Quaternary vegetation history in Hungary

Detailed information of SSR markers used for genotyping the RIL population. (XLSX 49 kb

Chemical-Vapor-Assisted Electrospray Ionization for Increasing Analyte Signals in Electrospray Ionization Mass Spectrometry

We report a chemical-vapor-assisted electrospray ionization (ESI) technique to improve the detection sensitivity of ESI mass spectrometry (MS). This simple technique involves introducing a chemical vapor into the sheath gas around the nano-ESI spray tip or through a tubing with its outlet placed close to the spray tip. A variety of chemical vapors were tested and found to have varying degrees of effects on analyte signal intensities. The use of benzyl alcohol vapors in ESI was found to increase signal intensities of standard peptides by up to 4-fold. When this technique was combined with capillary liquid chromatography tandem MS (LC-MS/MS), the number of unique peptides identified in the acid hydrolysate of alpha casein increased by 45% and the number of peptides and proteins identified in a tryptic digest of <i>E. coli</i> cell lysate increased by 13% and 14%, respectively, along with an increased average match score. This technique could also increase the analyte signals for some small molecules, such as phenylephrine, by up to 3-fold. The increased analyte signals observed in the chemical-vapor-assisted ESI process is related to the enhancement of the ionization efficiency in ESI. The method can be readily implemented to an existing ESI mass spectrometer at minimum cost for improving detection sensitivity

Critical Assessment of Time-Dependent Density Functional Theory for Excited States of Open-Shell Systems: II. Doublet-Quartet Transitions

Compared with closed-shell systems, open-shell systems place three additional challenges to time-dependent density functional theory (TD-DFT) for electronically excited states: (a) the spin-contamination problem is a serious issue; (b) the exchange-correlation (XC) kernel may be numerically instable; and (c) the single-determinant description of open-shell ground states readily becomes energetically instable. Confined to flip-up single excitations, the spin-contamination problem can largely be avoided by using the spin-flip TD-DFT (SF-TD-DFT) formalism, provided that a noncollinear XC kernel is employed. As for the numerical instabilities associated with such a kernel, only an ad hoc scheme has been proposed so far, viz., the ALDA0 kernel, which amounts to setting the divergent components (arising from density gradients and kinetic energy density) simply to zero. The ground-state instability problem can effectively be avoided by introducing the Tamm-Dancoff approximation (TDA) to TD-DFT. Therefore, on a general basis, the SF-TDA/ALDA0 Ansatz is so far the only promising means within the TD-DFT framework for flip-up single excitations of open-shell systems. To assess systematically the performance of SF-TDA/ALDA0, in total 61 low-lying quartet excited states of the benchmark set of 11 small radicals [<i>J. Chem. Theory Comput.</i> <b>2016</b>, <i>12</i>, 238] are investigated with various XC functionals. Taking the MRCISD+Q (multireference configuration interaction with singles and doubles plus the Davidson correction) results as benchmark, it is found that the mean absolute errors of SF-TDA/ALDA0 with the SAOP (statistical averaging of model orbital potentials), global hybrid, and range-separated hybrid functionals are in the range of 0.2–0.4 eV. This is in line not only with the typical accuracy of TD-DFT for singlet and triplet excited states of closed-shell systems but also with the gross accuracy of spin-adapted TD-DFT for spin-conserving excited states of open-shell systems

Combining Percolator with X!Tandem for Accurate and Sensitive Peptide Identification

In this work, Percolator was successfully interfaced with X!Tandem using a PHP program to generate an improved search platform, X!Tandem Percolator. In order to achieve the best classification performance of peptide identifications in Percolator, a set of experimentally validated spectral identifications (34,993 MS/MS spectra) were used to guide the development of discriminatory features from X!Tandem search results. By comparing the features (e.g., Log­(E) and mass error) of these experimentally validated peptide matches with those of false identifications, a comprehensive set of features can be chosen for Percolator in an objective and rational manner. The accuracy of X!Tandem Percolator was demonstrated by comparing the estimated q-value of the validated data set with the empirical q-value. By comparing the results from the X!Tandem Percolator and the original X!Tandem, superior sensitivity and specificity of the X!Tandem Percolator result was demonstrated on various shotgun proteomic data sets under different search conditions. In all of the cases studied in this work, X!Tandem Percolator could improve the number of peptide identifications at the same level of q-values

Nanoflow LC–MS for High-Performance Chemical Isotope Labeling Quantitative Metabolomics

Nanoflow liquid chromatography mass spectrometry (nLC–MS) is frequently used in the proteomics field to analyze a small amount of protein and peptide samples. However, this technique is currently not widespread in the metabolomics field. We report a detailed investigation on the development of an nLC–MS system equipped with a trap column for high-performance chemical isotope labeling (CIL) metabolomic profiling with deep coverage and high sensitivity. Experimental conditions were optimized for profiling the amine/phenol submetabolome with ¹³C-/¹²C-dansylation labeling. Comparison of analytical results from nLC–MS and microbore LC–MS (mLC–MS) was made in the analysis of metabolite standards and labeled human urine and sweat samples. It is shown that, with a 5-μL loop injection, 7 labeled amino acid standards could be detected with <i>S</i>/<i>N</i> ranging from 7 to 150 by nLC–MS with an injection of 5 nM solution containing a total of 25 fmol labeled analyte. For urine metabolome profiling where the sample amount was not limited, nLC–MS detected 13% more metabolites than mLC–MS under optimal conditions (i.e., 4524 ± 37 peak pairs from 26 nmol injection in triplicate vs 4019 ± 40 peak pairs from 52 nmol injection). This gain was attributed to the increased dynamic range of peak detection in nLC–MS. In the analysis of human sweat where the sample amount could be limited, nLC–MS offered the advantage of providing much higher coverage than mLC–MS. Injecting 5 nmol of dansylated sweat, 3908 ± 62 peak pairs or metabolites were detected by nLC–MS, while only 1064 ± 6 peak pairs were detected by mLC–MS. Because dansyl labeled metabolites can be captured on a reversed phase (RP) trap column for large volume injection and are well separated by RPLC, the CIL platform can be readily implemented in existing nLC–MS instruments such as those widely used in shotgun proteomics

