Journal: BMC Genomics

Article Title: Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene

doi: 10.1186/s12864-023-09750-2

Figure Lengend Snippet: Comparison between different bacteriocin gene clusters. The figure shows the homologies found when comparing contigs from different species that contain the Mr10EFGH transporter associated with the structural genes of enterocins MR10A/B (yellow and gold arrows), AS-48 (green arrow), carnocyclin A (gray arrow), uberolysin enterocin family (blue arrow) and orphans in bacteriocins. Similarities are conserved only at the ABC transporter level. From top to bottom, GenBank accession nos. NZ_CP035137.1 ( Enterococcus faecium SRCM103341), NZ_KB944733.1 ( Enterococcus faecalis EnGen0369 39 − 5), NZ_AYLU01000046.1 ( Enterococcus faecalis AZ19), NZ_PJXM01000014.1 ( Enterococcus faecalis EN19), NZ_SRYT01000012.1 ( Enterococcus faecalis NM58_B2-5) and NZ_KE352861.1 ( Enterococcus faecalis LA3B-2)

Article Snippet: In some cases, these orphan genomes were the result of incomplete contigs, e.g., NZ_LWHF01000045.1 ( Enterococcus faecium strain 17OM39), NZ_PKMN01000041.1 ( Enterococcus faecalis strain EN788), or NZ_LDND01000117.1 ( Enterococcus faecium strain KACC15711); however, they were evident orphan transporters in some cases, i.e., GenBank accession no. NZ_CABHDR010000049.1 ( Enterococcus faecium strain 4928STDY7387731), GenBank accession no. NZ_KE352861.1 ( Enterococcus faecalis LA3B-2 Scaffold63), or GenBank accession no. NZ_KB946329.1 ( Enterococcus phoeniculicola ATCC BAA-412 acvKl-supercont1.7).

Techniques: Comparison

Journal: BMC Genomics

Article Title: Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene

doi: 10.1186/s12864-023-09750-2

Figure Lengend Snippet: Comparison between contigs containing the transporter Mr10EFGH associated with the same bacteriocin. Panel A shows the comparison between contigs that contain the ABC transporter associated with structural genes of the uberolysin enterocin family (blue arrow). Panel B shows the similarity between contigs that contain the ABC transporter associated with structural genes of MR10A/B enterocins (yellow and gold arrows). In both cases, the homologies are maintained at the gene cluster level. The similarity varies between 63% (light red) and 100% (dark red). From top to bottom, GenBank accession nos. are NZ_AYLU01000046.1 ( Enterococcus faecalis AZ19), NZ_KB944733.1 ( Enterococcus faecalis EnGen0369 39 − 5), NZ_PJXM01000014.1 ( Enterococcus faecalis EN19), NZ_CP035137.1 ( Enterococcus faecium SRCM103341), NZ_SEHG01000089.1 ( Enterococcus durans OSY-EGY), and NZ_SRYT01000012.1 ( Enterococcus faecalis NM58_B2-5)

Article Snippet: In some cases, these orphan genomes were the result of incomplete contigs, e.g., NZ_LWHF01000045.1 ( Enterococcus faecium strain 17OM39), NZ_PKMN01000041.1 ( Enterococcus faecalis strain EN788), or NZ_LDND01000117.1 ( Enterococcus faecium strain KACC15711); however, they were evident orphan transporters in some cases, i.e., GenBank accession no. NZ_CABHDR010000049.1 ( Enterococcus faecium strain 4928STDY7387731), GenBank accession no. NZ_KE352861.1 ( Enterococcus faecalis LA3B-2 Scaffold63), or GenBank accession no. NZ_KB946329.1 ( Enterococcus phoeniculicola ATCC BAA-412 acvKl-supercont1.7).

Techniques: Comparison

Journal: Yeast (Chichester, England)

Article Title: Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

doi: 10.1002/yea.1672

Figure Lengend Snippet: Isolation of active mt nucleoids and identification of proteins associated with TAP–mtRNAP. (A) Purification of transcriptionally active mt nucleoids. Nucleoids were purified as described (see Materials and methods) and diluted as indicated. Transcription reactions were carried out for 20 min at 30 °C in the presence of 20 or 200 m m KCl or α-amanitin (AM). The labelled RNA products were resolved by electrophoresis in a 12% acrylamide denaturing gel, visualized by autoradiography and quantified by PhosphorImager analysis. The abundance of heterogeneous high molecular weight products under various conditions is shown; error bars represent standard error for mt nucleoid transcripts ( n = 3). (B) Rpo41p–TAP-associated proteins. Peptides eluted from the calmodulin–sepharose column were resolved on 4–12% bis-Tris NuPAGE Novex protein gels (Invitrogen) and visualized by silver staining (lane CE). Numbers indicate the molecular weights of protein markers in kDa (lane M). The bands from lane CE were submitted for LC–MS–MS analysis (Tufts University Core Facility). Arrows indicate silver-stained bands that match the molecular weight (MW) of major proteins identified in the gel slabs containing the bands. *Note that the actual MW of mature Pet127p may be smaller than predicted from its ORF as ∼90 N-terminal aa are likely to be absent in the mature form of Pet127p (Wiesenberger and Fox, ). (C) Example of LC–MS–MS spectra (Mss116p). Eleven peptides matching the Mss116p protein sequence were found as a result of an uninformed search against the S. cerevisiae protein database. S f , quality of match; TIC, total ion current; % TIC, signal or ion current for a given peptide; Sum TIC, sum of all TIC values for that protein identification—this exceeded the minimum required score in all cases. Additional information may be found at http://www.tucf.org . Complete LC–MS–MS scans are available upon request

Article Snippet: The washes were combined, polypeptides were precipitated with acetone/TCA at −20 °C for 3 h and separated by electrophoresis in 4–12% Bis–Tris NuPAGE Novex protein gels in an MES buffer system (Invitrogen).

