The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2'-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2'-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2'-deoxycytidine to 2'-deoxyguanosine and was determined directly using 2'-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2'-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.

The ionization mechanism of negative ion-direct analysis in real time (NI-DART) has been investigated using over 42 compounds, including fullerenes, perfluorocarbons (PFC), organic explosives, phenols, pentafluorobenzyl (PFB) derivatized phenols, anilines, and carboxylic acids, which were previously studied by negative ion-atmospheric pressure photoionization (NI-APPI). NI-DART generated ionization products similar to NI-APPI, which led to four ionization mechanisms, including electron capture (EC), dissociative EC, proton transfer, and anion attachment. These four ionization mechanisms make both NI-DART and NI-APPI capable of ionizing a wider range of compounds than negative ion-atmospheric pressure chemical ionization (APCI) or negative ion-electrospray ionization (ESI). As the operation of NI-DART is much easier than that of NI-APPI and the gas-phase ion chemistry of NI-DART is more easily manipulated than that of NI-APPI, NI-DART can be therefore used to study in detail the ionization mechanism of LC/NI-APPI-MS, which would be a powerful methodology for the quantification of low-polarity compounds. Herein, one such application has been further demonstrated in the detection and identification of background ions from LC solvents and APPI dopants, including water, acetonitrile, chloroform, methylene chloride, methanol, 2-propanol, hexanes, heptane, cyclohexane, acetone, tetrahydrofuran (THF), 1,4-dioxane, toluene, and anisole. Possible reaction pathways leading to the formation of these background ions were further inferred. One of the conclusions from these experiments is that THF and 1,4-dioxane are inappropriate to be used as solvents and/or dopants for LC/NI-APPI-MS due to their high reactivity with source basic ions, leading to many reactant ions in the background. [7] and atmospheric pressure photoionization (APPI) [8,9], these new ionization methods are especially successful in the analysis of compounds on a variety of surfaces, including concrete, human skin, currency, airline boarding passes, fruits, vegetables, cloth, drug tablets, and biological tissues without sample preparation. Of these new ionization methods, DART is particularly interesting due to its distinct ionization mechanism, while the others have close relationships with their respective traditional API methods.DART ionization begins with a stream of gas, usually helium, which is electrically discharged to produce ions, electrons, and metastable species. After heating and removal of charged particles, this stream of gas exits the DART source into the open air, and is able to ionize chemicals by instant contact, therefore permitting the analysis of gases, liquids, and solids. Although the DART ionization mechanisms are not yet fully understood, it has been proposed [1] that in the positive ion (PI) mode, metastable helium atoms induce Penning ionization of ambient water in the open air, generating protonated water clusters, mostly H 5 O 2 ϩ , which further ionize analytes through chemical reactions. In the negative ion (NI) mode; however,...

A transient microenvironment mechanism (TMEM) is proposed to address matrix effects for direct analysis in real time (DART). When the DART gas stream is in contact with the sample, a transient microenvironment (TME), which can shield analytes from direct ionization, may be generated through the desorption of the matrix containing the analyte. The DART gas stream can directly ionize the matrix molecules, but the analytes will be ionized primarily through gas-phase ion/molecule reactions with the matrix ions. Experimental results showed that as little as 10 nL of liquid or 10 microg of solid was able to generate an efficient TME. Generated TMEs were able to control the ionization of an analyte below an analyte-to-matrix ratio that was dependent on the DART temperature and the boiling points of the analyte and matrix. TMEs generated by common solvents were studied in detail. The ionization of both polar and nonpolar compounds, present in a solvent or another analyte below a ratio of 1:100, were found to be mainly controlled by the generated TMEs at a DART temperature of 300 degrees C.

Background: Traditional Chinese medicine uses various herbs for the treatment of various diseases for thousands of years and it is now time to assess the characteristics and effectiveness of these medicinal plants based on modern genetic and molecular tools. The herb Flos Lonicerae Japonicae (FLJ or Lonicera japonica Thunb.) is used as an anti-inflammatory agent but the chemical quality of FLJ and its medicinal efficacy has not been consistent. Here, we analyzed the transcriptomes and metabolic pathways to evaluate the active medicinal compounds in FLJ and hope that this approach can be used for a variety of medicinal herbs in the future.

