Additional file 1 of Recognizing puzzling PD1 + infiltrates in marginal zone lymphoma by integrating clonal and mutational findings: pitfalls in both nodal and transformed splenic cases

Additional file 1. Supplementary Materials. Methods. Histology and Immunohistochemistry. Cases were collected from the Peking University Cancer Hospital and reviewed by an expert panel (YF.S., YM.L., and XH.L.), with a consensus diagnosis of MZL or tSMZL. The study was approved by the ethical committee of the institution and followed the 1964 Helsinki Declaration. Hematoxylin and eosin (H&E) and immunohistochemistry-stained slides from each case were evaluated. Immunohistochemistry was performed on FFPE sections on a Ventana Benchmark automated immunostainer using UltraView detection kits. The panel of antibodies can be seen in Table S2. The interpretation of the PD1 staining pattern included both the predominant location of the PD1-positive cells (follicular or extrafollicular) and was considered “normal” if most PD-1-positive cells were confined to intrafollicular areas and concentrated in the light zone. In situ hybridization to detect EBV-encoded RNA. EBV status was determined by in situ hybridization (ISH) to detect EBV-encoded RNA 1 and 2 (EBER1/2s) using peroxidase-labeled probes (ISH-7001UM, Beijing Zhongshan Golden Bridge Biotechnology). Tissue from a known EBV-positive nasopharyngeal carcinoma was used as a positive control. The EBV status was considered positive if at least one definitive cell expressed EBER. All H&E, IHC and ISH slides were independently and dual assessed. Immunoglobulin Gene and T-Cell Receptor Gene Rearrangement Studies. DNA was extracted from FFPE tissue sections using a QIAGEN QIAamp DNA FFPE Tissue Kit according to the manufacturer’s protocol (QIAGEN, Germantown, MD). Polymerase chain reaction (PCR) for immunoglobulin gene (IGH and IGK loci) and T-cell receptor (TCR locus) rearrangements was performed using commercially available BIOMED-2 multiplex PCR kits (Righton Gene, Shanghai). PCR products were separated by capillary electrophoresis and subjected to GeneScan analysis for confirmation of the monoclonal character of the IG or TCR gene rearrangements on an ABI 9700 Genetic Analyzer (Applied Biosystems, Foster City, CA), and electropherograms were analyzed using GeneMapper software, version 4.0. Targeted exome sequencing (TES) and sequence data analysis. Genomic DNA (gDNA) extraction from FFPE tissues, library preparation, and target gene enrichment were performed according to the manufacturer’s protocol. The gDNA libraries were subjected to high-throughput sequencing with 150-bp paired-end reads on the NovaSeq 60,000 Sequencing System (Illumina, San Diego, CA) supported by a commercial vendor (Geneplus-Beijing, China). The average sequencing depth of tissues was ~500×. Sequence reads were aligned using BWA version 0.5.9 (Broad Institute). Single nucleotide variants (SNVs) were called using MuTect (version 1.1.4) and NChot. Small insertions and deletions (Indels) were determined by GATK. All final candidate variants were manually reviewed by using the IGV browser as reported previously [13]

Nature of Catalytic Behavior of Cobalt Oxides for CO₂ Hydrogenation

Cobalt oxide (CoOx) catalysts are widely applied in CO2 hydrogenation but suffer from structural evolution during the reaction. This paper describes the complicated structure–performance relationship under reaction conditions. An iterative approach was employed to simulate the reduction process with the help of neural network potential-accelerated molecular dynamics. Based on the reduced models of catalysts, a combined theoretical and experimental study has discovered that CoO(111) provides active sites to break C–O bonds for CH4 production. The analysis of the reaction mechanism indicated that the C–O bond scission of *CH2O species plays a key role in producing CH4. The nature of dissociating C–O bonds is attributed to the stabilization of *O atoms after C–O bond cleavage and the weakening of C–O bond strength by surface-transferred electrons. This work may offer a paradigm to explore the origin of performance over metal oxides in heterogeneous catalysis

Hydrophilic Indolium Cycloruthenated Complex System for Visual Detection of Bisulfite with a Large Red Shift in Absorption

Bisulfite, as an important additive in foodstuffs, is one of the most widely distributed environmental pollutants. The excessive intake of bisulfite may cause asthmatic attacks and allergic reactions. Therefore, the determination and visual detection of bisulfite are very important. Herein, a newly designed hydrophilic indolium cycloruthenated complex, [Ru­(mepbi)­(bpy)₂]⁺ [<b>1</b>; bpy = 2,2′-bipyridine and Hmepbi = 3,3-dimethyl-1-ethyl-2-[4-(pyridin-2-yl)­styryl]­benzo­[<i>e</i>]­indolium iodide (<b>3</b>)], was successfully synthesized and used as a bisulfite probe. The bisulfite underwent a 1,4-addition reaction with complex <b>1</b> in PBS buffer (10 mM, pH 7.40), resulting in a dramatic change in absorption spectra with a red shift of over 100 nm and a remarkable change in solution color from yellow to pink. It is worth noting that this obvious bathochromic shift is rarely observed in the detection of bisulfite through an addition reaction. The detection limit was calculated to be as low as 0.12 μM by UV–vis absorption spectroscopy. Moreover, complex <b>1</b> was also used to detect bisulfite in sugar samples (granulated and crystal sugar) with good recovery

