Rex1BD and the 14-3-3 protein control heterochromatin organization at tandem repeats by linking RNAi and HDAC.

Tandem DNA repeats are often organized into heterochromatin that is crucial for genome organization and stability. Recent studies revealed that individual repeats within tandem DNA repeats can behave very differently. How DNA repeats are assembled into distinct heterochromatin structures remains poorly understood. Here, we developed a genome-wide genetic screen using a reporter gene at different units in a repeat array. This screen led to identification of a conserved protein Rex1BD required for heterochromatin silencing. Our structural analysis revealed that Rex1BD forms a four-helix bundle structure with a distinct charged electrostatic surface. Mechanistically, Rex1BD facilitates the recruitment of Clr6 histone deacetylase (HDAC) by interacting with histones. Interestingly, Rex1BD also interacts with the 14-3-3 protein Rad25, which is responsible for recruiting the RITS (RNA-induced transcriptional silencing) complex to DNA repeats. Our results suggest that coordinated action of Rex1BD and Rad25 mediates formation of distinct heterochromatin structure at DNA repeats via linking RNAi and HDAC pathways.

Ultrasound targeted microbubble destruction assisted exosomal delivery of siHmox1 effectively inhibits doxorubicin-induced cardiomyocyte ferroptosis.

Ferroptosis, triggered by iron overload and excessive lipid peroxidation, plays a pivotal role in the progression of DOX-induced cardiomyopathy (DIC), and thus limits the use of doxorubicin (DOX) in clinic. Here, we further showed that cardiac ferroptosis induced by DOX in mice was attributed to up-regulation of Hmox1, as knockdown of Hmox1 effectively inhibited cardiomyocyte ferroptosis. To targeted delivery of siRNA into cardiomyocytes, siRNA-encapsulated exosomes were injected followed by ultrasound microbubble targeted destruction (UTMD) in the heart region. UTMD greatly facilitated exosome delivery into heart. Consistently, UTMD assisted exosomal delivery of siHomox1 nearly blocked the ferroptosis and the subsequent cardiotoxicity induced by doxorubicin. In summary, our findings reveal that the upregulation of HMOX1 induces ferroptosis in cardiomyocytes and UTMD-assisted exosomal delivery of siHmox1 can be used as a potential therapeutic strategy for DIC.

Targeted delivery of MerTK protein via cell membrane engineered nanoparticle enhances efferocytosis and attenuates atherosclerosis in diabetic ApoE-/- Mice.

BACKGROUND: Clearance of apoptotic cells by efferocytosis is crucial for prevention of atherosclerosis progress, and impaired efferocytosis contributes to the aggravated atherosclerosis. RESULTS: In this study, we found that diabetic ApoE-/- mice showed aggravated atherosclerosis as hyperglycemia damaged the efferocytosis capacity at least partially due to decreased expression of Mer tyrosine kinase (MerTK) on macrophages. To locally restore MerTK in the macrophages in the plaque, hybrid membrane nanovesicles (HMNVs) were thus developed. Briefly, cell membrane from MerTK overexpressing RAW264.7 cell and transferrin receptor (TfR) overexpressing HEK293T cell were mixed with DOPE polymers to produce nanovesicles designated as HMNVs. HMNVs could fuse with the recipient cell membrane and thus increased MerTK in diabetic macrophages, which in turn restored the efferocytosis capacity. Upon intravenous administration into diabetic ApoE-/- mice, superparamagnetic iron oxide nanoparticles (SMN) decorated HMNVs accumulated at the aorta site significantly under magnetic navigation, where the recipient macrophages cleared the apoptotic cells efficiently and thus decreased the inflammation. CONCLUSIONS:  Our study indicates that MerTK decrease in macrophages contributes to the aggravated atherosclerosis in diabetic ApoE-/- mice and regional restoration of MerTK in macrophages of the plaque via HMNVs could be a promising therapeutic approach.

Fluorinated covalent-organic polymers as stationary phase for analysis of organic fluorides by open-tubular capillary electrochromatography.

Fluorinated porous materials, which can provide specific fluorine-fluorine interaction, hold great promise for fluoride analysis. Here, a novel fluorinated covalent-organic polymer was prepared by using 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 2,3,5,6-tetrafluorotelephtal aldehyde as the precursors and introduced as stationary phase for open-tubular capillary electrochromatography. The as-synthesized fluorinated covalent-organic polymer and the modified capillary column were characterized by infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectrometry. Based on strong hydrophobic interaction and fluorine-fluorine interaction provided by fluorinated covalent-organic polymer coating layer, the modified column showed powerful separation selectivity toward hydrophobic compounds, organic fluorides, and fluorinated pesticides. Additionally, the fluorinated covalent-organic polymer with good porosity and regular shape was uniformly and tightly coated on the capillary inner wall. The obtained highest column efficiency could reach up to 1.2 × 105 plates⋅m-1 for fluorophenol. The loading capacity of the modified column can reach 141 pmol for trifluorotoluene. Besides, the relative standard deviations of retention times for intraday run (n = 5), interday run (n = 3), and between columns (n = 3) were all less than 2.55%. Significantly, this novel fluorinated material-based stationary phase shows great application potential in fluorides analysis.

A light-responsive molecular switch based on cucurbit[7]uril and 1,1'-bis(benzyl)-4-[2-(4-pyridyl)-vinyl]-pyridinium dibromide displaying white light emission.

By using 1H NMR, ESI-MS and UV spectra, a novel light-responsive molecular switch constructed using 1,1'-bis(benzyl)-4-[2-(4-pyridyl)-vinyl]-pyridinium (12+) and cucurbit[7]uril (Q[7]) is demonstrated. The E- to Z-isomerization of the double bond in 12+ results in the transition of the switching states from the 1 : 2 complex E-12+@Q[7]2 to the stable 1 : 1 complex Z-12+@Q[7]. In particular, both the 1 : 2 complex and the 1 : 1 complex can emit cold white fluorescence under UV light.

An inclusion complex of cucurbit[7]uril with benzimidazolyl benzyl viologen exhibits fluorescence and photochromic properties.

In order to investigate the photochromic mechanism of photochromic materials based on supramolecular host-guest systems, we designed and synthesized a unique viologen derivative (benzimidazolyl benzyl viologen, guest 1·Cl3), which does not contain oxygen atoms. The binding interaction of guest 13+ with host cucurbit[7]uril (Q[7]) was investigated by various techniques. The obtained supramolecular host-guest complex 13+@Q[7] exhibits interesting fluorescence emission and reversible photochromism. The ESR and XPS experimental data suggest that the photochromic process of the complex 13+@Q[7] comes from the electron transfer from the carbonyl O atoms of the host Q[7] to the bipyridinium N atoms of the guest 13+.

Selective Encapsulation of Therapeutic mRNA in Engineered Extracellular Vesicles by DNA Aptamer.

Extracellular vesicles (EV)-based delivery of therapeutic mRNAs is challenged by the low loading efficiency. In this study, we designed a DNA aptamer consisting of two parts: the single strand part recognized the AUG region of target mRNA, preventing mRNA from translation and ribosome assembly; and the double strand part containing the elements recognized by the CD9-ZF (zinc finger) motifs, sorting DNA aptamer-mRNA complex into CD9-ZF engineered EVs. In vitro and in vivo studies revealed that the system could efficiently load functional mRNAs to the EVs. Furthermore, adipose specific delivery of loaded Pgc1α mRNA via the strategy could efficiently induce white adipocyte browning. Similarly, delivery of interleukin-10 (Il-10) mRNA via the strategy had potent anti-inflammatory effect in inflammatory bowel disease (IBD) mouse model. Together, our study has proposed an efficient strategy to load therapeutic mRNAs of interest into EVs, which could be used as a promising strategy for gene therapy.

Porous layer open-tubular column with styrene and itaconic acid-copolymerized polymer as stationary phase for capillary electrochromatography-mass spectrometry.

