Profiling the role of m6A effectors in the regulation of pluripotent reprogramming.

The N6-methyladenosine (m6A) RNA modification plays essential roles in multiple biological processes, including stem cell fate determination. To explore the role of the m6A modification in pluripotent reprogramming, we used RNA-seq to map m6A effectors in human iPSCs, fibroblasts, and H9 ESCs, as well as in mouse ESCs and fibroblasts. By integrating the human and mouse RNA-seq data, we found that 19 m6A effectors were significantly upregulated in reprogramming. Notably, IGF2BPs, particularly IGF2BP1, were among the most upregulated genes in pluripotent cells, while YTHDF3 had high levels of expression in fibroblasts. Using quantitative PCR and Western blot, we validated the pluripotency-associated elevation of IGF2BPs. Knockdown of IGF2BP1 induced the downregulation of stemness genes and exit from pluripotency. Proteome analysis of cells collected at both the beginning and terminal states of the reprogramming process revealed that the IGF2BP1 protein was positively correlated with stemness markers SOX2 and OCT4. The eCLIP-seq target analysis showed that IGF2BP1 interacted with the coding sequence (CDS) and 3'UTR regions of the SOX2 transcripts, in agreement with the location of m6A modifications. This study identifies IGF2BP1 as a vital pluripotency-associated m6A effector, providing new insight into the interplay between m6A epigenetic modifications and pluripotent reprogramming.

CircARAP2 controls sMICA-induced NK cell desensitization by erasing CTCF/PRC2-induced suppression in early endosome marker RAB5A.

Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.

LncRNA Osilr9 coordinates promoter DNA demethylation and the intrachromosomal loop structure required for maintaining stem cell pluripotency.

Nuclear reprogramming of somatic cells into a pluripotent status has the potential to create patient-specific induced pluripotent stem cells for regenerative medicine. Currently, however, the epigenetic mechanisms underlying this pluripotent reprogramming are poorly understood. To delineate this epigenetic regulatory network, we utilized a chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to identify long noncoding RNAs (lncRNAs) embedded in the 3-dimensional intrachromosomal architecture of stem cell core factor genes. By combining CRIST-seq and RNA sequencing, we identified Oct4-Sox2 interacting lncRNA 9 (Osilr9) as a pluripotency-associated lncRNA. Osilr9 expression was associated with the status of stem cell pluripotency in reprogramming. Using short hairpin RNA (shRNA) knockdown, we showed that this lncRNA was required for the optimal maintenance of stem cell pluripotency. Overexpression of Osilr9 induced robust activation of endogenous stem cell core factor genes in fibroblasts. Osilr9 participated in the formation of the intrachromosomal looping required for the maintenance of pluripotency. After binding to the Oct4 promoter, Osilr9 recruited the DNA demethylase ten-eleven translocation 1, leading to promoter demethylation. These data demonstrate that Osilr9 is a critical chromatin epigenetic modulator that coordinates the promoter activity of core stem cell factor genes, highlighting the critical role of pluripotency-associated lncRNAs in stem cell pluripotency and reprogramming.

Control of Compensation Temperature in CoGd Films through Hydrogen and Oxygen Migration under Gate Voltage.

Electrical control of magnetic properties is crucial for low-energy memory and logic spintronic devices. We find that the magnetic properties of ferrimagnetic CoGd can be altered through ionic liquid gating. Gate voltages manipulate the opposite magnetic moments in Co and Gd sublattices and induce a giant magnetic compensation temperature change of more than 200 K in Pt/CoGd/Pt heterostructures. The electrically controlled dominant magnetic sublattice allows voltage-induced magnetization switching. Both experiments and theoretical calculations demonstrate that the significant modulations of compensation temperature are relevant to the reduced Gd moments due to the presence of hydrogen ions at positive voltages as well as the enhanced Co moments and reduced Gd moments due to the injection of oxygen ions at negative voltages. These findings expand the possibilities for all-electric and reversible magnetization control in the field of spintronics.

Electrical Control of Magnetism through Proton Migration in Fe3O4/Graphene Heterostructure.

Ion migration has direct and crucial bearing on the crystal lattice field, electron filling, orbital occupation and spin polarization, which in turn changes the physical properties. Electric field is an effective way to control ion migration, but it may include simultaneous movement of multiple ions and increase the complexity of the system. Therefore, controllable and selective single ion migration with an unambiguous mechanism is highly desired. Here, the magnetic moments of Fe3O4 could be reversibly controlled by ionic liquid gating on the basis of migration of pure protons. A bilayer graphene could serve as an ion sieve, allowing only protons rather than oxygen ions or hydroxyl groups to participate in the gating process, thus guaranteeing the reversibility of magnetic property changes. This work is expected to supply an ideal arena for electrically sketching the functionalities of solid state materials based on the selective ion migration.

Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

Oplr16 serves as a novel chromatin factor to control stem cell fate by modulating pluripotency-specific chromosomal looping and TET2-mediated DNA demethylation.

Formation of a pluripotency-specific chromatin network is a critical event in reprogramming somatic cells into pluripotent status. To characterize the regulatory components in this process, we used 'chromatin RNA in situ reverse transcription sequencing' (CRIST-seq) to profile RNA components that interact with the pluripotency master gene Oct4. Using this approach, we identified a novel nuclear lncRNA Oplr16 that was closely involved in the initiation of reprogramming. Oplr16 not only interacted with the Oct4 promoter and regulated its activity, but it was also specifically activated during reprogramming to pluripotency. Active expression of Oplr16 was required for optimal maintenance of pluripotency in embryonic stem cells. Oplr16 was also able to enhance reprogramming of fibroblasts into pluripotent cells. RNA reverse transcription-associated trap sequencing (RAT-seq) indicated that Oplr16 interacted with multiple target genes related to stem cell self-renewal. Of note, Oplr16 utilized its 3'-fragment to recruit the chromatin factor SMC1 to orchestrate pluripotency-specific intrachromosomal looping. After binding to the Oct4 promoter, Oplr16 recruited TET2 to induce DNA demethylation and activate Oct4 in fibroblasts, leading to enhanced reprogramming. These data suggest that Oplr16 may act as a pivotal chromatin factor to control stem cell fate by modulating chromatin architecture and DNA demethylation.

Long noncoding RNAs and their epigenetic function in hematological diseases.

Recent discoveries demonstrate the importance of long noncoding RNA (lncRNA) in the regulation of multiple major processes impacting development, differentiation, and metastasis of hematological diseases through epigenetic mechanisms. In contrast to genetic changes, epigenetic modification does not modify genes but is frequently reversible, thus providing opportunities for targeted treatment using specific inhibitors. In this review, we will summarize the function and epigenetic mechanism of lncRNA in malignant hematologic diseases.

Fluorometric determination of antioxidant capacity in human plasma by using upconversion nanoparticles and an inner filter effect mechanism.

The balance between free oxygen radicals and antioxidant defense systems is usually assessed by an antioxidant capacity assay. A rapid and sensitive antioxidant capacity assay is described here. It is making use of NaYF4:Yb/Er@NaYF4 core-shell upconversion nanoparticles (UCNPs) and potassium permanganate (KMnO4). In this strategy, added KMnO4 reduces the green (540 nm) emission of the UCNPs (under 980 nm photoexcitation) due to an inner filter effect. The antioxidants cysteine, ascorbic acid and glutathione (GSH) reduce the intense purple color of KMnO4 because it is reduced to Mn(II) ion. Hence, the green upconversion fluorescence is restored after the addition of antioxidants. Figures of merit for this assay (for the case of GSH) include a detection limit of 3.3 µM, a detection range that extends from 10 µM to 2.5 mM, and an assay time of a few seconds. The assay was applied to the evaluation of antioxidant capacity in human plasma samples spiked with GSH and gave satisfactory repeatability and specificity. Graphical abstract Schematic presentation of a fluorometric assay based on inner filter effect (IFE) between upconversion nanoparticles (UCNPs) and potassium permanganate (KMnO4) for the determination of antioxidant capacity in human plasma.

Acetone Sensing Properties and Mechanism of SnO2 Thick-Films.

In the present work, we investigated the acetone sensing characteristics and mechanism of SnO2 thick-films through experiments and DFT calculations. SnO2 thick film annealed at 600 °C could sensitively detect acetone vapors. At the optimum operating temperature of 180 °C, the responses of the SnO2 sensor were 3.33, 3.94, 5.04, and 7.27 for 1, 3, 5, and 10 ppm acetone, respectively. The DFT calculation results show that the acetone molecule can be adsorbed on the five-fold-coordinated Sn and oxygen vacancy (VO) sites with O-down, with electrons transferring from acetone to the SnO2 (110) surface. The acetone molecule acts as a donor in these modes, which can explain why the resistance of SnO2 or n-type metal oxides decreased after the acetone molecules were introduced into the system. Molecular dynamics calculations show that acetone does not convert to other products during the simulation.

