Despite the recent advances in single molecule manipulation techniques, purely mechanical approaches can not detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence which allowed us to examine the effect of sub-pN forces on the nanometer scale motion of the Holliday junction (HJ) at 100 Hz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.Many biological processes are dependent on tension. In recent years, single molecule force measurements have shown directly that biochemical reactions can be influenced by applied force (1). Yet, purely mechanical tools can not detect small scale conformational changes unless strong enough and persistent force is applied. At weak forces, the flexible tether connecting the mechanical probe to the biological molecule is not stretched enough to transmit small movements. This is unfortunate because weak and transient forces are likely more prevalent in vivo but the experimental limitations confine single molecule mechanical studies to examining the effect of relatively large forces. We aimed to study the effect of weak external forces on the biomolecular conformational dynamics by combining single molecule fluorescence resonance energy transfer (smFRET) (2-4) with manipulation using optical trap (5). smFRET has high spatial resolution (≤ 5 Å) (6, 7)) and can be measured at arbitrarily low forces. Previous attempts to combine FRET and optical trap using the DNA hairpin as a model system (8, 9) did not reveal new information because the hairpin unzips at high forces (~ 15 pico Newton (pN)), a regime that had been extensively investigated using force-based techniques (10, 11). Here, we report an approach to detect nanometer-* To whom correspondence should be addressed. (tjha@uiuc.edu). † Present Address: Department of Physics and Astronomy, Seoul National University, Seoul 151-742, Korea. scale motion at sub-pN forces. We used the approach to gain insight into the reaction landscape of the Holliday junction (HJ) by gently stretching it along different directions. NIH Public AccessThe HJ is a four-stranded DNA structure that forms as an intermediate during recombination (12). To understand the mechanisms of cellular enzymes that function with the HJ, a detailed description of the static and dynamic structural properties of the HJ itself is needed. In the absence of added ions the HJ adopts an open structure, where the four helical arms point toward the corners of a square (13, 14) (Fig. 1A). In the presence ...

U nlike most mature cells, smooth muscle cells (SMCs) are remarkably plastic and can dedifferentiate in response to environmental cues, 1,2 adding a layer of complexity to the regulation of gene expression. Although several transcription factors have been identified, a global mechanism that coordinately regulates SMC phenotype has yet to be uncovered. How SMC genes become silenced and then reactivated is unknown and is an area of intense investigation. Recent demonstration that the ten-eleven-translocation (TET) family of proteins is involved in DNA demethylation [3][4][5] prompted us to evaluate the role of the TET proteins in the modulation of SMC phenotype. Editorial see p 2002 Clinical Perspective on p 2057The TET proteins (TET1-TET3) are a recently discovered family of DNA demethylases. TET proteins oxidize 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), frequently called the sixth DNA base, in mammalian cells. 4,5 Through the base excision repair pathway, 5-hmC is then converted to unmethylated cytosine, leading to DNA demethylation and gene activation. [6][7][8] Therefore, the 5-hmC modification and the TET enzymes have emerged as key activators of gene expression. Studies of TET proteins and 5-hmC function in embryonic stem cells (ESCs) demonstrate that they play a major role in maintaining cellular pluripotency through the regulation of lineage-specific genes. 4,[9][10][11] In contrast to this role in ESC pluripotency, the TET proteins (and their 5-hmC products) have an opposing role in adult stem cells and somatic tissues. TET2 mutations have been described in several types of hematopoietic disorders in which the loss of TET2 has been shown to promote hematopoietic stem cell self-renewal.12 TET2 and 5-hmC levels are increased during neurogenesis, 13 and more recently, loss of TET2 and 5-hmC was demonstrated to be a key epigenetic event associated with Background-Smooth muscle cells (SMCs) are remarkably plastic. Their reversible differentiation is required for growth and wound healing but also contributes to pathologies such as atherosclerosis and restenosis. Although key regulators of the SMC phenotype, including myocardin (MYOCD) and KLF4, have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported. Methods and Results-Using human SMCs, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA-modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its product, 5-hydroxymethylcytosine (5-hmC), are enriched in contractile SMCs but reduced in dedifferentiated SMCs. TET2 knockdown inhibits expression of key procontractile genes, including MYOCD and SRF, with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, whereas TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regul...