Enhancing the specific surface area of stationary phase is important in chromatographic science, especially in open-tubular column in which the coating only exists on the inner surface. In this work, a porous layer open-tubular (PLOT) column with stationary phase of styrene and itaconic acid-copolymerized polymer was developed. Thermal-initiated polymerization method with strategies like controlling the ratio of reaction reagents to solvents and reaction time, confinement by the narrow inner diameter of capillary were used for preparing the stationary phase with uniform structure and relatively thick layer. Due to the high separation efficiency and capacity, the PLOT column was used for capillary electrochromatography (CEC) separation of multiple groups of analytes like alkylbenzenes, phenyl amines, phenols, vanillins, and sulfonamides with theoretical plates (N) up to 1,54,845 N/m. In addition, due to high permeability of the CEC column and large electroosmotic flow mobility generated by abundant carboxyl groups in the coating material, the PLOT-CEC column was successfully coupled with mass spectrometry (MS) through a sheath flow interface. The developed PLOT-CEC-MS method was used for the analysis of antiseptics like parabens and herbicides like pyridines.

Ultrasound triggered topical delivery of Bmp7 mRNA for white fat browning induction via engineered smart exosomes.

BACKGROUND: Efficient and topical delivery of drugs is essential for maximized efficacy and minimized toxicity. In this study, we aimed to design an exosome-based drug delivery platform endowed with the ability of escaping from phagocytosis at non-target organs and controllably releasing drugs at targeted location. RESULTS: The swtichable stealth coat CP05-TK-mPEG was synthesized and anchored onto exosomes through the interaction between peptide CP05 and exosomal surface marker CD63. Chlorin e6 (Ce6) was loaded into exosomes by direct incubation. Controllable removal of PEG could be achieved by breaking thioketal (TK) through reactive oxygen species (ROS), which was produced by Ce6 under ultrasound irradiation. The whole platform was called SmartExo. The stealth effects were analyzed in RAW264.7 cells and C57BL/6 mice via tracing the exosomes. To confirm the efficacy of the engineered smart exosomes, Bone morphogenetic protein 7 (Bmp7) mRNA was encapsulated into exosomes by transfection of overexpressing plasmid, followed by stealth coating, with the exosomes designated as SmartExo@Bmp7. Therapeutic advantages of SmartExo@Bmp7 were proved by targeted delivering Bmp7 mRNA to omental adipose tissue (OAT) of obese C57BL/6 mice for browning induction. SmartExo platform was successfully constructed without changing the basic characteristics of exosomes. The engineered exosomes effectively escaped from the phagocytosis by RAW264.7 and non-target organs. In addition, the SmartExo could be uptaken locally on-demand by ultrasound mediated removal of the stealth coat. Compared with control exosomes, SmartExo@Bmp7 effectively delivered Bmp7 mRNA into OAT upon ultrasound irradiation, and induced OAT browning, as evidenced by the histology of OAT and increased expression of uncoupling protein 1 (Ucp1). CONCLUSIONS: The proposed SmartExo-based delivery platform, which minimizes side effects and maximizing drug efficacy, offers a novel safe and efficient approach for targeted drug delivery. As a proof, the SmartExo@Bmp7 induced local white adipose tissue browning, and it would be a promising strategy for anti-obesity therapy.

Structural insights into chromosome attachment to the nuclear envelope by an inner nuclear membrane protein Bqt4 in fission yeast.

The dynamic association of chromosomes with the nuclear envelope (NE) is essential for chromosome maintenance. Schizosaccharomyces pombe inner nuclear membrane protein Bqt4 plays a critical role in connecting telomeres to the NE, mainly through a direct interaction with the telomeric protein Rap1. Bqt4 also interacts with Lem2 for pericentric heterochromatin maintenance. How Bqt4 coordinates the interactions with different proteins to exert their functions is unclear. Here, we report the crystal structures of the N-terminal domain of Bqt4 in complexes with Bqt4-binding motifs from Rap1, Lem2, and Sad1. The structural, biochemical and cellular analyses reveal that the N-terminal domain of Bqt4 is a protein-interaction module that recognizes a consensus motif and plays essential roles in telomere-NE association and meiosis progression. Phosphorylation of Bqt4-interacting proteins may act as a switch to regulate these interactions during cell cycles. Our studies provide structural insights into the identification and regulation of Bqt4-mediated interactions.

Ultrasound targeted microbubble destruction assisted exosomal delivery of miR-21 protects the heart from chemotherapy associated cardiotoxicity.

