here, we generated TMEM43 S358L mouse to explore the underlying mechanism This mouse strain showed the classic pathologies of ARVD patients, including structural abnormalities and cardiac fibrofatty TMEM43 S358L mutation led to hyper-activated nuclear factor κB (NF-κB) activation in heart tissues and primary cardiomyocyte cells Importantly, this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene, transforming growth factor beta (TGFβ1), and enhanced downstream signal, indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis Our study partially reveals the regulatory mechanism of ARVD development

Remodeling of host membranes during herpesvirus assembly and egress.

Abstract: Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human pathogens and cause many diseases. As large enveloped DNA viruses, herpesviruses undergo several complex steps to complete their life cycles and produce infectious progenies. Firstly, herpesvirus assembly initiates in the nucleus, producing nucleocapsids that are too large to cross through the nuclear pores. Nascent nucleocapsids instead bud at the inner nuclear membrane to form primary enveloped virions in the perinuclear space followed by fusion of the primary envelopes with the outer nuclear membrane, to translocate the nucleocapsids into the cytoplasm. Secondly, nucleocapsids obtain a series of tegument proteins in the cytoplasm and bud into vesicles derived from host organelles to acquire viral envelopes. The vesicles are then transported to and fuse with the plasma membrane to release the mature virions to the extracellular space. Therefore, at least two budding and fusion events take place at cellular membrane structures during herpesviruses assembly and egress, which induce membrane deformations. In this review, we describe and discuss how herpesviruses exploit and remodel host membrane structures to assemble and escape from the host cell.

Lipid droplets and mitochondria are anchored during brown adipocyte differentiation.

Abstract: Brown adipose tissue (BAT) acts as a site of non-shivering thermogenesis in mammals including adult humans (Nedergaard and Cannon, 2010). Prolonged cold exposure can induce the acquisition of thermogenic function in white adipose tissue (WAT) with the cells that have undergone the “browning” process being referred to as beige cells (Wu et al., 2013). Both mainly use mitochondrial β-oxidation of fatty acid (FA) from lipid droplets (LDs) in maintaining body temperature. The efficiency of hydrophobic FA transport between two organelles in aqueous cytoplasm has been challenged. Understanding the mechanism underlying the interaction between these two organelles is essential for animal physiology as well as for the development of therapies to treat metabolic diseases (Benador et al., 2018). Previously, we isolated LDs from BAT and found a tight contact between these two organelles (Yu et al., 2015). A recent study reported that LD-associated mitochondria can be separated from LDs by centrifugation at 9,000 ×g and termed them peridroplet mitochondria (PDM) (Benador et al., 2018). To reveal the nature of the interaction and avoid the impacts of housing temperature, we raised mice at their thermoneutral temperature (30 °C) and room temperature (23 °C), and analyzed their BAT. H&E staining demonstrated that most brown adipocytes from the 30 °C group were similar to white adipocytes with a unilocular LD (Fig. 1A, a and b) rather than multilocular LDs that are possessed in BATat 23 °C (Fig. 1A, d and e). There was a clear change in mitochondrial morphology from an elongated form at 30 °C (Fig. 1A, c) to sphere-shaped at 23 °C (Fig. 1A, f). More importantly, physical contact between LDs and mitochondria was still detected in the 30 °C BAT (Fig. 1A, c, arrows) with no obvious differences in the amount of contact seen in the 23 °C BAT (Fig. 1A, f, arrows). BAT LDs were then isolated and stained with LipidTOX Green for LDs (Fig. 1B, a and d) and MitoTracker Red for mitochondria (Fig. 1B, b and e) using our previously established methods (Yu et al., 2015). The MitoTracker signals were detected on the LipidTOX-stained spherical structures in isolated LDs from both 30 °C and 23 °C BAT (Fig. 1B, c and f, arrows), further confirming that the contact between LDs and mitochondria exists in mouse BAT even persists at 30 °C. Thus, we isolated LDs from mice housed at 30 °C using two centrifugal forces, 2,000 ×g (L) and 228,000 ×g (H), to determine if the contact between LDs and mitochondria could be broken by centrifugation as previously reported (Benador et al., 2018). Proteins from the cellular fractions were analyzed by silver staining (Fig. 1C, upper panel) and Western blotting (Fig. 1C, lower panel). Silver staining result presented that the protein profiles of LDs obtained by both centrifugations and cytosolic mitochondria (CM) were almost identical (Fig. 1C, upper panel, lanes 1–3). This similarity was confirmed by Western blotting in which mitochondrial proteins were detected at the same level between the two LD preparations and the isolated mitochondria, including VDAC, ATP5B, UCP1, TIM23, PROHIBITIN, and ATP5A (Fig. 1C, lower panel, lanes 1–3). These findings demonstrated that the contact between LDs and mitochondria in brown adipocytes existed at thermoneutral temperature, and that LD-anchored mitochondria (LDAM) could not be separated from LDs by high centrifugal force (228,000 ×g). These studies suggest that the tight contact between these two organelles is a native property of brown adipocytes. The tight contact/anchoring between LDs and mitochondria was also detected in our previous studies (Li et al., 2016; Zhang et al., 2011), andwas confirmed by the analysis of heart LD (Fig. S1A) and skeletal muscle LD (Fig. S1B) proteomes. To determine if the anchoring is restricted to oxidative tissues, mouse liver LDs were isolated and analyzed using the same methods. Figure 1D shows that liver LD fraction had a protein profile distinct from isolated mitochondria (Fig. 1D, upper panel, lanes 3 and 4) and lacked mitochondrial functional proteins (Fig. 1D, lower panel, lane 3). We further conducted similar experiments using rhesus monkey (Macaca mulatta) tissues. In agreement with the mouse model, LDs isolated from rhesus monkey BAT, heart, and muscles contained mitochondrial proteins but liver LDs did not (Fig. 1E). LD proteins of control (CK), obese (OB), and diabetic (TM) monkeys were variable, while LDAM proteins were consistent (Fig. 1E, lower panel). These results suggest that LDAM exist specifically in oxidative tissues in mice, rats, and monkeys. Since PLIN5 was a possible linker between the two organelles, especially in BAT (Benador et al., 2018; Olzmann and Carvalho, 2018), LDs were isolated from BAT of Plin5-

Core transcriptional signatures of phase change in the migratory locust

Abstract: Phenotypic plasticity plays fundamental roles in successful adaptation of animals in response to environmental variations. Here, to reveal the transcriptome reprogramming in locust phase change, a typical phenotypic plasticity, we conducted a comprehensive analysis of multiple phase-related transcriptomic datasets of the migratory locust. We defined PhaseCore genes according to their contribution to phase differentiation by the adjustment for confounding principal components analysis algorithm (AC-PCA). Compared with other genes, PhaseCore genes predicted phase status with over 87.5% accuracy and displayed more unique gene attributes including the faster evolution rate, higher CpG content and higher specific expression level. Then, we identified 20 transcription factors (TFs) named PhaseCoreTF genes that are associated with the regulation of PhaseCore genes. Finally, we experimentally verified the regulatory roles of three representative TFs (Hr4, Hr46, and grh) in phase change by RNAi. Our findings revealed that core transcriptional signatures are involved in the global regulation of locust phase changes, suggesting a potential common mechanism underlying phenotypic plasticity in insects. The expression and network data are accessible in an online resource called LocustMine (http://www.locustmine.org:8080/locustmine).

Single senescent cell sequencing reveals heterogeneity in senescent cells induced by telomere erosion

Abstract: Over a half-century ago, Dr. Leonard Hayflick described the phenotype of a finite lifespan for human fibroblasts being passaged in in vitro cell culture (Hayflick et al., 1961), a phenomenon today known as replicative cellular senescence. Cellular senescence has been defined as a state in which cells lose their potential to divide and are permanently arrested in either the G1, or arguably the G2 stage of the cell cycle (Mao et al., 2012). In addition to replicative cellular senescence—which is induced by large amounts of DNA damage at telomeres due to loss of the specialized T-loop structure—exogenous sublethal stresses such as ionizing radiation, genotoxic chemicals or hyper-activated oncogenes may also trigger a similar form of senescence, stress induced premature cellular senescence (SIPS). Cells in the states of replicative senescence and SIPS share a number of common features such as changes in morphology, secretion of inflammatory factors and an increase in βgalactosidase activity. The induction of cellular senescence is mediated by either Rb or p53, two critical tumor suppressors, and represents a potent anti-tumor mechanism (Campisi, 2013). Cellular senescence is also beneficial to injured tissues or organs by secreting inflammatory factors, termed the senescence associated secretory phenotype (SASP), to promote tissue repair and the clearance of senescent cells. Paradoxically, if senescent cells are not promptly removed by NK cells and macrophages (Krizhanovsky et al., 2008), the persistent secretion of SASP factors poses a great threat to tissue homeostasis and may promote tumorigenesis, angiogenesis and even metastasis (Rao et al., 2016). Additionally, the paracrine SASP signaling may also drive the neighboring, young, healthy cells into cellular senescence through the TGFbeta and IL-1 signaling (Acosta et al., 2013). Therefore, although senescent cells are relatively rare in vivo, they may cause tremendous damage to the surrounding tissues or organs. Indeed, recently developed methods of clearing senescent cells in vivo using mouse models have greatly improved the healthy lifespan of rodents, suggesting that targeting senescent cells holds the potential to delay the onset of various age-related pathologies (Xu et al., 2018). The clinical applications of senescent cell-targeting agents, termed senolytics, will be gated by the specificity, efficacy and toxicity of these compounds. Ideally, such chemicals should selectively ablate all types of senescent cells with minimal or no damage to young normal cells. However, although very promising, no senolytic compounds developed to date are able to eliminate all types of senescent cells; further they retain a low level of toxicity to young normal cells (Lamming et al., 2013). Therefore, understanding whether senescent cells induced by either exogenous or endogenous genotoxic stresses, or senescent cells induced by the same stress are identical or heterogeneous would help better achieve the goal of selectively eliminating senescent cells. Senescent cells are generally enlarged in volume, and flattened in morphology if they are fibroblast. They also retain a high level of DNA damage in the nuclei (Muñoz-Espín et al., 2014). At molecular level, p16 and SASP are often employed as markers to identify senescent cells. However, most previous research was conducted at the population level. Whether senescent cells vary between single cells remains to be further determined. Intriguingly, a recent report analyzing the expression of SASP at single cell level reveals that different senescent cells have distinct patterns of SASP expression (Wiley et al., 2017). The report indicates that the observed high level of SASP might result from a small population of senescent cells secreting large amounts of SASP, indicating that senescent cells might be a mixture of single cells with different expression signatures. Nevertheless, no study has yet been performed to systematically examine the heterogeneity of replicatively cellular senescent cells or SIPS cells at a single cell level. Here we employed droplet-based single-cell mRNA sequencing (Drop-seq) (Macosko et al., 2015), which is a powerful tool for analyzing the mRNA expression level of a large number of single cells, to compare the expression signatures at single cell level. To examine the change of heterogeneity during replicative cellular senescence, we cultured HCA2 fibroblasts to replicative senescence. Senescence was confirmed by increased number of β-gal positive cells and significantly reduced rate of EdU incorporation (Fig. S1A). Then we

Core pluripotency factors promote glycolysis of human embryonic stem cells by activating GLUT1 enhancer

Abstract: Human embryonic stem cells (hESCs) depend on glycolysis for energy and substrates for biosynthesis. To understand the mechanisms governing the metabolism of hESCs, we investigated the transcriptional regulation of glucose transporter 1 (GLUT1, SLC2A1), a key glycolytic gene to maintain pluripotency. By combining the genome-wide data of binding sites of the core pluripotency factors (SOX2, OCT4, NANOG, denoted SON), chromosomal interaction and histone modification in hESCs, we identified a potential enhancer of the GLUT1 gene in hESCs, denoted GLUT1 enhancer (GE) element. GE interacts with the promoter of GLUT1, and the deletion of GE significantly reduces the expression of GLUT1, glucose uptake and glycolysis of hESCs, confirming that GE is an enhancer of GLUT1 in hESCs. In addition, the mutation of SON binding motifs within GE reduced the expression of GLUT1 as well as the interaction between GE and GLUT1 promoter, indicating that the binding of SON to GE is important for its activity. Therefore, SON promotes glucose uptake and glycolysis in hESCs by inducing GLUT1 expression through directly activating the enhancer of GLUT1.

Chemical genomics reveals inhibition of breast cancer lung metastasis by Ponatinib via c-Jun

Abstract: Metastasis is the leading cause of human cancer deaths. Unfortunately, no approved drugs are available for anti-metastatic treatment. In our study, high-throughput sequencing-based high-throughput screening (HTS2) and a breast cancer lung metastasis (BCLM)-associated gene signature were combined to discover anti-metastatic drugs. After screening of thousands of compounds, we identified Ponatinib as a BCLM inhibitor. Ponatinib significantly inhibited the migration and mammosphere formation of breast cancer cells in vitro and blocked BCLM in multiple mouse models. Mechanistically, Ponatinib represses the expression of BCLM-associated genes mainly through the ERK/c-Jun signaling pathway by inhibiting the transcription of JUN and accelerating the degradation of c-Jun protein. Notably, JUN expression levels were positively correlated with BCLM-associated gene expression and lung metastases in breast cancer patients. Collectively, we established a novel approach for the discovery of anti-metastatic drugs, identified Ponatinib as a new drug to inhibit BCLM and revealed c-Jun as a crucial factor and potential drug target for BCLM. Our study may facilitate the therapeutic treatment of BCLM as well as other metastases.