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PDF file - 52K, Supplementary materials includes statistical analysis, antibodies and reagent information, supplementary references, and supplementary figure legend

PDF file - 36K, Specificity of qPCR primers for monitoring miR-103 and miR-107 expression, and effect of miR-103/107 on the expression of DAPK and KLF4 mRNAs

PDF file - 28K, Reporter analysis for examining the efficacies of miR-103 and miR-107 and their antagomirs

PDF file - 190K, miR-103/107 suppress the expression of epithelial markers but cannot induce EMT in CRC cells

PDF file - 51K, Re-expression of DAPK and KLF4 suppresses miR-103/107-induced migration and invasion in normoxia

PDF file - 91K, Supplementary Tables include miR-103/107 Expression Status with relation to tumor stage, as well as Multivariate Analysis

Sudden cardiac death (SCD) is an important cause of mortality worldwide. It accounts for approximately half of all deaths from cardiovascular disease. While coronary artery disease and acute myocardial infarction account for the majority of SCD in the elderly population, inherited cardiac diseases (inherited CDs) comprise a substantial proportion of younger SCD victims with a significant genetic component. Currently, the use of next-generation sequencing enables the rapid analysis to investigate relationships between genetic variants and inherited CDs causing SCD. Genetic contribution to risk has been considered an alternate predictor of SCD. In the past years, large numbers of SCD susceptibility variants were reported, but these results are scattered in numerous publications. Here, we present the SCD-associated Variants Annotation Database (SVAD) to facilitate the interpretation of variants and to meet the needs of data integration. SVAD contains data from a broad screening of scientific literature. It was constructed to provide a comprehensive collection of genetic variants along with integrated information regarding their effects. At present, SVAD has accumulated 2,292 entries within 1,239 variants by manually surveying pertinent literature, and approximately one-third of the collected variants are pathogenic/likely-pathogenic following the ACMG guidelines. To the best of our knowledge, SVAD is the most comprehensive database that can provide integrated information on the associated variants in various types of inherited CDs. SVAD represents a valuable source of variant information based on scientific literature and benefits clinicians and researchers, and it is now available on http://svad.mbc.nctu.edu.tw/.

Pathway enrichment analysis. (PDF 235Â kb)

Despite a general repression of translation under hypoxia, cells selectively upregulate a set of hypoxia-inducible genes. Results from deep sequencing revealed that Let-7 and miR-103/107 are hypoxia-responsive microRNAs (HRMs) that are strongly induced in vascular endothelial cells. In silico bioinformatics and in vitro validation showed that these HRMs are induced by HIF1α and target argonaute 1 (AGO1), which anchors the microRNA-induced silencing complex (miRISC). HRM targeting of AGO1 resulted in the translational desuppression of VEGF mRNA. Inhibition of HRM or overexpression of AGO1 without the 3' untranslated region decreased hypoxia-induced angiogenesis. Conversely, AGO1 knockdown increased angiogenesis under normoxia in vivo. In addition, data from tumor xenografts and human cancer specimens indicate that AGO1-mediated translational desuppression of VEGF may be associated with tumor angiogenesis and poor prognosis. These findings provide evidence for an angiogenic pathway involving HRMs that target AGO1 and suggest that this pathway may be a suitable target for anti- or proangiogenesis strategies.

Background— Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow–regulated KLF2. Methods and Results— Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3′ untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow. Conclusions— Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

Structural properties of single lambda DNA treated with anti-cancer drug cisplatin were studied with magnetic tweezers and AFM. Under the effect of low-concentration cisplatin, the DNA became more flexible, with the persistence length decreased significantly from approximately 52 to 15 nm. At a high drug concentration, a DNA condensation phenomenon was observed. Based on experimental results from both single-molecule and AFM studies, we propose a model to explain this kind of DNA condensation by cisplatin: first, di-adducts induce local distortions of DNA. Next, micro-loops of approximately 20 nm appear through distant crosslinks. Then, large aggregates are formed through further crosslinks. Finally, DNA is condensed into a compact globule. Experiments with Pt(dach)Cl(2) indicate that oxaliplatin may modify the DNA structures in the same way as cisplatin. The observed loop structure formation of DNA may be an important feature of the effect of platinum anti-cancer drugs that are analogous to cisplatin in structure.

