Deciphering the Prognostic and Therapeutic Significance of Cell Cycle Regulator CENPF: A Potential Biomarker of Prognosis and Immune Microenvironment for Patients with Liposarcoma.

Liposarcoma (LPS) is one of the most common subtypes of sarcoma with a high recurrence rate. CENPF is a regulator of cell cycle, differential expression of which has been shown to be related with various cancers. However, the prognostic value of CENPF in LPS has not been deciphered yet. Using data from TCGA and GEO datasets, the expression difference of CENPF and its effects on the prognosis or immune infiltration of LPS patients were analyzed. As results show, CENPF was significantly upregulated in LPS compared to normal tissues. Survival curves illustrated that high CENPF expression was significantly associated with adverse prognosis. Univariate and multivariate analysis suggested that CENPF expression could be an independent risk factor for LPS. CENPF was closely related to chromosome segregation, microtubule binding and cell cycle. Immune infiltration analysis elucidated a negative correlation between CENPF expression and immune score. In conclusion, CENPF not only could be considered as a potential prognostic biomarker but also a potential malignant indicator of immune infiltration-related survival for LPS. The elevated expression of CENPF reveals an unfavorable prognostic outcome and worse immune score. Thus, therapeutically targeting CENPF combined with immunotherapy might be an attractive strategy for the treatment of LPS.

Atom-based sensing technique of microwave electric and magnetic fields via a single rubidium vapor cell.

We present an atom-based approach for determining microwave electric and magnetic fields by using a single rubidium vapor cell in a microwave waveguide. For a 87Rb cascade three-level system employed in our experiment, a weak probe laser driving the lower transition, 5S1/2→5P3/2, is first used to measure the microwave magnetic field based on the atomic Rabi resonance. When a counter-propagating strong coupling laser is subsequently turned on to drive the Rydberg transition, 5P3/2→67D5/2, the same probe laser is then used as a Rydberg electromagnetically induced transparency (EIT) probe to measure the microwave electric field by investigating the resonant microwave dressed Autler-Townes splitting (ATS). By tuning the hyperfine transition frequency of the ground state using an experimentally feasible static magnetic field, we first achieved a measurement of the microwave electric and magnetic field strength at the same microwave frequency of 6.916 GHz. Based on the ideal relationship between the electric and magnetic field components, we obtained the equivalent microwave magnetic fields by fitting the inversion to the measured microwave electric fields, which demonstrated that the results were in agreement with the experimental measurement of the microwave magnetic fields in the same microwave power range. This study provides new experimental evidence for quantum-based microwave measurements of electric and magnetic fields by a single sensor in the same system.

Brain-targeted CRISPR/Cas9 nanomedicine for effective glioblastoma therapy.

CRISPR/Cas9 gene-editing technology shows great potential for treating a variety of diseases, such as glioblastoma multiforme (GBM). However, CRISPR components suffer from inherent delivery challenges, such as poor in vivo stability of Cas9 protein and gRNA, low blood-brain barrier (BBB) permeability and non-specific tissue or cell targeting. These defects have limited the application of Cas9/gRNA ribonucleoprotein (RNP) complexes for GBM therapy. Here, we developed a brain-targeted CRISPR/Cas9 based nanomedicine by fabricating an angiopep-2 decorated, guanidinium and fluorine functionalized polymeric nanoparticle with loading Cas9/gRNA RNP for the treatment of GBM. The guanidinium and fluorine domains of our polymeric nanoparticles were both capable of interacting with Cas9/gRNA RNP to stabilize it in blood circulation, without impairing its activity. Moreover, by leveraging angiopep-2 peptide functionality, the RNP nanoparticles efficiently crossed the BBB and accumulated in brain tumors. In U87MG cells, we achieved approximately 32% gene knockout and 67% protein reduction in the targeted proto-oncogene polo-like kinase 1 (PLK1). This was sufficient to suppress tumor growth and significantly improved the median survival time of mice bearing orthotopic glioblastoma to 40 days, while inducing negligible side or off-target effects. These results suggest that the developed brain-targeted CRISPR/Cas9 based nanomedicine shows promise for effective human glioblastoma gene therapy.

Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.

Cation-Free siRNA Micelles as Effective Drug Delivery Platform and Potent RNAi Nanomedicines for Glioblastoma Therapy.

Nanoparticle-based small interfering RNA (siRNA) therapy shows great promise for glioblastoma (GBM). However, charge associated toxicity and limited blood-brain-barrier (BBB) penetration remain significant challenges for siRNA delivery for GBM therapy. Herein, novel cation-free siRNA micelles, prepared by the self-assembly of siRNA-disulfide-poly(N-isopropylacrylamide) (siRNA-SS-PNIPAM) diblock copolymers, are prepared. The siRNA micelles not only display enhanced blood circulation time, superior cell take-up, and effective at-site siRNA release, but also achieve potent BBB penetration. Moreover, due to being non-cationic, these siRNA micelles exert no charge-associated toxicity. Notably, these desirable properties of this novel RNA interfering (RNAi) nanomedicine result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. Moreover, as a novel type of polymeric micelle, the siRNA micelle displays effective drug loading ability. When utilizing temozolomide (TMZ) as a model loading drug, the siRNA micelle realizes effective synergistic therapy effect via targeting the key gene (signal transducers and activators of transcription 3, STAT3) in TMZ drug resistant pathways. The authors' results show that this siRNA micelle nanoparticle can serve as a robust and versatile drug codelivery platform, and RNAi nanomedicine and for effective GBM treatment.

Polymeric Nanoparticles for Mitochondria Targeting Mediated Robust Cancer Therapy.

Despite all sorts of innovations in medical researches over the past decades, cancer remains a major threat to human health. Mitochondria are essential organelles in eukaryotic cells, and their dysfunctions contribute to numerous diseases including cancers. Mitochondria-targeted cancer therapy, which specifically delivers drugs into the mitochondria, is a promising strategy for enhancing anticancer treatment efficiency. However, owing to their special double-layered membrane system and highly negative potentials, mitochondria remain a challenging target for therapeutic agents to reach and access. Polymeric nanoparticles exceed in cancer therapy ascribed to their unique features including ideal biocompatibility, readily design and synthesis, as well as flexible ligand decoration. Significant efforts have been put forward to develop mitochondria-targeted polymeric nanoparticles. In this review, we focused on the smart design of polymeric nanosystems for mitochondria targeting and summarized the current applications in improving cancer therapy.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Oocyte IVM or vitrification significantly impairs DNA methylation patterns in blastocysts as analysed by single-cell whole-genome methylation sequencing.

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1.

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.

ROS-Responsive Polymeric siRNA Nanomedicine Stabilized by Triple Interactions for the Robust Glioblastoma Combinational RNAi Therapy.

Small interfering RNA (siRNA) holds inherent advantages and great potential for treating refractory diseases. However, lack of suitable siRNA delivery systems that demonstrate excellent circulation stability and effective at-site delivery ability is currently impeding siRNA therapeutic performance. Here, a polymeric siRNA nanomedicine (3I-NM@siRNA) stabilized by triple interactions (electrostatic, hydrogen bond, and hydrophobic) is constructed. Incorporating extra hydrogen and hydrophobic interactions significantly improves the physiological stability compared to an siRNA nanomedicine analog that solely relies on the electrostatic interaction for stability. The developed 3I-NM@siRNA nanomedicine demonstrates effective at-site siRNA release resulting from tumoral reactive oxygen species (ROS)-triggered sequential destabilization. Furthermore, the utility of 3I-NM@siRNA for treating glioblastoma (GBM) by functionalizing 3I-NM@siRNA nanomedicine with angiopep-2 peptide is enhanced. The targeted Ang-3I-NM@siRNA exhibits superb blood-brain barrier penetration and potent tumor accumulation. Moreover, by cotargeting polo-like kinase 1 and vascular endothelial growth factor receptor-2, Ang-3I-NM@siRNA shows effective suppression of tumor growth and significantly improved survival time of nude mice bearing orthotopic GBM brain tumors. New siRNA nanomedicines featuring triple-interaction stabilization together with inbuilt self-destruct delivery ability provide a robust and potent platform for targeted GBM siRNA therapy, which may have utility for RNA interference therapy of other tumors or brain diseases.