Performance of TD-DFT for Excited States of Open-Shell Transition Metal Compounds

Time-dependent density functional theory (TD-DFT) has been very successful in accessing low-lying excited states of closed-shell systems. However, it is much less so for excited states of open-shell systems: unrestricted Kohn–Sham based TD-DFT (U-TD-DFT) often produces physically meaningless excited states due to heavy spin contaminations, whereas restricted Kohn–Sham based TD-DFT often misses those states of lower energies. A much better variant is the explicitly spin-adapted TD-DFT (X-TD-DFT) [<i>J. Chem. Phys.</i> <b>2011</b>, <i>135</i>, 194106] that can capture all the spin-adapted singly excited states yet without computational overhead over U-TD-DFT. While the superiority of X-TD-DFT over U-TD-DFT has been demonstrated for open-shell systems of main group elements, it remains to be seen if this is also the case for open-shell transition metal compounds. Taking as benchmark the results by MS-CASPT2 (multistate complete active space second-order perturbation theory) and ic-MRCISD (internally contracted multireference configuration interaction with singles and doubles), it is shown that X-TD-DFT is indeed superior to U-TD-DFT for the vertical excitation energies of ZnH, CdH, ScH₂, YH₂, YO, and NbO₂. Admittedly, there exist a few cases where U-TD-DFT appears to be better than X-TD-DFT. However, this is due to a wrong reason: the underestimation (due to spin contamination) and the overestimation (due to either the exchange-correlation functional itself or the adiabatic approximation to the exchange-correlation kernel) happen to be compensated in the case of U-TD-DFT. As for [Cu­(C₆H₆)₂]²⁺, which goes beyond the capability of both MS-CASPT2 and ic-MRCISD, X-TD-DFT revises the U-TD-DFT assignment of the experimental spectrum

Electrophoresis of PCR products of the samples and genotyping of the <i>IL</i>-8 +781C/T SNP.

<p>(A) <i>IL</i>-8 +781C/T SNP: lane M, 100 bp marker ladder, lanes 1–4, samples, the 203 bp bands are the PCR products. (B) genotyping of the <i>IL</i>-8 +781C/T SNP: lane M, 100 bp marker ladder; lanes 1 and 2, CT genotype (203-, 189- and 19-bp); lanes 3, TT genotype (203 bp); and lanes 4, CC genotype (184- and 19-bp). The 19 bp fragment was invisible in the gel owing to its fast migration speed.</p

<i>Obol</i> is maternally supplied and expressed in PGCs.

<p>(A-I) Chemical WISH, showing maternal inheritance (A and C) and PGC expression (D-I) of <i>Obol</i>, <i>Odazl</i> and <i>Olvas</i>. PGCs are seen in two clusters bilateral to the body axis. (J-M) Dual color fluorescent SISH of <i>Obol</i> and <i>Odazl</i>. (N-P) Dual color fluorescent SISH of <i>Obol</i> and <i>Olvas</i>. At stage 27, <i>Obol</i>, <i>Odazl</i> and <i>Olvas</i> RNAs colocalize in gondal PGCs in two clusters. so, somites; no, notochord. (A-C) Top view. (D-P) Lateral view. The anterior is to the left.</p

Nucleotide sequence of the medaka <i>boule</i> cDNA.

<p>O<i>bol</i> and its deduced protein OBol: shown in bold are the translation start codon, stop codon and putative poly-adenylation signal. Highlighted are RRM motif (Turquoise) and DAZ motif (light grey). Underlined are primer sequences for 5′ RACE (dash) and RT-PCR (solid, fragment for RNA probe) with arrows depicting their extension directions.</p

Phylogeny and ontogeny of DAZ family genes.

<p>Left, Phylogeny and sex-specificity. The ancient member <i>boule</i> exists in all metazoans, whereas <i>dazl</i> is in vertebrates and <i>daz</i> is restricted to human and certain primates. Evolutionary branching and two gene duplication events (R1 and R2) are indicated. Sex specificity of expression is indicated in different colors. Right, Ontogenic expression. Expression pattern of each member is indicated by extent of horizontal lines. Drawings are originals by author or redrawn based the references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Houston1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Xu1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Cheng1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Xu3" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Ruggiu1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Mita1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Maruyama1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Niederberger1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Houston2" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Brekhman1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006097#pone.0006097-Huang1" target="_blank">[46]</a>. Genes (in italics) refer to RNA expression while Proteins represent the protein expression profiles. Major stages of germline development are diagramed as a timeline for DAZ family gene expression. Expression of <i>dazl</i> is detected in many or all stages of germline development in both sexes. Daz has premeiotic male expression. Meiotic expression of Boule occurs in male fly, mouse and human (most abundantly in primary spermatocytes) and mitotic and meiotic in female worm. Data obtained in this study from medaka clearly demonstrate that <i>boule</i> is also expressed in embryos at the earliest stage, in primordial germ cells (PGCs) and adult premeiotic and meiotic germ cells of both sexes. The expression of medaka <i>boule</i> is similar to that of <i>dazl</i> in medaka and other organisms, despite of clear differences as detailed in the text.</p