Eleven ionic species and fine and coarse particle mass concentrations of fine (PM2.5) and coarse (PM10-2.5) particulate matter were investigated in Kaohsiung, southern Taiwan. The PM2.5 and PM10-2.5 particulate concentrations were 49-64 and 34-37 microg x m(-3), respectively. Fifty-five to 64% of the particulate matter less than 10 microm in aerodynamic diameter (PM10) mass was attributed to the PM2.5. PM2.5 concentrations at Daliao (a rural and industrial complex area) were higher than at Tzuoying (an urban and industrial complex area). Ionic species contributed 45-53% and 42-45% of PM2.5 and PM10, respectively. Potassium ions (K+), sulfate (SO4(2-)), and ammonium (NH4+) were predominant in PM2.5, whereas sodium, calcium, and magnesium ions were foremost in PM10-2.5. Nitrate (NO3-) existed in both the PM2.5 and PM10-2.5. Chloride (Cl-), NO3-, and NH4+ concentrations were higher at night than during the day, and they were easily transferred into the gas phase by photochemical reactions and temperature-induced volatilization. The NH4+/SO4(2-) ratios were 2.6 and 2.5 at Daliao and Tzuoying, respectively, which indicated that both sampling sites were rich in NH4+. Therefore, ammonium nitrate would be present in the area.

In 1998, the U.S. Environmental Protection Agency (EPA) initiated a major air quality program known as the Particulate Matter (PM) Supersites Program. The Supersites Program was a multiyear, $27 million air quality monitoring program consisting of eight regional air quality projects located throughout the United States, each with differing atmospheric pollution conditions resulting from variations in source emissions and meteorology. The overall goal of the program was to elucidate source-receptor relationships and atmospheric processes leading to PM accumulation on urban and regional scales; thus providing the scientific underpinning for modeling and data analysis efforts to support State Implementation Plans and more effective risk management approaches for PM. The program had three main objectives: (1) conduct methods development and evaluation, (2) characterize ambient PM, and (3) support health effects and exposure research. This paper provides a synthesis of key scientific findings from the Supersites Program and related studies. EPA developed 16 science/policy-relevant questions in conjunction with state and other federal agencies, Regional Planning Organizations, and the private sector. These questions were addressed to the extent possible, even given the vast amount of new information available from the Supersites Program, in a series of papers published as a special issue of the Journal of Air & Waste Management Association (February 2008). This synthesis also includes discussions of: (1) initial Supersites Program support for air quality management efforts in specific locations throughout the United States; (2) selected policy-relevant insights, based on atmospheric sciences findings, useful to air quality managers and decision makers planning emissions management strategies to address current and future PM National Ambient Air Quality Standards (NAAQS) and network planning and implementation; (3) selected health-relevant insights interpreted from atmospheric sciences findings in light of future directions for health and exposure scientists planning studies of the effects of PM on human health; and (4) selected knowledge gaps to guide future research. Finally, given the scope and depth of research and findings from the Supersites Program, this paper provides a reference source so readers can glean a general understanding of the overall research conducted and its policy-relevant insights. Supporting details for the results presented are available through the cited references. An annotated table of contents allows readers to easily find specific subject matter within the text.

A mathematical model is proposed to illustrate the activation of STIM1 (stromal interaction molecule 1) protein, the assembly and activation of calcium-release activated calcium (CRAC) channels in T cells. In combination with De Young-Keizer-Li-Rinzel model, we successfully reproduce a sustained Ca(2+) oscillation in cytoplasm. Our results reveal that Ca(2+) oscillation dynamics in cytoplasm can be significantly affected by the way how the Orai1 CRAC channel are assembled and activated. A low sustained Ca(2+) influx is observed through the CRAC channels across the plasma membrane. In particular, our model shows that a tetrameric channel complex can effectively regulate the total quantity of the channels and the ratio of the active channels to the total channels, and a period of Ca(2+) oscillation about 29 s is in agreement with published experimental data. The bifurcation analyses illustrate the different dynamic properties between our mixed Ca(2+) feedback model and the single positive or negative feedback models.