Bioreducible Zinc(II)-Dipicolylamine Functionalized Hyaluronic Acid Mediates Safe siRNA Delivery and Effective Glioblastoma RNAi Therapy.

RNA interference (RNAi) is an emerging therapeutic modality for tumors. However, lack of a safe and efficient small interfering RNA (siRNA) delivery system limits its clinical application. Here, we report a bioreducible and less-cationic siRNA delivery carrier by conjugating Zn(II)-dipicolylamine complexes (Zn-DPA) onto hyaluronic acid (HA) via a redox-sensitive disulfide (-SS-) linker. Such polymer conjugates can formulate stable siRNA nanomedicines via coordination between zinc ions of DPA and the anionic phosphate of siRNA. After the conjugates are taken up by cells, intracellular reduction stimulus subsequently triggers the release of siRNAs and elucidates the desired RNAi effect. Our studies showed the formulated siRNA nanomedicines can be efficiently delivered into tumor cells/tissues and mediates less cytotoxicities both in vitro and in vivo. More importantly, when applied in a xenograft glioblastoma tumor model, this siRNA nanomedicine demonstrated significantly enhanced antitumor ability comparing to naked siRNA. This work demonstrates that such bioreducible Zn-DPA-functionalized HA conjugates without using cationic material as a siRNA carrier represents a promising direction for RNAi-based cancer therapy.

Stilbenes from Veratrum maackii Regel Protect against Ethanol-Induced DNA Damage in Mouse Cerebellum and Cerebral Cortex.

Ethanol is a principle ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol-associated oxidative DNA damage in the central nervous system is well documented. Natural source compounds may offer new options to protect the brain against ethanol-induced genotoxicity. Veratrum maackii Regel is a toxic rangeland plant linked to teratogenicity which is also used in traditional Chinese medicine as "Lilu" and is reported to contain a family of compounds called stilbenes that can have positive biological activity. In this study, nine stilbenes were isolated from the aerial parts of V. maackii Regel, and their structures were identified as cis-mulberroside A (1), resveratrol-4,3'- O-ß-d-diglucopyranoside (2), mulberroside A (3), gentifolin K (4), resveratrol-3,5- O-ß-d-diglucopyranoside (5), oxyresveratrol- 4'- O-ß-d-glucopyranoside (6), oxyresveratrol-3- O-ß-d-glucopyranoside (7), oxyresveratrol (8), and resveratrol (9) using ESI-MS and NMR techniques. The total concentration of extracted compounds 2-9 was 2.04 mg/g, suggesting that V. maackii Regel is a novel viable source of these compounds. In an in vivo comet assay, compounds 1-9 were observed to decrease DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration. Histological observation also revealed decreased brain injury in mice administered with compounds 1-9 after acute ethanol administration. The protective effects of compound 6 were associated with increasing T-SOD and GSH-PX activities and a decrease in NO and MDA concentrations. These findings suggest that these compounds are potent inhibitors of ethanol-induced brain injury possibly via the inhibition of oxidative stress and may be valuable leads for future therapeutic development.

DNA nanoclew templated spherical nucleic acids for siRNA delivery.

