Table_1_Critical closing pressure as a new hemodynamic marker of cerebral small vessel diseases burden.DOC

PurposeTo investigate cerebrovascular hemodynamics, including critical closing pressure (CrCP) and pulsatility index (PI), and their independent relationship with cerebral small vessel disease (CSVD) burden in patients with small-vessel occlusion (SVO).MethodsWe recruited consecutive patients with SVO of acute cerebral infarction who underwent brain magnetic resonance imaging (MRI), transcranial Doppler (TCD) and CrCP during admission. Cerebrovascular hemodynamics were assessed using TCD. We used the CSVD score to rate the total MRI burden of CSVD. Multiple regression analysis was used to determine parameters related to CSVD burden or CrCP.ResultsNinety-seven of 120 patients (mean age, 64.51 ± 9.99 years; 76% male) completed the full evaluations in this study. We observed that CrCP was an independent determinant of CSVD burden in four models [odds ratio, 1.41; 95% confidence interval (CI), 1.17–1.71; P ConclusionsCrCP representing cerebrovascular tension is an independent determinant and predictor of CSVD burden. It was significantly correlated with age, BMI and systolic blood pressure. These results provide new insights in the mechanism of CSVD development.</p

Table_2_Critical closing pressure as a new hemodynamic marker of cerebral small vessel diseases burden.XLS

PurposeTo investigate cerebrovascular hemodynamics, including critical closing pressure (CrCP) and pulsatility index (PI), and their independent relationship with cerebral small vessel disease (CSVD) burden in patients with small-vessel occlusion (SVO).MethodsWe recruited consecutive patients with SVO of acute cerebral infarction who underwent brain magnetic resonance imaging (MRI), transcranial Doppler (TCD) and CrCP during admission. Cerebrovascular hemodynamics were assessed using TCD. We used the CSVD score to rate the total MRI burden of CSVD. Multiple regression analysis was used to determine parameters related to CSVD burden or CrCP.ResultsNinety-seven of 120 patients (mean age, 64.51 ± 9.99 years; 76% male) completed the full evaluations in this study. We observed that CrCP was an independent determinant of CSVD burden in four models [odds ratio, 1.41; 95% confidence interval (CI), 1.17–1.71; P ConclusionsCrCP representing cerebrovascular tension is an independent determinant and predictor of CSVD burden. It was significantly correlated with age, BMI and systolic blood pressure. These results provide new insights in the mechanism of CSVD development.</p

Determination of Sulfonamides in Chicken Muscle by Pulsed Direct Current Electrospray Ionization Tandem Mass Spectrometry

A simple and rapid approach for the simultaneous detection of trace amounts of six sulfonamides in chicken muscle was developed using pulsed direct current electrospray ionization tandem mass spectrometry (pulsed-dc ESI-MS/MS). The pretreatment of chicken muscle samples consisted of two steps: acetonitrile extraction and <i>n</i>-hexane delipidation. Sulfonamides do not need to be derivatized or chromatographed prior to pulsed-dc ESI-MS/MS. The factors affecting the performance of pulsed-dc ESI-MS/MS were studied. Under optimum conditions, the quantitative performance of pulsed-dc ESI-MS/MS was validated according to European Union Decision 2002/657/EC, and the sensitivity of pulsed-dc ESI-MS/MS was 3 times higher than that of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The limits of detection obtained by pulsed-dc ESI-MS/MS were in the range of 0.07–0.11 μg/kg. The proposed method was simple, rapid, and sensitive, and was successfully used for quantitation and rapid screening of sulfonamides in real chicken muscle samples

Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome, Translation, and RNA Processing System Homologs

<div><p>While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of <i>Haloferax volcanii</i>) that is essential for maintaining cellular pools of thiolated tRNA^Lys_UUU and for growth at high temperature. When purified from <i>Hfx. volcanii</i>, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA^Lys_UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.</p></div

HvJAMM1 (desampylase) collapses SAMP2-NcsA conjugates.

<p>NcsA-StrepII fractions were purified from <i>ΔncsA</i> and <i>ΔubaA</i> strains, incubated with HvJAMM1 in the presence and absence of EDTA, and analyzed by IB as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099104#s4" target="_blank">Methods</a> section for details.</p

Multiple amino acid sequence alignment of <i>Hfx. volcanii</i> NcsA (HVO_0580) with ANH superfamily members including proteins of <i>Saccharomyces cerevisiae</i> (ScNcs6, GI:50593215), <i>Homo sapiens</i> (HsNcs6, GI:74713747), <i>Pyrococcus horikoshii</i> (PH1680, GI:14591444; PH0300, GI:14590222), <i>Thermus thermophilus</i> (TTHA0477 or TtuA, GI: 55980446), <i>Salmonella typhimurium</i> (StTtcA, GI:16764998), and <i>Escherichia coli</i> (EcTtcA, GI:85674916).

