Transcriptome Analysis and Identification of Cadmium-Induced Oxidative Stress Response Genes in Different Meretrix meretrix Developmental Stages.

Cadmium (Cd) is one of the major pollutants in the aquatic environment, and it can easily accumulate in aquatic animals and result in toxic effects by changing the metabolism of the body, causing a serious impact on the immune system, reproductive system, and the development of offspring. The clam Meretrix meretrix is one of the commercially important species that is cultivated in large-scale aquaculture in China. To elucidate the underlying molecular mechanisms of Cd2+ in the developmental processes, fertilized eggs and larvae of M. meretrix at different developmental stages were exposed to Cd2+ (27.2 mg L-1 in natural seawater) or just natural seawater without Cd2+ (control), and high-throughput transcriptome sequencing and immunohistochemistry techniques were used to analyze the toxic effects of Cd on larvae at different early developmental stages. The results revealed 31,914 genes were differentially expressed in the different stages of M. meretrix development upon treatment with Cd2+. Ten of these genes were differentially expressed in all stages of development examined, but they comprised only six unigenes (CCO, Ndh, HPX, A2M, STF, and pro-C3), all of which were related to the oxidative stress response. Under Cd exposure, the expression levels of CCO and Ndh were significantly upregulated in D-shaped and pediveliger larvae, while pro-C3 expression was significantly upregulated in the fertilized egg, D-shaped larva, and pediveliger. Moreover, HPX, A2M, and STF expression levels in the fertilized egg and pediveliger larvae were also significantly upregulated. In contrast, CCO, Ndh, HPX, A2M, STF, and pro-C3 expression levels in the postlarva were all downregulated under Cd exposure. Besides the genes with changes in expression identified by the transcriptome, the expression of two other oxidative stress-related genes (MT and Nfr2) was also found to change significantly in the different developmental stages of M. meretrix upon Cd exposure, confirming their roles in combating oxidative stress. Overall, the findings of this study indicated that Cd would interfere with cellular respiration, ion transport, and immune response through inducing oxidative stress, and changes in the expression of oxidative stress-related genes might be an important step for M. meretrix to deal with the adverse effects of Cd at different stages of its development.

MiniDBG: A Novel and Minimal De Bruijn Graph for Read Mapping.

The De Bruijn graph (DBG) has been widely used in the algorithms for indexing or organizing read and reference sequences in bioinformatics. However, a DBG model that can locate each node, edge and path on sequence has not been proposed so far. Recently, DBG has been used for representing reference sequences in read mapping tasks. In this process, it is not a one-to-one correspondence between the paths of DBG and the substrings of reference sequence. This results in the false path on DBG, which means no substrings of reference producing the path. Moreover, if a candidate path of a read is true, we need to locate it and verify the candidate on sequence. To solve these problems, we proposed a DBG model, called MiniDBG, which stores the position lists of a minimal set of edges. With the position lists, MiniDBG can locate any node, edge and path efficiently. We also proposed algorithms for generating MiniDBG based on an original DBG and algorithms for locating edges or paths on sequence. We designed and ran experiments on real datasets for comparing them with BWT-based and position list-based methods. The experimental results show that MiniDBG can locate the edges and paths efficiently with lower memory costs.

Role of polyunsaturated phospholipids in liquid-ordered and liquid-disordered phases.

Polyunsaturated phospholipids interact complexly with other membrane components. We have examined pair interactions among ternary lipid bilayers composed of saturated DPPC, polyunsaturated PLePC, and cholesterol in the liquid-ordered and the liquid-disordered phases by all-atom molecular dynamics simulations. The results show that PLePC exhibits strong repulsion with DPPC and cholesterol in the liquid-disordered phase. When the bilayer changes to the liquid-ordered phase, the repulsion of PLePC with DPPC and cholesterol reduces significantly. The phase state of the bilayer which affects the order of acyl tails as well as their density distributions along the bilayer normal is a key factor regulating the role of PLePC in lipid mixtures. Polyunsaturated phospholipids play a strong repulsive role in the liquid-disordered phase but a weak role in the liquid-ordered phase.