A superior biocompatible spherical nucleic acid (SNA) conjugate was fabricated by grafting siRNA onto the surface of a core composed of a spherical DNA nanostructure that we have termed a DNA nanoclew (DC). After uptake by cultured cancer cells, SNA nanoparticles release engrafted siRNAs by cleavage of the intracellular Dicer enzyme. Moreover, in vitro experiments reveal that such SNAs demonstrate potent gene knockdown at both mRNA and protein levels, while with negligible cytotoxicity.

Chemical template-assisted synthesis of monodisperse rattle-type Fe3O4@C hollow microspheres as drug carrier.

A chemical template strategy was put forward to synthesize monodisperse rattle-type magnetic carbon (Fe3O4@C) hollow microspheres. During the synthesis procedure, monodisperse Fe2O3 microspheres were used as chemical template, which released Fe3+ ions in acidic solution and initiated the in-situ polymerization of pyrrole into polypyrrole (PPy) shell. With the continual acidic etching of Fe2O3 microspheres, rattle-type Fe2O3@PPy microspheres were generated with the cavity appearing between the PPy shell and left Fe2O3 core, which were then transformed into Fe3O4@C hollow microspheres through calcination in nitrogen atmosphere. Compared with traditional physical template, the shell and cavity of rattle-type hollow microspheres were generated in one step using the chemical template method, which obviously saved the complex procedures including the coating and removal of middle shells. The experimental results exhibited that the rattle-type Fe3O4@C hollow microspheres with different parameters could be regulated through controlled synthesis of the intermediate Fe2O3@PPy product. Moreover, when the rattle-type Fe3O4@C hollow microspheres were investigated as drug carrier, they manifested sustained-release behaviour of doxorubicin, justifying their promising applications as carriers in drug delivery. STATEMENT OF SIGNIFICANCE: The aim of the present study was first to synthesize rattle-type Fe3O4@C hollow microspheres through a simple synthesis method as a drug carrier. Here a chemical template synthesis of rattle-type hollow microspheres was developed, which saved the complex procedures including the coating and removal of middle shells in traditional physical template. Second, all the influence factors in the reaction processes were systematically investigated to obtain rattle-type Fe3O4@C hollow microspheres with controlled parameters. Third, the rattle-type Fe3O4@C hollow microspheres were studied as drug carriers and the influences of their structural parameters on drug loading and releasing performance were investigated.

Template-etching route to construct uniform rattle-type Fe3O4@SiO2 hollow microspheres as drug carrier.

Template-etching strategy was put forward to synthesize rattle-type magnetic silica (Fe3O4@SiO2) hollow microspheres in a controlled way. During the experiment, monodisperse Fe2O3 microspheres were fabricated as physical template to generate uniform Fe2O3@SiO2 with controlled shell thicknesses through sol-gel method, and the subsequent Fe2O3 template etching process created variable space between Fe2O3 core and SiO2 shell, and the final calcination process transformed rattle-type Fe2O3@SiO2 hollow microspheres into corresponding Fe3O4@SiO2 product in hydrogen/nitrogen atmosphere. Compared with traditional physical template, here template-etching synthesis of rattle-type hollow microspheres saved the insertion of middle shells and their removal, which simplified the synthesis process with controllable core size and shell thickness. The rattle-type Fe3O4@SiO2 hollow microspheres as drug carrier show efficient doxorubicin (DOX) loading, and the release rate of DOX loaded the rattle-type Fe3O4@SiO2 hollow microspheres exhibit a surprising shell-thickness-dependent and a pH responsive drug release features. Additionally, MTT assays in HeLa cells demonstrated that the Fe3O4@SiO2 nanocarriers were non-toxic even at the concentration of 250µgmL-1 for 48h. Thus, our results revealed that the Fe3O4@SiO2-DOX could play an important role in the development of intracellular delivery nanodevices for cancer therapy.

Transgenesis of Tol2-mediated seamlessly constructed BAC mammary gland expression vectors in Mus musculus.

Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300 kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100 kb of sequence from the BAC 5' end (173 kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.

Effect of trichostatin a on SGC-7901 gastric cancer cells.