The compaction of single DNA molecules by purified histones is studied using magnetic tweezers. The compaction rate increases rapidly when the histone concentration is increased from 0.002 to 0.2 mmol/L, and saturates when the concentration is beyond 0.2 mmol/L. The time course of compaction is exponential at low histone concentrations. It becomes sigmoidal at high concentrations. Cooperativity between the histones bound to DNA is proposed to be responsible for the transition. The histones are loaded onto DNA randomly at low concentrations. They tend to bind DNA cooperatively at high concentrations because the structural torsions of DNA induced by the bound histones become overlapping so that the binding of one histone facilitates the binding of others. Under very large forces, the compacted histone-DNA complex can be disrupted in a discrete manner with a step size of ∼60 nm. But the histones cannot be completely stripped off DNA, as is revealed by the lowered B-S transition plateau of the histone-bound DNA.

Coarse and fine particulate matter (PM) were taken by a dichotomous sampler, and gas precursors were determined by a denuder sampler at two stations in central Taiwan. Water-soluble ionic constituents of PM and their precursor gases were analyzed by ionic chromatograph. In summer, the daytime/nighttime PM10 concentrations were 37 ± 10/41 ± 18 μg m−3 and 36 ± 14/34 ± 18 μg m−3 for Xitun and Jhushan, respectively. Average PM10 concentration in winter was 1.55 and 1.76 times that of summer for Xitun and Jhushan, respectively. PM mass concentrations were similar for both stations, although one station is located in the downtown area of Taichung, and the other is in a rural area with no heavy pollution sources. Water-soluble ionic species content was 38–53 % of PM2.5 and 43–48 % of PM10 mass concentration. HNO3, HCl, and SO2 were high in the daytime; the daytime-to-nighttime concentration ratio was 3.75–6.88 for HNO3,1.7–7.8 for HCl, and 1.45–2.77 for SO2. High NH3 levels were determined in the area, especially in winter, which could be a precursor of NH4 + to form particulate matter. In Xitun, motor vehicles downtown and in the industrial district could be sources of air pollution. In contrast, there are few industrial sources at Jhushan; therefore, the transport of air pollutants from upwind of other regions and the accumulation of pollutants could be important PM sources at Jhushan.

The effects of monovalent (Na+, K+) and divalent (Mg2+, Ca2+, Mn2+) ions on the interaction between DNA and histone are studied using the molecular combing technique. Lamda-DNA molecules and DNA-histone complexes incubated with metal cations (Na+, K+, Mg2+, Ca2+, Mn2+) are stretched on hydrophobic surfaces, and directly observed by fluorescence microscopy. The results indicate that when these cations are added into the DNA solution, the fluorescence intensities of the stained DNA are reduced differently. The monovalent cations (Na+, K+) inhibit binding of histone to DNA. The divalent cations (Mg2+, Ca2+, Mn2+) enhance significantly the binding of histone to DNA and the binding of the DNA-histone complex to the hydrophobic surface. Mn2+ also induces condensation and aggregation of the DNA-histone complex., Comment: 11 pages, 7 figures

RecQ helicase–DNA interactions were directly visualized with fluorescence microscopy. DNA–RecQ complexes formed in binding and unwinding reaction were stretched onto the hydrophobic surface by molecular combing method. The complexes can be observed with fluorescence microscope because the DNA molecules were labeled with dye molecules of YOYO-1. The DNA binding and unwinding activity of RecQ helicase leads to reduced lengths of the observed DNA molecules. More direct observations with atomic force microscopy were also made. It was seen that RecQ is mainly monomeric both in solution and after binding to DNA.

Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses and mediating important proinflammatory functions in asthma. This cytokine exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high-affinity binding subunit. Soluble IL-4R (sIL-4R) lacks the transmembrane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralizing its activity, the high specificity and affinity of sIL-4R make it ideal as an IL-4 antagonist. In this study, a sIL-4R cDNA encoding the extracellular domain of IL-4R alpha chain was cloned into a Streptomyces-Escherichia coli shuttle plasmid pSGLgpp and expressed secretly in Streptomyces lividans TK 24. On SDS-PAGE gel, the expressed sIL-4R protein showed a Mw of 24 kDa, agreeable with the predicted size. The N-terminal sequence of the protein was also determined, confirming its identity and indicating that no degradation occurred at the N-terminus. With DEAE-Sepharose Fast Flow and Superdex HR 75 columns, the protein was purified and used on HPLC analysis, the purity reaching about 90%. The results of the ligand-binding blot and ELISA showed that such protein has biological activity of binding with ligand IL-4.

Ohmic and microwave heating were employed to investigate electro-thermal effects on apple juice yield by preheating apple samples to 40 or 50 °C. Cylindrical raw and pretreated samples from fresh apple were wrapped in a non-absorbent cloth and pressed to produce juice. Data on juice yield and applied force were recorded and compared with the raw material. The results showed that apple juice yield was strongly improved by both thermal pretreatments, and pretreated samples showed less resistance during extraction. The thermal effect of ohmic pretreatment on juice yield increased with the pretreatment temperature, while for microwave pretreatment, the low pretreatment temperature resulted in high juice yield. Ohmic heating as a pretreatment increased juice yield and required less input work, compared to microwave heating; thus frequency might play a major role in this effect. The thermal effect difference could be due to increased migration of cellular moisture during ohmic pretreatment. These observations indicate ohmic pretreatment has the potential to enhance juice yield and save energy.

The crowded intracellular environment influences the diffusion-mediated cellular processes, such as metabolism, signaling, and transport. The hindered diffusion of macromolecules in heterogeneous cytoplasm has been studied over years, but the detailed diffusion distribution and its origin still remain unclear. Here, we introduce a novel method to map rapidly the diffusion distribution in single cells based on single-particle tracking (SPT) of quantum dots (QDs). The diffusion map reveals the heterogeneous intracellular environment and, more importantly, an unreported compartmentalization of QD diffusions in cytoplasm. Simultaneous observations of QD motion and green fluorescent protein-tagged endoplasmic reticulum (ER) dynamics provide direct evidence that the compartmentalization results from micron-scale domains defined by ER tubules, and ER cisternae form perinuclear areas that restrict QDs to enter. The same phenomenon was observed using fluorescein isothiocyanate-dextrans, further confirming the compartmentalized diffusion. These results shed new light on the diffusive movements of macromolecules in the cell, and the mapping of intracellular diffusion distribution may be used to develop strategies for nanoparticle-based drug deliveries and therapeutics.

Eighty-one stool samples from Taiwanese were collected for analysis of the association between the gut flora and obesity. The supervised analysis showed that the most, abundant genera of bacteria in normal samples (from people with a body mass index (BMI)≤24) wereBacteroides(27.7%),Prevotella(19.4%),Escherichia(12%),Phascolarctobacterium(3.9%), andEubacterium(3.5%). The most abundant genera of bacteria in case samples (with a BMI≥27) wereBacteroides(29%),Prevotella(21%),Escherichia(7.4%),Megamonas(5.1%), andPhascolarctobacterium(3.8%). A principal coordinate analysis (PCoA) demonstrated that normal samples were clustered more compactly than case samples. An unsupervised analysis demonstrated that bacterial communities in the gut were clustered into two main groups: N-like and OB-like groups. Remarkably, most normal samples (78%) were clustered in the N-like group, and most case samples (81%) were clustered in the OB-like group (Fisher’sP value=1.61E-07). The results showed that bacterial communities in the gut were highly associated with obesity. This is the first study in Taiwan to investigate the association between human gut flora and obesity, and the results provide new insights into the correlation of bacteria with the rising trend in obesity.

