Journal: bioRxiv

Article Title: Global 5-methylcytosine-RNA disruption reduces the vectorial competence to DENV2 of heatwave-exposed Aedes aegypti mosquitoes

doi: 10.1101/2024.03.14.585075

Figure Lengend Snippet: 5mC content in total RNA from A. aegypti challenged with DENV2 and exposed to heatwave or azacytidine treatment. Total RNA was extracted from 10 specimens per condition and time. 2 µg of total RNA was derivatized with 2-bromoacetophenone and fluorometrically detected at 306/378 nm excitation/emission by HPLC-FLD. The percentage of 5mC was calculated relative to total cytidines in RNA. (A) 5mC content in total RNA from A. aegypti midgut at 1 dpi and 4 dpi. Representation of the arithmetic mean ± SEM of the overall 5mC percentage of three to five biological replicates performed in duplicates. ANOVA and Tukey’s multiple comparison test. Only p-values < 0.05 are shown. (B) 5mC content in total RNA from abdomen, thorax and head of A. aegypti 7 dpi. Plot of the arithmetic mean ± SEM of the overall 5mC percentage of three to five biological replicates performed in duplicates. ANOVA and Tukey’s multiple comparison test. Only p-values < 0.05 are shown. Transcriptional response of A. aegypti challenged with DENV2 and exposed to heatwave or azacytidine treatment. Heat map of transcriptional expression of dnmt2 , hsp70 , r2d2 , dicer1 , dicer2 and ago1 by qPCR. The color gradient represents the ratio (fold change) between the expression of the treated and control condition. Only genes with a fold change ≥ 2 or ≤ -2 were included in the heat maps; genes not meeting this criterion are shown in white boxes. (C) Relative expression of dnmt2 , hsp70 and antiviral iRNA pathway genes in midguts of A. aegypti 1 dpi and 4 dpi by qPCR. Representation of the mean of three biological replicates of 30 midguts each and two technical replicates. (D) Relative expression of dnmt2 , hsp70 and antiviral iRNA pathway genes in abdomen, thorax and head of A. aegypti 7 dpi by qPCR. Representation of the mean of three biological replicates of 30 specimens each and two technical replicates.

Article Snippet: The chromatography was carried out in an Agilent Series 1100 HPLC system with a Sorbax C18 (250 x 4.6 mm, 5 µm) column set to 30°C and a Supleco precolumn.

Techniques: Comparison, Expressing

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 1. Silver-stained 16.4% (w/v) T (total acrylamide), 1.9% (w/v) C (bisacrylamide) Tricine SDS-polyacrylamide gel show- ing the LPS profiles of E. coli K12 strains. 1, x705; 2, x711; 3, x711(pJB2); 4, Y10; 5, D21e7 (rfa-1); 6, CS2051 (has a deletion elimi- nating rfaG, rfaP, rfaM, rfaN, and rfaB); 7, D31m4 (rfa-229, rfa-230).

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 2. Physical map of the lpcA region. Vector sequences are not shown. pE4021 is a cosmid clone containing chromosomal DNA from E. coli W3110, including the region from 4.9 to 5.8 min. RNHQ, PEPD, GPTA, PHOE, and PROAB indicate the location of sequenced genes. pJB1 contains a 14-kb EcoRI fragment cloned from pE4021. pJB2 and pJB8 contain a 3-kb BamHI fragment cloned from pJB1 into different vectors. pJB2-9 to pJB2-34 indicate the various deletions of pJB2 span- ning the lpcA region. pJB15 indicates the DNA insert used for construc- tion of DIG-labeled riboprobes. ORF1 and ORF2 are two open reading frames found on opposite strands of the DNA. The direction of tran- scription of lpcA is indicated by the arrow beneath ORF2. The comple- mentation of the novobiocin supersensitivity phenotype by the deletion clones is indicated: R, successful complementation; S, unsuccessful complementation. Restriction enzymes indicated are: A, AvaII; B, BamHI; Bs, BstEII; E, EcoRI; Ev, EcoRV; Hc, HincII; P, PvuI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Plasmid Preparation, Clone Assay, Labeling

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 4. A schematic representation of the proposed events leading to the chromosomal deletion of the lpcA locus in E. coli strain x711. Panel A, chromosomal map of E. coli K12 strain x705. RNHQ, LPCA, PROAB, IS30A, IS5A, IS1B, and IS30 indicate the location of sequenced genes. Panel B, Southern blot showing chromo- somal DNA profiles of E. coli strains x711 and x705 probed with a 14-kb EcoRI DIG-11-dUTP-labeled DNA probe. M, l HindIII molecular weight markers; 1, x711 DNA digested with EcoRI; 2, x705 DNA di- gested with EcoRI; 3, pJB2 digested with BamHI; 4, pJB1 digested with EcoRI. Panel C, restriction maps of pJB1 and pJB16 showing identical nucleotide sequence (hatched box) and the IS5 element (open box). Panel D, transposition of the IS5A insertion element from approximately 5.9 min to 5.2 min followed by replication of the element, and chromosomal map of E. coli strain x711 showing the resulting deletion of the lpcA locus. Restriction endonucleases indicated are: E, EcoRI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Southern Blot, Labeling, Molecular Weight, Sequencing

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 5. Reversed-phase high performance liquid chromatogra- phy analyses of carbohydrates synthesized by E. coli strains x711 and x711(pJB2) cell extracts following incubation with 1.0 mmol of sedoheptulose 7-phosphate. Panel A, x711 incubated 60 min. Panel B, x711(pJB2) incubated 2 min. Panel C, x711(pJB2) incu- bated 60 min without sedoheptulose 7-phosphate. Panel D, x711(pJB2) boiled extract incubated 60 min. Large arrow indicates the retention peak of the phosphorylated product. Small arrow indicates the reten- tion peak of sedoheptulose 7-phosphate.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Synthesized, Incubation

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 6. Effect of alkaline phosphatase treatment in the reac- tion products analyzed by reversed-phase high performance liquid chromatography. Upper panel, HPLC profile of x711(pJB2) extract incubated with 1.0 mmol of sedoheptulose 7-phosphate (SED- 7-P) and treated with alkaline phosphatase (4 units) prior to derivat- ization with ABEE. Arrow indicates the location of the reaction peak of the reaction product in the absence of alkaline phosphatase treatment. Lower panel, HPLC profile of authentic glyceromannoheptose derivat- ized with ABEE. ABEE, p-aminobenzoic ethyl ester; AP, alkaline phos- phatase; GMH, glyceromannoheptose.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: High Performance Liquid Chromatography, Incubation