USP7 negatively controls global DNA methylation by attenuating ubiquitinated histone-dependent DNMT1 recruitment.

Previous studies have implicated an essential role for UHRF1-mediated histone H3 ubiquitination in recruiting DNMT1 to replication sites for DNA maintenance methylation during S phase of the cell cycle. However, the regulatory mechanism on UHRF1-mediated histone ubiquitination is not clear. Here we present evidence that UHRF1 and USP7 oppositely control ubiquitination of histones H3 and H2B in S phase of the cell cycle and that DNMT1 binds both ubiquitinated H3 and H2B. USP7 knockout markedly increased the levels of ubiquitinated H3 and H2B in S phase, the association of DNMT1 with replication sites and importantly, led to a progressive increase of global DNA methylation shown with increased cell passages. Using DNMT3A/DNMT3B/USP7 triple knockout cells and various DNA methylation analyses, we demonstrated that USP7 knockout led to an overall elevation of DNA methylation levels. Mechanistic study demonstrated that USP7 suppresses DNMT1 recruitment and DNA methylation through its deubiquitinase activity and the interaction with DNMT1. Altogether our study provides evidence that USP7 is a negative regulator of global DNA methylation and that USP7 protects the genome from excessive DNA methylation by attenuating histone ubiquitination-dependent DNMT1 recruitment.

Exploiting the higher specificity of silver amalgamation: selective detection of mercury(II) by forming Ag/Hg amalgam.

Heavy metal ion pollution poses severe risks in human health and the environment. Driven by the need to detect trace amounts of mercury, this article demonstrates, for the first time, that silver/mercury amalgamation, combining with DNA-protected silver nanoparticles (AgNPs), can be used for rapid, easy and reliable screening of Hg(2+) ions with high sensitivity and selectivity over competing analytes. In our proposed approach, Hg(2+) detection is achieved by reducing the mercury species to elemental mercury, silver atoms were chosen as the mercury atoms' acceptors by forming Ag/Hg amalgam. To signal fluorescently this silver amalgamation event, a FAM-labeled ssDNA was employed as the signal reporter. AgNPs were grown on the DNA strand that resulted in greatly quenching the FAM fluorescence. Formation of Ag/Hg amalgam suppresses AgNPs growth on the DNA, leading to fluorescence signal increase relative to the fluorescence without Hg(2+) ions, as well as marked by fluorescence quenching. This FAM fluorescence enhancement can be used for detection of Hg(2+) at the a few nanomolar level. Moreover, due to excellent specificity of silver amalgamation with mercury, the sensing system is highly selective for Hg(2+) and does not respond to other metal ions with up to millimolar concentration levels. This sensor is successfully applied to determination of Hg(2+) in tap water, spring water and river water samples. The results shown herein have important implications in the development of new fluorescent sensors for the fast, easy, and selective detection and quantification of Hg(2+) in environmental and biological samples.

Nucleic acid-modulated silver nanoparticles: a new electrochemical platform for sensing chloride ion.

Inorganic nanomaterials have generated considerable interest in connection to the design of biosensors. Here we exploit the DNA-induced generation of silver nanoparticles for developing an electrical biosensing protocol for chloride ions. Conjugated with thiol modified oligonucleotide, silver nanoparticles were template-synthesized and immobilized on gold electrode. During cyclic voltammogram (CV) scans, the silver nanoparticles were oxidized at high potential to form a layer of Ag/AgCl complex in the presence of Cl(-), giving off sharp solid state redox signals. Under the optimum condition, the electrode responded to Cl(-) over a dynamic range of 2.0 × 10(-5)-0.01 M, with a detection limit of 5.0 × 10(-6) M. Moreover, the specific solubility product constant-based anion recognition made the electrode applicable at a wide pH range and in complex biological systems. To demonstrate the analytical applications of this sensor in real samples, the Cl(-) concentrations in human urine were measured without any sample pretreatment. Urinary Cl(-) detected by the proposed sensor ranged from 110 to 200 mM, which was comparable to the results obtained by standard silver titration.

Design of aptamer-based sensing platform using triple-helix molecular switch.