Techniques: Isolation, Purification, Electrophoresis, Autoradiography, High Molecular Weight, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Staining, Molecular Weight, Sequencing

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: Effect of FLT3 inhibition on DC differentiation and surface expression of costimulatory molecules. DCs generated from murine bone marrow cells were differentiated in the presence of 100 nM gilteritinib, midostaurin or quizartinib and were gated using forward and sideward scatter. Dead cells were excluded by life/dead staining. Proportion of double-positive CD11c+/CD11b+ cells is markedly reduced after differentiation in the presence of midostaurin ( a ). Human PBMCs were cultured to obtain DCs as described. Midostaurin-induced monocyte differentiation indicated by reduced CD1a and increased CD14 expression in the presence of midostaurin ( p < 0.0001) and gilteritinib ( p < 0.05) ( b ). Differentiation of murine bone marrow cells in the presence of midostaurin (M1 = 1 nM; M10 = 10 nM; M100 = 100 nM), gilteritinib (G1 = 1 nM; G10 = 10 nM; G100 = 100 nM) or quizartinib (Q1 = 1 nM; Q10 = 10 nM; Q100 = 100 nM) in different concentrations did not induce apoptosis ( c ). The mean fluorescence intensity of CD40 and CD86 on bmDCs after LPS stimulation was reduced in a dose-dependent manner by midostaurin ( p < 0.0001) and gilteritinib ( p < 0.05) exposure ( d ). MoDCs showed reduced CD80, CD83 and CD86 expression after midostaurin treatment with 100 nM. The effect was less pronounced after gilteritinib (100 nM) and only minimal after quizartinib (100 nM) treatment. MFI and rates of positive cells are depicted in the right upper corner ( e ). ns not significant, * p < 0.05, **** = p < 0.0001.

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Inhibition, Expressing, Generated, Staining, Cell Culture, Fluorescence

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: FTL3 inhibition suppresses cytokine secretion by dendritic cells. After 6 days of differentiation in the presence of FLT3i at 100 nM concentration, cytokine release of bmDCs was measured 18 h after LPS stimulation. IL-12 levels were only reduced after midostaurin treatment ( p < 0.01), while IL-6 ( p < 0.01 for midostaurin and p < 0.001 for gilteritinib) and CCL-2 levels ( p < 0.0001 for both FLT3i) were also negatively affected by gilteritinib treatment. TNFα secretion was not affected. The generation of bmDCs is illustrated. Created with BioRender.com/j07f023 ( a ). For moDCs, midostaurin (100 nM) and gilteritinib (100 nM) treatment reduces TNFα ( p < 0.0001), IL-6 ( p < 0.01) and IL-12 levels ( p < 0.0001), quizartinib (100 nM) treatment did not affect TNFα levels but lowered IL-12 ( p < 0.01) and IL-6 ( p < 0.05) levels. Schematic illustration of moDC generation created with BioRender.com/u60g276 ( b ). ns not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Inhibition, Concentration Assay

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: RNA sequencing reveals inhibition of important immune response pathways by FLT3i, which is supported by protein expression analysis. BmDCs were cultured in the presence of DMSO or FLT3i. After 6 days, cells were stimulated with LPS, when indicated, and RNA was harvested 18 h later. Using hierarchical clustering, the top 20 differentially expressed genes found in RNA sequencing analysis reveal specific clustering of DMSO control samples and after exposure to midostaurin but not for other treatment groups. Expression values are depicted from low (blue) to high (red) ( a ). Concomitantly, principal component analysis (PCA) based on all analyzed gene sets also showed high similarity of midaustaurin-treated samples ( b ). Log-fold change in gene expression of midostaurin-, gilteritinib- and quizartinib-treated moDCs after LPS maturation is depicted in a heatmap. Up- (blue) and downregulation (red) are color-coded ( c ). To prove the inhibition of relevant pathways, protein was extracted from human moDCs after FLT3i treatment and LPS stimulation. Stat3 and Stat5 phosphorylation was impaired by midostaurin treatment ( d ). Inhibition of Akt phosphorylation was observed after midostaurin and quizartinib treatment ( e ). The NFκB pathway was inhibited by midostaurin and gilteritinib treatment as cRel and relB expression were reduced. Quizartinib did not affect relB or cRel expression ( f ). The uncropped bolts are shown in .

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: RNA Sequencing, Inhibition, Expressing, Cell Culture, Control, Gene Expression, Phospho-proteomics

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: KEGG analysis of midostaurin-treated bmDCs (selected pathways).

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Cell Differentiation

Journal: The EMBO Journal

Article Title: LARP1 binds ribosomes and TOP mRNAs in repressed complexes

doi: 10.1038/s44318-024-00294-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Samples were boiled at 95 °C for 5 min and loaded into 4–12% Criterion XT-Bis-Tris polyacrylamide gels (Bio-Rad 3450125).

Techniques: Over Expression, Recombinant, Luciferase, Sequencing, Protease Inhibitor, SYBR Green Assay, BIA-KA, Membrane, Blocking Assay, Reporter Assay, Lysis, Marker, Software, Chromatography, Amplification, Imaging