Chemotherapy related cardiotoxicity is now becoming one of the biggest hurdles for the prognosis of cancer patients. Therapeutically delivering protective small RNAs holds promise for the cardiotoxicity prevention and therapy. However, heart is intrinsically refractory to the nanoparticle-mediated drug delivery. In this study, we found that the exosome-mediated miRNA delivery into the heart could be significantly augmented with the aid of ultrasound targeted microbubble destruction (UTMD). Moreover, we found that UTMD assisted exosomal miR-21 delivery into the heart significantly decreased the cell death, and restored the cardiac function in a doxorubicin induced cardiotoxicity mouse model. Our study here not only provides a promising strategy to protect the heart from the chemotherapy related cardiotoxicity, but also sheds light on gene therapy of other heart diseases.

Symmetrical-Tetramethyl-Cucurbit[6]uril-Driven Movement of Cucurbit[7]uril Gives Rise to Heterowheel [4]Pseudorotaxanes.

Two novel heterowheel [4]pseudorotaxanes consisting of cucurbit[7]uril (Q[7]) and symmetrical-tetramethyl-cucurbit[6]uril (TMeQ[6]) were constructed via the multirecognition mechanism, in which Q[7] can rotate freely around the horizontal axis, while TMeQ[6] cannot. In the construction process, due to strong repulsive forces between carbonyl portals of two neighboring wheels, the dethreading and movement of the wheels along the axle was observed. The dissociation of the [4]pseudorotaxanes was also discussed.

Identification of a novel melatonin-binding nuclear receptor: Vitamin D receptor.

Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.

In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9.

In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Host-guest complexation of di-cyclohexanocucurbit[6]uril and hexa-cyclohexanocucurbit[6]uril with alkyldiammonium ions: a comparative study.

The host-guest complexation of symmetrical di-cyclohexanocucurbit[6]uril (Cy2Q[6]) and hexa-cyclohexanocucurbit[6]uril (Cy6Q[6]) with a series of alkyldiammonium ions (H(3+)N(CH(2))nNH(3+), n = 2-8) has been studied both in solution and in the gas phase. (1)H NMR data indicate that all alkyldiammonium ions have inclusion interactions with both hosts except for the ethanediammonium ion. In addition, if the alkyl chain of the alkyldiammonium ion is longer than n = 5 methylene groups, compressed conformation may occur, which depends on the cavity shape of the hosts and the length of the alkyl chain. Isothermal titration calorimetry (ITC) studies point out that the host-guest complexations of both hosts with the latter five alkyldiammonium ions are enthalpically driven. The comparison of the thermodynamic data reveals that the enthalpies of the van der Waals interactions contribute more to the host-guest complexation enthalpy than the ion-dipole interactions. The enthalpic gain arises from the van der Waals interactions and the reduction of entropy upon the host-guest complexation is strongly affected by the cavity shape of the host. Gas phase structures of long alkyldiammonium guests within both hosts are completely different from those in aqueous solution.

Phylogenetic and biological characterization of three K1203 (H5N8)-like avian influenza A virus reassortants in China in 2014.

Three H5N8 avian influenza viruses isolated from domestic geese in China in 2014 were characterized phylogenetically and biologically. Phylogenetic analysis of the complete genomic sequences of the three isolates from this study and those of 61 other H5N8 viruses retrieved from the GISAID platform indicated that, chronologically and geographically, all H5N8 viruses of the Asian H5N1 HA lineage of clade 2.3.4.4 are the direct descendents of the K1203 (H5N8)-like viruses first isolated in China in 2010. The three viruses from this study shared high sequence similarity in all eight gene segments with three other isolates from China in 2013, and two Korean isolates were distinct from the recently circulating reassortants causing outbreaks in Asia, Europe and the United States in 2014 and 2015. In vitro viral growth curves indicated that these H5N8 viruses replicated to high titers in CEF, DEF, MDCK and A549 cells but to significantly lower titers in Vero cells. Pathogenicity studies in vivo indicated that these viruses were all highly virulent to chickens and mallard ducks, while they varied from moderate to high virulence in mice. Additionally, hemagglutination assays using α-2,3-sialidase-treated goose red blood cells and solid-phase direct binding assays with different glycans demonstrated that the three viruses could bind to both avian-type SAα-2,3Gal and human-type SAα-2,6Gal receptors. Our findings confirmed the progenitor nature of the K1203-like viruses in generating recent prevalent clade 2.3.4.4 H5N8 reassortants, which have caused tremendous damage to the poultry industry and are a potential threat to public health.