<p>Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.</p

Proteins Identified by LC-MS/MS proteomic analysis^a.

a<p>MS-identified proteins with coverage above 25% are reported according to the <i>Hfx. volcanii</i> gene locus tag from the National Center for Biotechnology Information and were unique to samples prepared from strain <i>ΔncsA</i> expressing the FLAG-tagged SAMP1 in tandem with StrepII-tagged NcsA, FLAG-tagged SAMP2 in tandem with StrepII-tagged NcsA, or StrepII-tagged NcsA alone compared to the vector alone. Theoretical molecular mass (M_r) estimated from deduced polypeptide based on <i>Hfx. volcanii</i> DS2 genome sequence.</p

Model of NcsA activity and sampylation (panel A) as well as its association with protein partners (panel B).

<p>Panel A, NcsA is proposed to catalyze the formation of 2-thiouridine (s²U) at the wobble uridine position of tRNAs specific for lysine (tRNA^Lys_UUU), glutamate (tRNA^Glu_UUC), and glutamine (tRNA^Gln_UUG) via an adenylated tRNA intermediate using thiocarboxylated SAMP2 as a source of activated sulfur. The E1-like UbaA adenylates the C-terminal α-carboxylate group of SAMP2. This modification readies the Ub-fold SAMP2 for either thiocarboxylation via an enzyme (cysteine desulfurase or rhodanese) catalyzed persulfide sulfur or protein modification via formation of a UbaA-SAMP2 thioester intermediate. Polymeric chains of SAMP2 are formed on an NcsA lysine residue via isopeptide linkages that are cleaved by HvJAMM1 protease. Whether additional factors are needed to provide specificity to the sampylation system is unclear, as E2 and E3 homologs are not predicted based on genome sequence. Panel B, NcsA is found isopeptide linked to SAMP2 and non-covalently associated with various proteins, as noted by dotted red lines. NcsA partners include the E1-like UbaA and Ub-fold SAMP2 of the tRNA thiolation and sampylation pathways. NcsA is also found associated with EF-1α that binds aminoacylated-tRNAs and mediates translation elongation, PAN-A/1 (an AAA+ATPase associated with energy-dependent proteolysis by proteasomes (20S core particles or CPs) and protein remodeling, and the β-CASP ribonuclease homolog of the aCPSF1 family thought to cleave mRNA and/or tRNA. RNAP, RNA polymerase.</p

NcsA is required for growth of <i>Hfx. volcanii</i> at an elevated temperature (50°C).

<p><i>Hfx. volcanii</i> H26 (wt, parent), <i>Δsamp2</i>, <i>ΔncsA</i>, and <i>trans</i> complemented <i>ΔncsA</i> strains were grown in ATCC 974 medium. Freshly isolated colonies were inoculated into 3 ml medium (in 13×100 mm culture tubes) and thrice subcultured at 42°C. Cells grown to logarithmic phase at 42°C from these subcultures were used as inoculum for monitoring growth at 50°C as presented in panel A. Cells grown to stationary-phase from these 50°C cultures were used as an inoculum for monitoring long-term growth at 50°C as presented in panel B. Inoculum was at 0.02 OD₆₀₀ with rotary shaking (200 rpm) in 20 ml medium in 250 ml baffled flasks for the growth assays presented in panels A and B. For panel C, cell cultures, as indicated above each plate, were diluted to 0.1 OD₆₀₀ and then spot-plated on solid agar ATCC 974 medium in serial dilutions as indicated. Plates were incubated at 50°C. Control experiments performed at 42°C are presented in Figure S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099104#pone.0099104.s001" target="_blank">File S1</a>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099104#s4" target="_blank">Methods</a> section for details.</p

NcsA is covalently associated with SAMP2 through a UbaA-dependent mechanism.

<p>A) NcsA StrepII-affinity purified fractions purified from <i>ΔncsA</i> strains expressing NscA-StrepII with and without Flag-SAMP1/2 proteins were separated by reducing SDS-PAGE and analyzed by Coomassie blue stain (upper panel) and α-StrepII and α-Flag IB (middle and bottom panels, respectively) as indicated. B) α-StrepII immunoblot of NcsA-StrepII purified from H26 (wt, parent), <i>ΔubaA</i>, and <i>Δsamp2</i>. C) α-Flag immunoblot of NcsA-StrepII purified from H26 (wt, parent) and <i>ΔubaA</i> strains co-expressing Flag-SAMP2. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 1 L cultures. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099104#s4" target="_blank">Methods</a> section for details.</p