For successful assay development of an aptamer-based biosensor, various design principles and strategies, including a highly selective molecular recognition element and a novel signal transduction mechanism, have to be engineered together. Herein, we report a new type of aptamer-based sensing platform which is based on a triple-helix molecular switch (THMS). The THMS consists of a central, target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP). The STP is doubly labeled with pyrene at the 5'- and 3'-end, respectively, and initially designed as a hairpin-shaped structure, thus, bringing the two pyrenes into spacer proximity. Bindings of two arm segments of the aptamer with the loop sequence of STP enforce the STP to form an "open" configuration. Formation of aptamer/target complex releases the STP, leading to new signal readout. To demonstrate the feasibility and universality of our design, three aptamers which bind to human α-thrombin (Tmb), adenosine triphosphate (ATP), and L-argininamide (L-Arm), respectively, were selected as models. The universality of the approach is achieved by virtue of altering the aptamer sequence without change of the triple-helix structure.

Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes.

The stem cell protein Lin28 functions to inhibit the biogenesis of a group of miRNAs but also stimulates the expression of a subset of mRNAs at the post-transcriptional level, the underlying mechanism of which is not yet understood. Here we report the characterization of the molecular interplay between Lin28 and RNA helicase A (RHA) known to play an important role in remodeling ribonucleoprotein particles during translation. We show that reducing Lin28 expression results in decreased RHA association with polysomes while increasing Lin28 expression leads to elevated RHA association. Further, the carboxyl terminus of Lin28 is necessary for interaction with both the amino and carboxyl termini of RHA. Importantly, a carboxyl terminal deletion mutant of Lin28 that retains RNA-binding activity fails to interact with RHA and exhibits dominant-negative effects on Lin28-dependent stimulation of translation. Taken together, these results lead us to suggest that Lin28 may stimulate translation by actively recruiting RHA to polysomes.

New strategy for label-free and time-resolved luminescent assay of protein: conjugate Eu3+ complex and aptamer-wrapped carbon nanotubes.

We report here a carbon nanotube-based approach for label-free and time-resolved luminescent assay of lysozyme (LYS) by engineering an antilysozyme aptamer and luminescent europium(III) (Eu(3+)) complex. The sensing mechanism of the approach is based on the exceptional quenching capability of carbon nanotubes for the proximate luminescent Eu(3+) complex and different propensities of single-stranded DNA and the DNA/protein complex to adsorb on carbon nanotubes. The luminescence of a mixture of chlorosulfonylated tetradentate ß-diketone-Eu(3+) and the antilysozyme aptamer was efficiently quenched by single-walled carbon nanotubes (SWNTs) unless the aptamer interacted with LYS. Due to the highly specific recognition ability of the aptamer for the target and the powerful quenching property of SWNTs for luminescence regents, this proposed approach has a good selectivity and high sensitivity for LYS. In the optimum conditions described, >700-fold signal enhancement was achieved for micromolar LYS, and a limit of detection as low as 0.9 nM was obtained, which is about 60-fold lower than those of commonly used fluorescent aptamer sensors. Moreover, due to the much longer lifetime of the Eu(3+) luminescence than those of the ubiquitous endogenous fluorescent components, the time-resolved luminescence technique could be conveniently used for application in complicated biological samples. LYS concentrations in human urine were thus detected using time-resolved luminescence measurement with satisfactory recoveries of 95-98%.

High-efficiency siRNA-based gene knockdown in human embryonic stem cells.

Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.

Silver ions-mediated conformational switch: facile design of structure-controllable nucleic acid probes.

Conformationally constraint nucleic acid probes were usually designed by forming an intramolecular duplex based on Watson-Crick hydrogen bonds. The disadvantages of these approaches are the inflexibility and instability in complex environment of the Watson-Crick-based duplex. We report that this hydrogen bonding pattern can be replaced by metal-ligation between specific metal ions and the natural bases. To demonstrate the feasibility of this principle, two linear oligonucleotides and silver ions were examined as models for DNA hybridization assay and adenosine triphosphate detection. The both nucleic acids contain target binding sequences in the middle and cytosine (C)-rich sequences at the lateral portions. The strong interaction between Ag(+) ions and cytosines forms stable C-Ag(+)-C structures, which promises the oligonucleotides to form conformationally constraint formations. In the presence of its target, interaction between the loop sequences and the target unfolds the C-Ag(+)-C structures, and the corresponding probes unfolding can be detected by a change in their fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using Ag(+) ion complexes instead of traditional Watson-Crick-based duplex. In particular, the intrinsic feature of the metal-ligation motif facilitates the design of functional nucleic acids probes by independently varying the concentration of Ag(+) ions in the medium.