AIM: To explore Trichostatin A (TSA) effect on SGC-7901 gastric cancer cells. METHODS: MTT, fluorescence microscopy, and flow cytometry were used to assess TSA effect on cell growth and apoptosis in SGC-7901. Immunocytochemistry was used to evaluate the expression of acetylated histone H4 in SGC-7901 cells.Gene expression profile was determined by microarray assays. Glycoprotein hormones alpha subunit (CGA) gene and protein expressions in SGC-7901 cells were evaluated by Real-time PCR and Western blot, respectively. In addition, CGA protein levels in gastric adenocarcinoma and normal adjacent tissues were assessed by immunohistochemistry. RESULTS: TSA inhibited SGC-7901 cell growth. In addition, cell proliferation was significantly decreased (P = 0.02) in TSA treatment groups (0.93 ± 0.07) compared with controls (1.15 ± 0.07). Apoptosis related morphological changes, including nuclear chromatin condensation and fluorescence strength, were observed by fluorescence microscopy. These findings corroborated the increased expression of acetylated histone H4 observed in TSA treated cells compared to controls, as determined by immunocytochemistry. Interestingly, treatment of SGC-7901 cells with TSA (75 ng/ml) resulted in CGA gene down-regulation (P = 0.0381). Accordingly, CGA protein levels were decreased in TSA treated SGC-7901 cells. Finally, immunohistochemistry analysis showed that CGA expression was significantly higher in gastric adenocarcinoma tissues than normal adjacent tissues (P = 0.001). CONCLUSION: TSA induces cell apoptosis and increases the levels of acetylated histone H4 in SGC-7901 cells. In addition, TSA treatment decreases the expression in gastric cancer cells of the CGA gene, which is upregulated in gastric adenocarcinoma tissues.

Differential expression of glycoprotein non-metastatic melanoma protein B (GPNMB) involved in trichostatin A-induced apoptosis in gastric cancer.

In this study, we investigated the effect of trichostatin A (TSA) on the gastric cancer cell line BGC-823. The effect of TSA on growth inhibition and apoptosis of BGC-823 cells was examined. The gene expression profile was determined by microarray. Western blotting was used to study the levels of acetylated histone H4 and Glycoprotein non-metastatic melanoma protein B (GPNMB) proteins. GPNMB gene expression was measured by real-time PCR. GPNMB protein levels in gastric adenocarcinoma tissues and adjoining normal tissues were detected by immunohistochemistry. The results showed that a significant decrease in cell population following treatment with 75 ng/mL TSA for 48 h (0.87 ± 0.04) as compared to control (1.14 ± 0.06) (P = 0.02). Apoptotic cells were increased in TSA (75 ng/mL for 48 h) treated group as compared to the control group (from 2.02% to 19.74%) by flow cytometry. The expression of acetylated histone H4 was increased in TSA treated (75 ng/mL for 48 h) group (from 1.00 ± 0.26 to 1.87 ± 0.33, F = 5.862, P = 0.0038) as compared to the control group by Western blotting. After 48 h TSA treatment (75 ng/mL), BGC-823 cells showed decrease in GPNMB gene expression (from 1.00 ± 0.21 to 0.59 ± 0.11, F = 6.214, P = 0.0018). Immunohistochemistry showed that GPNMB expression in gastric adenocarcinoma was significantly higher than the adjoining normal tissues (P = 0.000). To conclusion, our results support that TSA can induce apoptosis, and increase acetylated histone H4 in BGC-823 cells. GPNMB expression is decreased in BGC-823 cells after TSA treatment. GPNMB is overexpressed in gastric adenocarcinoma tissue. GPNMB involved in TSA-induced apoptosis might participate in gastric cancer.

Telomere reprogramming and maintenance in porcine iPS cells.

Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

Higher methylation in genomic DNA indicates incomplete reprogramming in induced pluripotent stem cells.

Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.