Cylindrical samples of carrot, potato and yam were dried in a hot-air dehydrator after preheating to 50C or 80C by three different heating methods (conventional, microwave and ohmic). The results showed that enhancement of drying rate increased with pretreatment temperature. Ohmic pretreatment increased the drying rate more than conventional and microwave heating. Desorption isotherms showed that in the low a w range, desorption data of preheated and raw materials were similar. However, the isotherms of preheated samples shifted when a w was high, which indicated that thermal pretreatments altered the structure, and apparently, the water distribution within these materials. For all samples, ohmic pretreatment showed stronger influences on isotherms than microwave heating, while the pretreatment effect of conventional heating was only observed for potato tissue.

Electrical conductivity (σ) is the most important parameter in ohmic heating. Although data exist on its changes during ohmic heating, limited information is available about preheated foods. In this study, the conductivity changes of raw vegetable samples (potato, carrot, and yam) in cyclic ohmic heating and samples preheated by conventional heating prior to ohmic heating were investigated. In cyclic ohmic heating, cylindrical vegetable samples were subjected to three repeated cycles of ohmic heating (40 V/cm, 60Hz) to 80C, and cooling to 25C. Fresh samples were also preheated by conventional heating to 80C, then subjected to ohmic heating for comparison. Specific heats changed by cycles, although moisture content remained constant in all cases. The results show that in cyclic ohmic heating, the heating rate increased by cycles. Samples preheated by either conventional or ohmic heating showed a higher heating rate than raw materials. Electrical conductivity data during ohmic heating showed that preheated vegetables have higher conductivities than fresh ones, and a tendency of increase by cycles was found.

The effect of starch gelatinization on electrical conductivity (σ) was investigated in this study. Suspensions of corn and potato starch were prepared with a starch:water ratio of 1:5 (w/w) by mixing with the appropriate amount of water. Suspensions were ohmically heated with agitation to 90 °C by AC at 60 Hz, and a voltage gradient of 20 V/cm. Partially gelatinized and fully gelatinized samples, prepared by preheating suspensions to or beyond the gelatinization ranges, were also tested. Gelatinization energy (ΔHG) and degree of gelatinization (%SG) of each sample were determined by differential scanning calorimetry (DSC) at a scanning rate of 10 °C/min. The results showed that endothermic gelatinization peaks were found on both DSC thermograms and electrical conductivity curves, with similar shapes and temperature ranges. Data of %SG obtained from conductivity curves and DSC thermograms were in agreement in low and mid-gelatinization ranges but were different when %SG was high, due to high ohmic heating rate. Electrical conductivity increased with temperature, but decreased with degree of gelatinization, apparently caused by structural changes and an increase in bound water.

Background— The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2). Methods and Results— Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell–specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas. Conclusions— Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.

Cisplatin is the main platinum antitumor drug applied in clinical settings. However, its trans isomer, transplatin, is known to have an ineffective antitumor activity. Despite intensive studies in this field, the structural and biophysical properties of DNA molecules reacting with these two platinum complexes have not been fully elucidated. In the present study, we observed that transplatin made efficient cross-linking of DNA in the vicinity of cisplatin adducts. High-resolution atomic force microscopy studies revealed that the transplatin-induced cross-linkings of nucleotides flanking cisplatin adducts were characterized by kinked-loop structures with rod-like shapes of nanometer scales (similar to 10-60 nm). The results were further confirmed by denaturing gel electrophoresis and single-molecule experiment using magnetic tweezers. In vivo studies revealed that transplatin and cisplatin co-treatment could induce a considerable amount of kinked loops with smaller sizes (similar to 15 nm) in cellular DNA. Furthermore, compared with cisplatin treatment alone, the co-treatment resulted in enhanced cytotoxicity, increased amount of interstrand cross-links, and cell lesions more reluctant to cellular repair system. The results of the present study provide a new clue for understanding the stepwise reactions of DNA with platinum drugs and might serve as a basis for the development of a new antitumor strategy. (C) 2013 Elsevier Inc. All rights reserved.

Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introduced to quantitatively study the dynamics of endocytic trafficking. Compared with the control cells, the velocity of directed motion was reduced by 30% due to the suppression of high speed movements of EGF-QDs along the microtubules in PTX-treated cells. The endocytic trafficking in PTX-treated cells was mainly via super-diffusive mode of motion, whereas in control cells, it was mostly via sub-diffusive mode of motion. Moreover, PTX shortened endosomal trafficking and prevented EGF-QDs from moving to the perinuclear area via the rapid delivery of EGF-QDs into the peripheral lysosomes. The present study may shed light on the mechanism of the effect of PTX on the treatment of lung cancer.

Metastasis is the major cause of poor prognosis in colorectal cancer (CRC), and increasing evidence supports the contribution of miRNAs to cancer progression. Here, we found that high expression of miR-103 and miR-107 (miR-103/107) was associated with metastasis potential of CRC cell lines and poor prognosis in patients with CRC. We showed that miR-103/107 targeted the known metastasis suppressors death-associated protein kinase (DAPK) and Krüppel-like factor 4 (KLF4) in CRC cells, resulting in increased cell motility and cell–matrix adhesion and decreased cell–cell adhesion and epithelial marker expression. miR-103/107 expression was increased in the presence of hypoxia, thereby potentiating DAPK and KLF4 downregulation and hypoxia-induced motility and invasiveness. In mouse models of CRC, miR-103/107 overexpression potentiated local invasion and liver metastasis effects, which were suppressed by reexpression of DAPK or KLF4. miR-103/107–mediated downregulation of DAPK and KLF4 also enabled the colonization of CRC cells at a metastatic site. Clinically, the signature of a miR-103/107 high, DAPK low, and KLF4 low expression profile correlated with the extent of lymph node and distant metastasis in patients with CRC and served as a prognostic marker for metastasis recurrence and poor survival. Our findings therefore indicate that miR-103/107–mediated repression of DAPK and KLF4 promotes metastasis in CRC, and this regulatory circuit may contribute in part to hypoxia-stimulated tumor metastasis. Strategies that disrupt this regulation might be developed to block CRC metastasis. Cancer Res; 72(14); 3631–41. ©2012 AACR.

Magnetic tweezers were employed to investigate the interactions between DNA and cisplatin (transplatin). Cisplatin shortened DNA and reduced its persistence length more significantly than transplatin due to the formation of many more diadducts than those formed by transplatin. Interestingly, the presence of transplatin could enhance the ability of cisplatin in shortening DNA. An optimal concentration ratio of transplatin to cisplatin existed at which cisplatin showed the highest ability in shortening DNA. Moreover, abrupt length changes were also observed when DNA was treated with a mixture of cisplatin and transplatin at high concentrations. A model was proposed to qualitatively explain well these results.

Summary Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato ( Pst ). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNA is miR393b ∗ , which targets a Golgi-localized SNARE gene, MEMB12 . Pst infection downregulates MEMB12 in a miR393b ∗ -dependent manner. Loss of function of MEMB12 , but not SYP61 , another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein, PR1. Overexpression of miR393b ∗ resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b ∗ to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393 ∗ /miR393 represent an example of a miRNA ∗ /miRNA pair that functions in immunity through two distinct AGOs: miR393 ∗ through AGO2 and miR393 through AGO1.

Diffusion coefficients and equilibrium distribution coefficient were determined for infusion of salt solution into vegetable (potato) tissue. Samples were infused with aqueous NaCl solutions of various concentrations, and concentrations in solid and liquid were determined over time using titration methods. Equilibrium distribution coefficients were dependent on salt concentration. Values of diffusion coefficient were found to be within the ranges reported in the literature. For the conditions studied, the diffusion coefficient was unaffected by brine concentration. An analytical model for concentration profiles was found to be in good agreement with experimental results.

BACKGROUND: Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (-) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection. CONCLUSIONS/SIGNIFICANCE: The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.