To better guide the development of liquid chromatography/electron capture-atmospheric pressure photoionization-mass spectrometry (LC/EC-APPI-MS) in analysis of low polarity compounds, the ionization mechanism of 19 compounds was studied using dopant assisted negative ion-APPI. Four ionization mechanisms, i.e., EC, dissociative EC, proton transfer, and anion attachment, were identified as being responsible for the ionization of the studied compounds. The mechanisms were found to sometimes compete with each other, resulting in multiple ionization products from the same molecule. However, dissociative EC and proton transfer could also combine to generate the same [M Ϫ H] Ϫ ions. Experimental evidence suggests that O 2 Ϫ· , which was directly observed in the APPI source, plays a key role in the formation of [M Ϫ H] Ϫ ions by way of proton transfer. Introduction of anions more basic than O 2 Ϫ· , i.e., C 6 H 5 CH 2 Ϫ , into the APPI source, via addition of di-tert-butyl peroxide in the solvent and/or dopant, i.e., toluene, enhanced the deprotonation ability of negative ion-APPI. Although the use of halogenated solvents could hinder efficient EC, dissociative EC, and proton transfer of negative ion-APPI due to their EC ability, the subsequently generated halide anions promoted halide attachment to compounds that otherwise could not be efficiently ionized. With the four available ionization mechanisms, it becomes obvious that negative ion-APPI is capable of ionizing a wider range of compounds than negative ion chemical ionization (NICI), negative ion-atmospheric pressure chemical ionization (negative ion-APCI) or negative ion-electrospray ionization (negative ion-ESI). (J Am Soc Mass Spectrom 2007, 18, 1789 -1798) © 2007 American Society for Mass Spectrometry I t is well known that liquid chromatography mass spectrometry (LC/MS) favors the analysis of polar compounds, as acid-base solution reactions are the most commonly observed ionization mechanism when using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Traditionally, gas chromatography mass spectrometry (GC/MS) using both electron ionization (EI) and chemical ionization (CI) is employed for the analysis of volatile and thermostable nonpolar compounds. However, it can become difficult to analyze nonvolatile and/or thermo-labile nonpolar compounds via hyphenated techniques of chromatography and MS.In the year 2000, atmospheric pressure photoionization (APPI) [1] was developed as a complement to LC/ESI-MS and LC/APCI-MS. Presently, two fundamentally different APPI sources are commercially available [1,2]. Syagen Technology (Tustin, CA) produces PhotoMate, and Applied Biosystems/MDS SCIEX (Concord, Ontario, Canada) markets PhotoSpray. In both designs, photons emitted by a krypton discharge lamp are used to initiate a series of gas-phase reactions, which finally lead to analyte ionization. However, they differ in that PhotoMate utilizes a design to enhance direct APPI, while PhotoSpray implements a design using a dopant, usually tolu...

Two high molecular weight copolymers of poly(N-isopropylacrylamide) (PNIPAM) densely grafted with short poly(ethylene oxide) (PEO) chains (PNIPAM-g-PEO) were studied by NMR and laser light scattering. The long PNIPAM chains with densely grafted PEO branches had a random coil conformation at very dilute concentrations and low temperatures (i.e., T ≤ 30 °C). When the temperature was increased above 31 °C, the copolymers could undergo a broad “coil-to-globule” transition. The collapsed copolymer chains had a 〈R g〉/〈R h〉 value of about 1.0 with PNIPAM chains inside the core and the hydrophilic PEO chains on the surface. This kind of PNIPAM-g-PEO copolymers was studied as a DNA separation medium in capillary electrophoresis. Several advantages of the copolymers as a separation medium for DNA fragments were achieved, such as an automatic coating ability for the capillary inner wall, an easier injection into the capillary channel due to the slightly adjustable viscosity with temperature (up to 31 °C), a high resolution (i.e., one base pair resolution), and fast separation time. In contrast, the homo-PNIPAM or PEO showed worse DNA separation efficiency under similar conditions. The high DNA separation efficiency of the PNIPAM-g-PEO copolymers is related to the polymer chain conformation. The long copolymer chains in a random coil conformation with densely grafted PEO branches could form a physical network with a relatively stable and uniform pore size at high concentrations (i.e., ≥10 wt %). The separation medium has a high sieving ability for DNA separation in terms of DNA migration mechanisms. The collapsed copolymer chains in the globule state could destroy the chain network and thus lose the DNA separation ability.

Isothiocyanates (ITCs) are a class of well-known cancerpreventive phytochemicals, but are primarily disposed of and concentrated in the urine as N-acetylcysteine conjugates (NAC-ITCs) in vivo. Because human urinary bladder cancers occur almost exclusively in the bladder epithelium, which is directly exposed to the urine stored in the bladder, we undertook to examine the anti-cancer activity of NAC-ITCs in cultured human bladder cancer cells. In this paper, we report that the NAC conjugates of four naturally occurring ITCs, including allyl ITC, benzyl ITC (BITC), phenethyl ITC and sulforaphane, potently inhibited the growth of cells derived from both low-grade superficial and high-grade invasive human bladder cancers and drug-resistant bladder cancer cells. Moreover, the growth-inhibitory potencies were similar between the conjugates and their parent compounds. Further study of NAC-BITC and BITC as model compounds showed that both compounds accumulated in cells predominantly as the glutathione conjugate of BITC, but the accumulation of the former was slower. Moreover, both compounds also demonstrated the same anti-proliferative mechanisms: causing the cleavage of the same set of caspases (caspase-3, -8 and -9) in apoptosis induction, arresting cells in the same phases (S and G2/M) and targeting the same cell cycle regulator (Cdc25C), although a longer treatment time or slightly higher doses were needed for NAC-BITC to achieve the same effect as BITC, presumably due to slower cellular uptake of NAC-BITC. These data show that the NAC-ITCs are biologically similar to their parent compounds and are highly effective against human bladder cancer cells.