An Ultrahigh Sensitivity Acetone Sensor Enhanced by Light Illumination.

Au:SmFe0.9Zn0.1O3 is synthesized by a sol-gel method and annealed at 750 °C. Through XRD, SEM and XPS analysis methods, the microstructure of the material has been observed. The average particle size is about 50 nm. The sensor shows a high sensitivity toward acetone vapor. As the relative humidity increases, the resistance and sensitivity of the sensor decline. To obtain a low optimum operating temperature, light illumination with different wavelengths has been introduced. The sensitivity toward acetone is improved at lower operating temperature when the sensor is irradiated by light. The smaller the wavelengths, the better the sensitivity of the sensor. Compared with other gases, the sensor shows excellent selectivity to acetone vapor, with better sensitivity, selectivity and stability when under light illumination.

UV Light Illumination Can Improve the Sensing Properties of LaFeO3 to Acetone Vapor.

The synthesized LaFeO3 nanocrystalline sensor powders show positive response to sensing acetone vapor at 200 °C. The responses to acetone vapor (at 0.5, 1, 2, 5, 10 ppm) are 1.18, 1.22, 1.89, 3.2 and 7.83. To make the sensor operate at a lower optimum temperature, UV light illumination 365 nm is performed. Response of the sensor has a larger improvement under 365 nm UV light illumination than without it. The responses to acetone vapor (at 0.5, 1, 2, 5, 10 ppm) are 1.37, 1.85, 3.16, 8.32 and 14.1. Furthermore, the optimum operating temperature is reduced to 170 °C. As the relative humidity increases, the resistance and sensitivity of sensor are reduced. The sensor shows good selectivity toward acetone when compared with other gases. Since the detection of ultralow concentrations of acetone vapor is possible, the sensor can be used to preliminarily judge diabetes in the general public, as a high concentration of acetone is exhaled in breath of diabetic patients. The sensor shows a good stability, which is further enhanced under UV light illumination. The sensor shows better stability when under 365 nm UV light illumination. Whether under light illumination or not. The LaFeO3 material shows good performance as a sensor when exposed to acetone vapor.

Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.

BACKGROUND/AIMS: Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. METHODS: We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. RESULTS: We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. CONCLUSION: The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL.

Optical-magnetic bifunctional properties and mechanistic insights on upconversion of NaYF4:Yb,Ho,Tm@NaGdF4 with a tunable nanodumbbell morphology.

The energy-upconversion of lanthanide-doped nanoparticles with a core-shell structure can be utilized to enhance and tune optical properties and can generate multifunctionality in a single system. Herein, the core-shell nanoparticles NaYF4:Yb,Ho,Tm@NaGdF4 were prepared by thermally decomposing lanthanide acetylacetonate precursors. Through modifying the molar ratio of the core and shell, nanodumbbell-shaped particles with different sizes and morphologies were precisely synthesized. The formation mechanism and the heterogeneous epitaxial growth process of the nanodumbbell-shaped particles were studied. After coating the shell layer, upconversion luminescence intensities, spectral purity and fluorescence lifetimes were improved. Furthermore, the magnetic performance of the core-shell nanoparticles was characterized. The optical-magnetic bifunctional upconversion core-shell particles with programmable shape and multiple properties provide an ideal platform for the preparation of nanodumbbell-shaped particles and the promotion of upconversion materials for biomedical research.

Valproic Acid Enhances iPSC Induction From Human Bone Marrow-Derived Cells Through the Suppression of Reprogramming-Induced Senescence.

Reprogramming of human somatic cells into pluripotent cells (iPSCs) by defined transcription factors is an extremely inefficient process. Treatment with the histone deacetylase inhibitor valproic acid (VPA) during reprogramming can improve the induction of iPSCs. To examine the specific mechanism underlying the role of VPA in reprogramming, we transfected human bone marrow-derived cells (HSC-J2 and HSC-L1) with lentiviruses carrying defined factors (OCT4, SOX2, KLF4, and c-MYC, OSKM) in the presence of VPA. We found that, OSKM lentiviruses caused significant senescence in transfected cells. Administration of VPA, however, significantly suppressed this reprogramming-induced stress. Notably, VPA treatment improved cell proliferation in the early stages of reprogramming, and this was related to the down-regulation of the activated p16/p21 pathway. In addition, VPA also released the G2/M phase blockade in lentivirus-transfected cells. This study demonstrates a new mechanistic role of the histone deacetylase inhibitor in enhancing the induction of pluripotency. J. Cell. Physiol. 231: 1719-1727, 2016. © 2015 Wiley Periodicals, Inc.

Converting Skin Fibroblasts into Hepatic-like Cells by Transient Programming.

Transplantation of hepatocytes is a promising therapy for end-stage liver disease, but the availability of functional cells currently precludes its clinical application. We now report a simple transient reprogramming approach to convert fibroblasts into hepatic-like cells. Human skin fibroblasts were treated with fish egg extracts to become the transiently remodeled cells (TRCs). After infected with retroviral EGFP, they were directly injected into the fetal monkey liver, where they underwent in situ differentiation in the hepatic niche. The hepatic-like cells were functional as shown by the synthesis of hepatic markers in vivo, including albumin, cytokeratin-18, and hepatic serum antigen. Similarly, when implanted in the mouse liver, the TRCs were differentiated into hepatic-like cells that synthesize albumin and CK18 and became completely integrated into the liver parenchyma. The potency of TRCs was mechanistically related to the activation of several signal pathways, which reactivate endogenous genes related to cell potency. This study demonstrates the feasibility of a simple and inexpensive epigenetic remodeling approach to convert human fibroblasts into therapeutic hepatic-like cells for the treatment of end-stage liver disease.

Promoter histone H3K27 methylation in the control of IGF2 imprinting in human tumor cell lines.

Aberrant imprinting of the insulin-like growth factor II (IGF2) gene is a molecular hallmark of many tumors. Reactivation of the normally suppressed maternal allele leads to upregulation of the growth factor that promotes tumor growth. However, the mechanisms underlying the loss of imprinting (LOI) remain poorly defined. We examined the epigenotypes at the gene promoters that control IGF2 allelic expression. Using chromatin immunoprecipitation, we found that in cells characterized by maintenance of IGF2 imprinting, three IGF2 promoters were differentially modified, with the suppressed allele heavily methylated at histone H3K27 while the active allele was unmethylated. In the LOI tumors, however, both alleles were unmethylated, and correspondingly there was no binding of SUZ12, the docking factor of the polycomb repressive complex 2 (PRC2), and of the zinc finger-containing transcription factor (CTCF) that recruits the PRC2. Using chromatin conformation capture, we found that the CTCF-orchestrated intrachromosomal loop between the IGF2 promoters and the imprinting control region was abrogated in cells with LOI. SUZ12, which docks the PRC2 to IGF2 promoters for H3K27 methylation, was downregulated in LOI cells. These data reveal a new epigenetic control pathway related to the loss of IGF2 imprinting in tumors.

Restoration of IGF2 imprinting by polycomb repressive complex 2 docking factor SUZ12 in colon cancer cells.

The insulin-like growth factor II (IGF2) gene is aberrantly expressed in tumors as a result of loss of imprinting (LOI). Reactivation of the normally-suppressed maternal allele may lead to IGF2 upregulation and increased tumor growth, particularly in colon cancer. However, the mechanisms underlying IGF2 LOI in tumors are poorly defined. In this report, we identified polycomb repressive complex 2 (PRC2) docking factor SUZ12 as a critical factor in regulating IGF2 imprinting in tumors. Human colon cancer cell lines (HRT18 and HT29) show loss of IGF2 imprinting. Ectopic expression of SUZ12 restored normal monoallelic expression of IGF2 in these two colon cancer cell lines. Using chromatin immunoprecipitation (ChIP) and chromatin conformation capture (3C), we found that the virally-expressed SUZ12 bound to IGF2 promoters, coordinating with endogenous CTCF to orchestrate a long range intrachromosomal loop between the imprinting control region (ICR) and the IGF2 promoters. The histone methyltransferase EZH2 was recruited to the IGF2 promoters, where it induced H3K27 hypermethylation, suppressing one allele, leading to the restoration of IGF2 imprinting. These data demonstrate that SUZ12 is a key molecule in the regulation of monoallelic expression of IGF2, suggesting a novel epigenetic therapeutic strategy for modulating IGF2 production in human tumors.

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress.

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency.

A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies.

Dysregulation of the insulin-like growth factor type I receptor (IGF1R) has been implicated in the progression and therapeutic resistance of malignancies. In acute myeloid leukemia (AML) cells, IGF1R is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in cancer. We discovered a novel intragenic long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter. The IRAIN lncRNA was expressed exclusively from the paternal allele, with the maternal counterpart being silenced. Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction. In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients. These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.