Recent research has found that long noncoding RNAs (lncRNAs) were involved in various human cancers. However, the role of these lncRNAs in cervical cancer remains unexplored. Therefore, we aimed to investigate the biological function of maternally expressed gene 3 (MEG3), a cancer-related lncRNA, and its underlying mechanism in cervical cancer. In this study, MEG3 expression of 108 patients' cervical cancer tissues and adjacent normal tissues was detected by quantitative real-time PCR analysis (qRT-PCR), and the functional effect of MEG3 was determined in vitro assays. We observed that MEG3 was downregulated in cervical cancer tissues, compared to the adjacent normal tissues, and was negatively related with FIGO stages, tumor size, lymphatic metastasis, HR-HPV infection and the expression of homo sapiens microRNA-21 (miR-21). Furthermore, we focused on the function and molecular mechanism of MEG3, finding that overexpression of MEG3 reduced the level of miR-21-5p expression, causing inhibition of proliferation and increased apoptosis in cervical cancer cells. In summary, our findings indicate that MEG3 function as a tumor suppressor by regulating miR-21-5p, resulting in the inhibition of tumor growth in cervical cancer. As a result, this study improves our understanding of the function of MEG3 in cervical cancer and will help to provide new potential target sites for cervical cancer treatment.Abbreviations: lncRNA, long noncoding RNA; MEG3, maternally expressed gene 3; qRT-PCR, quantitative real-time PCR analysis; miR-21, homo sapiens microRNA-21; ncRNA, noncoding RNA; NSCLC, nonsmall cell lung cancer; miRNA, MicroRNA; PDCD4, programmed cell death 4; CASC2, cancer susceptibility candidate 2; GAS5, growth arrest specific 5; LVSI, Lymphatic vascular space invasion; MDM2, mouse double minute 2 homolog; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; CCK-8, Cell Counting Kit-8.

Resveratrol is a naturally occurring polyphenol that exhibits pleiotropic health beneficial effects, including anti-inflammatory, cardio-protective, and cancer-protective activities. It is recognized as one of the more promising natural molecules in the prevention and treatment of chronic inflammatory and autoimmune disorders. Ulcerative colitis is an idiopathic, chronic inflammatory disease of the colon associated with a high colon cancer risk. Here, we used a dextran sulfate sodium (DSS) mouse model of colitis, which resembles human ulcerative colitis pathology. Resveratrol mixed in food ameliorates DSS-induced colitis in mice in a dose-dependent manner. Resveratrol significantly improves inflammation score, downregulates the percentage of neutrophils in the mesenteric lymph nodes and lamina propria, and modulates CD3 + T cells that express tumor necrosis factor-α and IFN-γ. Markers of inflammation and inflammatory stress (p53 and p53-phospho-Ser 15) are also downregulated by resveratrol. Because chronic colitis drives colon cancer risk, we carried out experiments to determine the chemopreventive properties of resveratrol. Tumor incidence is reduced from 80% in mice treated with azoxymethane (AOM) + DSS to 20% in mice treated with AOM + DSS + resveratrol (300 ppm). Tumor multiplicity also decreased with resveratrol treatment. AOM + DSS-treated mice had 2.4 ± 0.7 tumors per animal compared with AOM + DSS + 300 ppm resveratrol, which had 0.2 ± 0.13 tumors per animal. The current study indicates that resveratrol is a useful, nontoxic complementary and alternative strategy to abate colitis and potentially colon cancer associated with colitis. Cancer Prev Res; 3(4); 549-59. ©2010 AACR.

While substrate permeation through monomeric pores of aquaporins is well characterized, little is known about the possible tetrameric pore. AQP1 has been suggested to function as an ion channel upon cGMP activation, although this idea has been controversial. Taking a theoretical and experimental approach, we demonstrate that the current might arise through the tetrameric pore and propose a plausible mechanism for conduction and gating. In response to simulated ion permeation, immediate hydration of the putative central pore was facilitated by moderate conformational changes of pore-lining residues. cGMP is found to interact with an unusually arginine-rich, cytoplasmic loop (loop D) facilitating its outward motion, which is hypothesized to trigger the opening of a cytoplasmic gate. Physiological analyses of wild-type AQP1 and a designed mutant in which two arginines of the gating loop are replaced by alanine provide experimental support for identifying a key component of the proposed mechanism.

Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3'-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3'-end of the HBx is often deleted. HBx-human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.

Novartis Pharmaceuticals Corporation.

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