Using Dicationic Ion-Pairing Compounds To Enhance the Single Cell Mass Spectrometry Analysis Using the Single-Probe: A Microscale Sampling and Ionization Device

A unique mass spectrometry (MS) method has been developed to determine the negatively charged species in live single cells using the positive ionization mode. The method utilizes dicationic ion-pairing compounds through the miniaturized multifunctional device, the single-probe, for reactive MS analysis of live single cells under ambient conditions. In this study, two dicationic reagents, 1,5-pentanediyl-bis­(1-butylpyrrolidinium) difluoride (C₅(bpyr)₂F₂) and 1,3-propanediyl-bis­(tripropylphosphonium) difluoride (C₃(triprp)₂F₂), were added in the solvent and introduced into single cells to extract cellular contents for real-time MS analysis. The negatively charged (1– charged) cell metabolites, which form stable ion-pairs (1+ charged) with dicationic compounds (2+ charged), were detected in positive ionization mode with a greatly improved sensitivity. We have tentatively assigned 192 and 70 negatively charged common metabolites as adducts with (C₅(bpyr)₂F₂) and (C₃(triprp)₂F₂), respectively, in three separate SCMS experiments in the positive ion mode. The total number of tentatively assigned metabolites is 285 for C₅(bpyr)₂F₂ and 143 for C₃(triprp)₂F₂. In addition, the selectivity of dicationic compounds in the complex formation allows for the discrimination of overlapped ion peaks with low abundances. Tandem (MS/MS) analyses at the single cell level were conducted for selected adduct ions for molecular identification. The utilization of the dicationic compounds in the single-probe MS technique provides an effective approach to the detection of a broad range of metabolites at the single cell level

Suppression of Graft Regeneration, Not Ischemia/Reperfusion Injury, Is the Primary Cause of Small-for-Size Syndrome after Partial Liver Transplantation in Mice

<div><p>Background</p><p>Ischemia/reperfusion injury (IRI) is commonly considered to play a crucial role in the pathogenesis of small-for-size syndrome (SFSS) after liver transplantation. Rapid regeneration is also considered essential for the survival of SFS grafts.</p><p>Methods</p><p>Mouse models of full-size orthotopic liver transplantation, 50% partial liver transplantation and 30% partial liver transplantation were established. Survival rate and serum alanine aminotransferase were observed. IRI was assessed by hepatic pathologic alterations, apoptosis and necrosis. Regeneration response was detected by mitotic index, BrdU incorporation and PCNA, Cyclin D1 and Cyclin E expression. The expression of mTOR, AKT, ERK, JNK2 and p70S6K, also involved in regeneration signaling pathways, were analyzed as well.</p><p>Results</p><p>30% partial liver graft resulted in a significantly low 7-day survival rate (P = 0.002) with no marked difference in tissue injury compared with the 50% partial graft group. Serum alanine aminotransferase levels were not significantly different between partial transplantation and full-size transplantation. Western blot analysis of caspase-3 and TUNEL staining also indicated no significant difference in apoptosis response between 30% partial transplantation and half-size or full-size transplantation (P = 0.436, P = 0.113, respectively). However, liver regeneration response indicators, mitotic index (P<0.0001) and BrdU (P = 0.0022), were markedly lower in 30% LTx compared with 50% LTx. Suppressed expression of PCNA, cyclin D1, cyclin E, mTOR, JNK2, AKT, ERK and p70S6K was also detected by western blot.</p><p>Conclusions</p><p>Liver regeneration is markedly suppressed in SFSS, and is more likely the primary cause of SFSS, rather than ischemia/reperfusion injury. Therapy for recovering graft regeneration could be a potentially important strategy to reduce the incidence of SFSS.</p></div

ALT levels in mice receiving liver grafts of various sizes.

<p>Before sacrificing the mice for histology, blood was collected for determination of serum alanine aminotransferase levels. Means + SD of six mice each are given.</p

Decreased survival rate after transplantation of small-for-size liver grafts.

<p>Mice were observed 7 days postoperatively for survival. Group sizeswere 8–10 each in the FSG, HSG and TSG groups, respectively. P<0.05 by the Kaplan-Meier test. At 48h post-transplantation, when the samples were taken, the 1-day survival rate of TSG was 100%.</p

Additional file 1: of Oral health knowledge, behaviors and parental practices among rural–urban migrant children in Guangzhou: a follow-up study

Questionnaire of Oral Health. We design this questionnaire of oral health to investigate oral health knowledge, behaviors and parental practices among rural–urban migrant children. The same questionnaire was sent to children in the baseline and final survey. (DOC 78 kb

Detection of BrdU-labeled hepatocytes.

<p>Liver grafts were recovered 48-positive and negative cells in 10 random fields per slide were counted under a microscope with a 40× objective lens. The percentage of BrdU-labeled hepatocytesis plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093636#pone-0093636-g006" target="_blank">figure 6</a>.</p

H&E staining for Mitotic Index.

<p>Sham-operation and varying size graft LTx were performed as described in METHODS. Livers were harvested at 48 h after surgery and stained with hematoxylin and eosin to assess necrosis. Representative images are shown (bar = 100 μm). Ten random fields per slide were captured in a blinded manner using a 10× objective lens. Mitotic hepatocytes were counted in 10 random fields per H&E stained liver section and plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093636#pone-0093636-g003" target="_blank">figure 3</a>.</p

Western Blot for expression of caspase-3.

<p>Alterations of caspase-3 expression after liver transplantation. Livers were collected 48 h post-transplantation. Caspase-3 was detected by immunoblotting. Representative gels are shown.</p

TUNEL staining for liver grafts 48 hours after implantation.

<p>Liver grafts were fixed and sectioned, and apoptotic cells were detected by immunohistochemical DNA strand break labeling. Ten fields were captured at random under a 10× objective lens. TUNEL-positive and -negative cells were counted.</p

Western Blot results of the four groups.

<p><b>A:</b> Livers were recovered at 48-transplantation. PCNA, cyclin D1 (CyD1) and cyclin E (Cy E) were detected by immunoblotting. Representative gels of 4 livers/group are shown. <b>B:</b>Alterations of EGFR downstream signaling molecules after liver transplantation. Livers were collected 48 h post-transplantation. Phospho-Akt, phospho-mTOR, phospho-p70S6 kinase (p-p70S6K), phospho-ERK, phospho-JNK and actin were detected by immunoblotting. Representative gels are shown.</p