Modulating molecular level space proximity: a simple and efficient strategy to design structured DNA probes.

To construct efficient oligonucleotide probes, specific nucleic acid is designed as a conformationally constrained form based on the formation of a Watson-Crick-based duplex. However, instability of Watson-Crick hydrogen bonds in a complex biological environment usually leads to high background signal from the probe itself and false positive signal caused by nonspecific binding. To solve this problem, we propose a way to restrict the labeled-dyes in a hydrophobic cavity of cyclodextrin. This bounding, which acts like extra base pairs to form the Watson-Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. To demonstrate the feasibility of the design, a stem-containing oligonucleotide probe (P1) for DNA hybridization assay and a stemless one (P2) for protein detection were examined as models. Both oligonucleotides were doubly labeled with pyrene at the 5'- and 3'- ends, respectively. It is the cyclodextrin/pyrene inclusion interaction that allows modulating the degree of conformational constraints of P1 and P2 and thus their background signals and selectivity. Under the optimal conditions, the ratio of signal-to-background of P1/gamma-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of gamma-CD. In addition, the usage of gamma-CD shifts the melting temperature of P1 from 57 to 68 degrees C, which is reasonable for improving target-binding selectivity. This approach is simple in design, avoiding any variation of the stem's length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes.

Design of bis-spiropyran ligands as dipolar molecule receptors and application to in vivo glutathione fluorescent probes.

Despite considerable efforts toward the development of various sophisticated spiropyrans for metal ion sensing, less attention has been paid to organic molecule sensing. One of the major difficulties for detection of organic molecules using a spiropyran is the weak and nonspecific interaction between the spiropyran and the target. Here, we report the synthesis and molecular recognition characterization of two bis-spiropyrans for dipolar molecules and their application to in vivo glutathione (GSH) fluorescent probes. Unlike the mono-spiropyrans, the newly designed bis-spiropyran molecules feature a rigidly maintained molecular cleft and two spiropyran units as binding modules. The molecular recognition is based on multipoint electrostatic interactions and structure complementarity between the opened merocyanine form of the spiropyran and the analyte. It was observed that the spiropyran 1a binds GSH in aqueous solution with high affinity (K = (7.52 +/- 1.83) x 10(4) M(-1)) and shows strong fluorescence emission upon binding. Remarkably, fluorescence output of 1a is not significantly affected by other amino acids and peptides, especially, structurally similar compounds, such as cysteine and homocysteine. Furthermore, fluorescence anisotropy and confocal fluorescent microscopy confirmed that spiropyran 1a is a comparatively good candidate for intracellular delivery and can be accumulated intensively into cells. Thus, 1a can be utilized in vivo as a GSH probe or as a marker to show the level of intracellular GSH.

Strategy for molecular beacon binding readout: separating molecular recognition element and signal reporter.

A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. Hg2+ was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of Hg2+ between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing Hg2+ ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 microM, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter.

Fluorescent assay of DNA hybridization with label-free molecular switch: reducing background-signal and improving specificity by using carbon nanotubes.

Using classic DNA-intercalating dye and carbon nanotubes, a simple, but efficient, method for fluorescent detection of DNA hybridization has been developed.

Reversible molecular switching of molecular beacon: controlling DNA hybridization kinetics and thermodynamics using mercury(ii) ions.

We report that the hydrogen-bonding pattern in a molecular beacon can be replaced by metal-dependent pairs of Hg(2+) and DNA thymine (T) bases. A molecular beacon based on T-Hg(2+)-T exhibits a lower background signal and higher thermostability than regular molecular beacons.

Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization.

We report an effective, novel self-assembled single-wall carbon nanotube (SWNT) complex with an oligonucleotide and demonstrate its feasibility in recognizing and detecting specific DNA sequences in a single step in a homogeneous solution. The key component of this complex is the hairpin-structured fluorescent oligonucleotide that allows the SWNT to function as both a "nanoscaffold" for the oligonucleotide and a "nanoquencher" of the fluorophore. Given this functionality, this carbon nanotube complex represents a new class of universal fluorescence quenchers that are substantially different from organic quenchers and should therefore have many applications in molecular engineering and biosensor development. Competitive binding of a DNA target and SWNTs with the oligonucleotide results in fluorescence signal increments relative to the fluorescence without a target as well as in marked fluorescence quenching. In contrast to the common loop-and-stem configuration of molecular beacons (MBs), this novel fluorescent oligonucleotide needs only one labeled fluorophore, yet the emission can be measured with little or no background interference. This property greatly improves the signal-to-background ratio compared with those for conventional MBs, while the DNA-binding specificity is still maintained by the MB. To test the interaction mechanisms of the fluorescent oligonucleotide with SWNTs and target DNA, thermodynamic analysis and fluorescence anisotropy measurements, respectively, were applied. Our results show that MB/SWNT probes can be an excellent platform for nucleic acid studies and molecular sensing.

Europium(III) complex-based luminescent sensing probes for multi-phosphate anions: modulating selectivity by ligand choice.

Four tetradentate beta-diketonate-Eu3+ complexes were developed as probes for the luminescent sensing of multi-phosphates. By using an appropriate ligand, the pyrophosphate ion (PPi) could be selectively and sensitively detected.

Gold nanoparticle-based colorimetric and "turn-on" fluorescent probe for mercury(II) ions in aqueous solution.

An approach for visual and fluorescent sensing of Hg2+ in aqueous solution is presented. This method is based on the Hg(2+)-induced conformational change of a thymine (T)-rich single-stranded DNA (ssDNA) and the difference in electrostatic affinity between ssDNA and double-stranded (dsDNA) with gold nanoparticles. The dye-tagged ssDNA containing T-T mismatched sequences was chosen as Hg2+ acceptor. At high ionic strength, introduction of the ssDNA to a colloidal solution of the aggregates of gold nanoparticles results in color change, from blue-gray to red of the solution, and the fluorescence quenching of the dye. Binding of Hg2+ with the ssDNA forms the double-stranded structure. This formation of dsDNA reduces the capability to stabilize bare nanoparticles against salt-induced aggregation, remaining a blue-gray in the color of the solution, but fluorescence signal enhancement compared with that without Hg2+. With the optimum conditions described, the system exhibits a dynamic response range for Hg2+ from 9.6 x 10(-8) to 6.4 x 10(-6) M with a detection limit of 4.0 x 10(-8) M. Both the color and fluorescence changes of the system are extremely specific for Hg2+ even in the presence of high concentrations of other heavy and transition metal ions, which meet the selective requirements for biomedical and environmental application. The combined data from transmission electron microscopy, fluorescence anisotropy measurements, and dialysis experiments indicate that both the color and the fluorescence emission changes of the DNA-functioned gold nanoparticles generated by Hg2+ are the results of the metal-induced formation of dsDNA and subsequent formation of nanoparticle aggregates.

Synthesis and biological activity of some novel derivatives of 4-amino-3-(D-galactopentitol-1-yl)-5-mercapto-1,2,4-triazole.

To discover new 1,2,4-triazole derivatives which may possess significant biological activities, we synthesized a series of novel 6-aryl-3-(D-galactopentitol-1-yl)-7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines and 4-(arylmethylidene)amino-5-(D-galactopentitol-1-yl)-3-mercapto-4H-1,2,4-triazoles from 4-amino-3-(D-galactopentitol-1-yl)-5-mercapto-1,2,4-triazole. All the title compounds were characterized by elemental analysis, IR, 1H- and 13C-NMR. Plant growth-regulating activity tests showed that these compounds have remarkable effects on the growth of radish and wheat.