Background MicroRNAs (miRNAs), small non-coding RNAs of 19 to 25 nt, play important roles in gene regulation in both animals and plants. In the last few years, the oligonucleotide microarray is one high-throughput and robust method for detecting miRNA expression. However, the approach is restricted to detecting the expression of known miRNAs. Second-generation sequencing is an inexpensive and high-throughput sequencing method. This new method is a promising tool with high sensitivity and specificity and can be used to measure the abundance of small-RNA sequences in a sample. Hence, the expression profiling of miRNAs can involve use of sequencing rather than an oligonucleotide array. Additionally, this method can be adopted to discover novel miRNAs. Results This work presents a systematic approach, miRExpress, for extracting miRNA expression profiles from sequencing reads obtained by second-generation sequencing technology. A stand-alone software package is implemented for generating miRNA expression profiles from high-throughput sequencing of RNA without the need for sequenced genomes. The software is also a database-supported, efficient and flexible tool for investigating miRNA regulation. Moreover, we demonstrate the utility of miRExpress in extracting miRNA expression profiles from two Illumina data sets constructed for the human and a plant species. Conclusion We develop miRExpress, which is a database-supported, efficient and flexible tool for detecting miRNA expression profile. The analysis of two Illumina data sets constructed from human and plant demonstrate the effectiveness of miRExpress to obtain miRNA expression profiles and show the usability in finding novel miRNAs.

We report observations of in vitro DNA compaction into toroids in the absence of any condensing agent. The DNA toroid formation is induced by geometry confinement from microdroplets on mica surfaces. With AFM imaging we show that the confined DNA molecules may take the form of random coils or semiordered folded loops with large microdroplets, while they readily take the form of compact and ordered toroids when the microdroplet sizes are small enough. To better understand these phenomena, we carried out coarse-grained Brownian dynamics simulation, obtaining results that were in good agreement with the experimental observations. The simulation reveals that the toroid formation is sensitive to not only the microdroplet size, but also the DNA stiffness.

Spraying treatments with trisodium phosphate (TSP) and cetylpyridinium chloride (CPC) were evaluated for their effectiveness in reducing Salmonella typhimurium attached to chicken skins. Chicken skins with an area of 38.5 cm 2 were cut from the breast areas of pre-chill chicken carcasses, mounted in a plastic holder, and inoculated with S. typhimurium. The inoculated skins were sprayed with tap water, 10% (wt/vol) TSP, or 0,1% CPC solutions at 10, 35, or 60°C and 206.8, 413.7, 620.5, 827.4, or 1034.2 kPa for 30 s. After spraying, each skin was rinsed with tap water, transferred to a plastic bag containing 50 ml buffered peptone water, and stomached for I min. The stomaching water was collected, diluted serially, plated on xylose lysine tergitol 4 (XLT4) agar and Petrifilm aerobic count plates, and incubated for 18 to 24 h at 37°C. The results showed that tap water spraying reduced S. typhimurium by 0.7 to 1.6 log, while the reduction ranges for TSP and CPC spraying treatments were 1.6 to 2.3 and 1.5 to 2.5 log, respectively. Greater reductions in the numbers of S. typhimurium were obtained in TSP spraying treatments in the high pressure range (620.5 to 1034.2 kPa) and in CPC spraying treatments at 10°C.

To investigate the morbidity of congenital hearing impairment among high-risk newborns and the relevant pathogenic factors.Initial screening of hearing by otoacoustic emission (OAE) was conducted among 208 high-risk newborns, 130 males and 78 females after they were born and the second screening was conducted by OAE was conducted 42 days after those with positive results were examined by auditary brainstem response (ABR).All of the 208 newborns received the initial screening with a screening rate of 100%, and 130 newborns received the second screening with a screening rate of 62.5%. The positive rates in the initial screening and second screening were 34.61% and 7.14% respectively. Six newborns were examined by ABR and 2 of them were diagnoses as with hearing impairment with a prevalence rate of 1.78%. The factors, such as sex, age, and Apgar score affected the initial screening results.The most proper time for initial hearing screening is around the 42nd day after birth. High-risk newborns should receive regular test even though they pass the initial screening. A special follow-up system should be constructed for the high-risk newborns.