Relationship between the TRIM24 expression and the clinicopathological factors.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085462#pone-0085462-t001" target="_blank">Table 1:</a> a. Missing data for 2 patients. b. Missing data for 3 patients. c. Missing data for 1 patient. d. Missing data for 10 patients; 7 patients were excluded according to the inclusion criteria. *.We defined that those patients whose recurrence time is less than 6 months as intrahepatic metastasis.</p

The Kaplan-Meier survival curves of patients with positive TRIM24 expression and negative TRIM24 expression.

<p>The Kaplan-Meier survival curves of patients with positive TRIM24 expression and negative TRIM24 expression.</p

Depletion of TRIM24 reduces cell proliferation.

<p>4(A). Western blotting analysis of the cell-cycle related proteins showed the expression of Cyclin D1 and CDK4 were decreased but showed no significant change in p21 after knockdown TRIM24 in HepG2 cells; 4(B). Cell cycle analyses showed that the percentage of G1 phase was increased in siTRIM24 group (P<0.05), whereas the percentages of S phase (P<0.05) and G2 phase (P<0.05) were decreased in the TRIM24 knockdown cells compared with control cells; 4(C) CCK-8 assay suggested that cell proliferation of HepG2 after TRIM24 silencing was reduced compared with the control group.</p

Immunohistochemical staining of TRIM24 in tissue sections.

<p>A. Negative staining in normal liver tissue. B. Negative staining in benign liver lesions tissues (Hepatic hemangioma). C. Negative TRIM24 staining in an AFP>400 ug/L, well differentiated HCC tissue. D. Positive TRIM24 staining in an AFP<400 ug/L, moderate differentiated HCC tissue. E. Negative TRIM24 staining in an AFP<400 ug/L,well differentiated HCC tissue. F&G. Positive TRIM24 staining in an AFP>400 ug/L, poor differentiated HCC tissue. H. Negative control using antibody diluent.</p

Depletion of TRIM24 inhibits the process of EMT.

<p>5(A). Western blotting analysis of the cell-apoptosis related proteins showed the expression of E-cadherin was increased and Snail, Slug, Vimentin, andβ-catenin were decreased after knockdown TRIM24 in HepG2 cells; 5(B). Reduced migration and invasion ability of HepG2 cells at 48 h post-transfection with siTRIM24 (P<0.05, compared with controls).</p

Transposase-mediated excision of the <i>TcBuster</i> transposon in HEK-293 cells.

<p>(<b>a</b>) An agarose gel of the excision PCR. Plasmid DNA was extracted from transfected HEK-293 cells and used as a template for nested PCR to detect the excision of the transposon DNA. Lane 1, 1 kb ladder; lane 2, PCR reaction without any DNA template added; lanes 3–7, PCR on extracts from cells transfected with either 1 µg of the transposon plasmid pTcBNeo (lane 3), 867 ng transposon pTcBNeo and 133 ng pCMVGFP negative control (lane 4), or 867 ng transposon pTcBNeo and 133 ng pXL-CMV-TcBuster_CO transposase plasmid (three separate transfections, lanes 5–7). (<b>b</b>) The three PCR bands shown in (<b>a</b>) were gel-purified and TOPO-cloned. Clones were sequenced to determine the exact excision junction. The sequence flanking the transposon in pTcBNeo is shown at the top. The TAAAG homology region is shown in blue. Mismatches are shown in lowercase pink. Dashes are used to maintain alignment and if pink, indicate a missing bp. The sequences are ranked according to (1) incidence (# of clones) followed by (2) number of bp not matching the highest incidence clone (# bp mismatches).</p

The effect of transposon and transposase plasmid dose on the number of drug-resistant colonies formed.

<p>(<b>a</b>) HEK-293 cells in 6-well plates were transfected in triplicate with either 12.5 ng (light grey bars), 50 ng (dark grey bars), or 500 ng (black bars) of pTcBNeo carrying the neomycin-resistance transposon and 0 ng, 100 ng, 250 ng, or 500 ng of pCMV-<i>TcBuster</i> expressing the transposase. (<b>b</b>) HEK-293 cells in 6-well plates were transfected in triplicate with 500 ng of pTcBNeo plasmid carrying the neomycin-resistance transposon and the indicated amount of pCMV-<i>TcBuster</i> (0.5 ng, 1 ng, 5 ng, 10 ng, 25 ng, 50 ng, 100 ng, 250 ng, or 500 ng). In both <b>a</b> and <b>b</b>, pUC19 was used as filler DNA to increase the total amount of DNA transfected to 1 µg. Cells were diluted 1∶750 in selection media and grown for two weeks to allow drug-resistant cells to multiply and form colonies. The colonies were fixed, stained, and counted. The mean and standard error of the mean (SEM; n = 3) are shown.</p