Journal: Medicina

Article Title: The Impact of MGMT Promoter Methylation and Temozolomide Treatment in Serbian Patients with Primary Glioblastoma

doi: 10.3390/medicina55020034

Figure Lengend Snippet: Determination of the methylation status of MGMT promoter in primary glioblastoma by A methylation-specific polymerase chain reaction (MSP). S: DNA standard 100 bp ladder; K-: negative control; K1: unmethylated human control DNA; K2: unmethylated and bisulfite-converted human control DNA; K3: methylated and bisulfite-converted human control DNA; M: Polymerase chain reaction (PCR) reaction with primers specific for methylated MGMT promoter; U: PCR reaction with primers specific for unmethylated MGMT promoter; PD: primer dimers; 1–25: bisulfite-converted DNA isolated from patients with primary glioblastoma; patients denoted as 1, 2, 5, 8, 9, 15, 16, 17, 19, and 25 were treated with RT+TMZ (defined as Group 1 in ); patients marked as 4, 7, 10, 12, 18, 22, 23, and 24 were treated with RT+PCV (defined as Group 2 in ); patients designated as 3, 6, 11, 13, 14, 20, and 21 were treated with RT+BCNU (defined as Group 3 in .)

Article Snippet: The amplification reaction was carried out in a Mastercycler Gradient (Eppendorf) using the following program: 95 °C for 15 min, then 35 cycles of 95 °C for 50 s, 59 °C for 50 s and 72 °C for 50 s, and final extension at 72 °C for 10 min. Control PCR reactions were performed using EpiTect PCR Control DNA set (Qiagen, Hilden, Germany) consisting of: - unmethylated and unconverted human DNA (genomic DNA purified from a human colorectal cancer cell line HCT116 DKO with double knockouts of both DNA methyltransferases (DNMT1 (-/-) and DNMT3b (-/-)) (K1 in ); - unmethylated and bisulfite-converted human DNA (genomic DNA originated from the same HCT116 DKO cell line as K1 DNA, but modified by sodium bisulfite upon isolation; as a result of bisulfite conversion non-methylated cytosines were turned to uracils) (K2 in ); - methylated and bisulfite-converted human DNA (genomic DNA derived from HCT116 DKO cell line which was in vitro methylated at all cytosine positions comprising CpG dinucleotides by M.SssI methyltransferase and then treated with sodium bisulfite; the final outcome of the bisulfite treatment was that 5-methylcytosines were left unaffected) (K3 in ).

Techniques: Methylation, Polymerase Chain Reaction, Negative Control, Isolation

Journal: Science immunology

Article Title: Human Innate Lymphoid Cell Precursors Express CD48 that Modulates ILC Differentiation through 2B4 Signaling

doi: 10.1126/sciimmunol.aay4218

Figure Lengend Snippet: (A) Freshly isolated UCB-derived CD34+ HSCs were stained for CD244 and CD48 (representative, n=4). (B) CD244 in CD34+CD48− cells (aqua) and in CD34+CD48+ cells (red) for day 1 and day 3 UCB-derived CD34+ HSCs (representative, n=4). (C) SAP mRNA expression in day 5 CD34+CD48− and CD34+CD48+ cells (n=4/group). (D and E) Anti-CD244 and anti-CD48 blocking antibodies or isotype IgG controls? were added to differentiating CD34+α4β7+CD48+ cultures to block CD244 signaling. (F and G) CD34+α4β7+CD48+ cells were plated on anti-CD244 or IgG coated plates twice for 4 days prior to 21 days of differentiation to activate CD244 signaling via cross-linking. Cells were stained for ILCs, and the percentage (D and F) or absolute number (E and G) of CD94+ NK cells, CD294+ ILC2 and CD117+ ILC3 are shown (n=4/group). (H) CD244+ cultures are shown for cells transfected with control or CD244 gRNA (n=3/group). (I) Representative ILC2 (CD294) staining for cultures transfected with control or CD244 gRNA. (J) Dot plot showing generation of ILC2s from cultures that express (control gRNA) or lack (CD244 gRNA) CD244. (C-H) Mean ± SD, paired t-tests (C, F, G and H) or one-way ANOVA (D and E) and p-values are depicted.

Article Snippet: The Alt-R Cas9 Nuclease V3, universal tracrRNA, CD244 gRNA and non-human control gRNA were purchased from Integrated DNA Technologies.

Techniques: Isolation, Derivative Assay, Staining, Expressing, Blocking Assay, Transfection

Journal: bioRxiv

Article Title: Detection of race-specific resistance against Puccinia coronata f. sp. avenae in Brachypodium species

doi: 10.1101/281568

Figure Lengend Snippet: Colonization of Brachypodium accessions by P. coronata f. sp. avenae . A . Fungal growth estimates in foliar tissue for isolates 12SD80, 203, and 12NC29 depicted by the percentage of colonized area at 14 dpi. Crown rust susceptible oat ( A. sativa ) variety Marvelous serves as positive experimental control. Results from two independent experiments for each isolate are shown with distinct color intensities and each bar represents a mean value in one independent experiment (biological replicate). Error bars represent standard error of mean of three leaves (technical replicates) within one independent experiment. B . Quantification of fungal DNA for each rust isolate in B. distachyon accessions ABR6 (blue line) and Bd21 (orange line). Error bars represent standard error of mean of four independent experiments (biological replicates).

Article Snippet: The relative abundance of fungal DNA was measured using the Femto™ Fungal DNA Quantification Kit (Zymo Research) based on quantification of ITS regions using ITS-specific primers and fungal DNA Standards provided by the manufacturer.

Techniques: