Journal: Gels

Article Title: Characterization of Sugar Reduction in Model Confectionary Gels Using Descriptive Analysis

doi: 10.3390/gels8100644

Figure Lengend Snippet: Model confectionary gel attributes, definitions, reference brand, reference preparation and reference intensity developed by descriptive analysis panel. Reference intensities were iteratively calculated as the average intensity rating of the group using 16-point category scale from 0 to 15.

Article Snippet: , Chewy , Resistance when chewing , Welch’s Mixed Fruit Fruit Snacks (Welch Foods Inc., Concord, MA, USA) , Two strawberry gummies (in 29.5 mL cup) , 9.3.

Techniques: Chocolate, Cream, Residue

Journal: Nature Communications

Article Title: mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

doi: 10.1038/s41467-023-42386-0

Figure Lengend Snippet: a Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor site; SAS, splice acceptor site; pA, polyadenylation signal. b Confocal images of HEK293 cells transfected with v1 and v2 containing different BDs. Scale bar: 50 µm. c RT-PCR of reconstituted full-length cerulean. GAPDH served as input reference. Sequencing result of the splicing junction shown below. bp, base pairs. d Western blots of transfected cells shown in ( b ). α-cer, anti-cerulean antibody; α-tub, anti-β-tubulin antibody; kDa, kilodalton. e Quantification of reconstitution efficiency relative to cis-ctrl using western blots (WB, minimum of n = 3) or flow cytometry (FC, n = 3). f Western blot of cells transfected with v1 and v2 containing different human ( RHO intron 2-derived, BD 5) or bacterial ( lacZ -derived, BD 9–12) binding domains. g Ratiometric quantification of reconstitution efficiency of the transfections shown in ( f ) relative to cis-ctrl. n = 3. h Western blots of cells transfected with v1 and v2 containing different SDS or SAS. i Ratiometric quantification of reconstitution efficiencies relative to cis-ctrl. n = 3. Welch’s ANOVA with Dunnett T3 (SAS) or Kruskal-Wallis test with Dunnett T3 (SDS) was used. j Western blots of cells transfected with versions of v1 containing (+pA) or lacking (−pA) a polyadenylation signal and with versions of v2 containing (+prom) or lacking (−prom) a promoter. k Ratiometric quantification of reconstitution efficiencies relative to cis-ctrl. n = 5 (pA), n = 3 (prom). Two-tailed Mann–Whitney test was used. Scatter plots show mean ± SEM. For all co-transfections equimolar amounts have been used (1 µmol v1 + 1 µmol v2, corresponding to 1 µmol cis-ctrl). All ‘n’ in Fig. 1 represent independent transfections. All source data are provided as a Source Data file.

Article Snippet: Welch’s ANOVA with Dunnett T3 (SAS) or Kruskal-Wallis test with Dunnett T3 (SDS) was used. j Western blots of cells transfected with versions of v1 containing (+pA) or lacking (−pA) a polyadenylation signal and with versions of v2 containing (+prom) or lacking (−prom) a promoter. k Ratiometric quantification of reconstitution efficiencies relative to cis-ctrl. n = 5 (pA), n = 3 (prom).

Techniques: Plasmid Preparation, Positive Control, Binding Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Flow Cytometry, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

doi: 10.1038/s41467-023-42386-0

Figure Lengend Snippet: a Upper panel, dCas9-VPR size and the split site used for REVeRT. Lower panel, REVeRT vector cassettes expressing split dCas9-VPR and three sgRNAs (g1–g3) targeting the promoter region of Myo7b . Titer-matched dual REVeRT AAV8(Y733F) vectors were injected via different routes of administration shown in ( c , f , h ). b dCas9-VPR targeting the Myo7b promoter. c Experimental design for ( d , e ). OD, Oculus dexter ; OS, Oculus sinister . Contralateral eyes injected with AAV formulation buffer served as reference. d qRT-PCR from injected retinas. Shown are the relative expression levels of reconstituted dCas9-VPR (left) and the transactivated Myo7b (right). n = 5 retinas. e Western blot from the retinas of two mice (#1 and #2) injected with REVeRT AAVs or buffer. f Experimental design for ( g ). g qRT-PCR from injected hippocampi. Shown are the relative expression levels of reconstituted dCas9-VPR (left) and transactivated Myo7b (right). Untreated (untrd) hippocampi served as reference. n = 4 hippocampi (REVeRT), n = 6 hippocampi (untrd). h Experimental design for ( i ). i qRT-PCR from organs or tissues harvested from intraperitoneally injected mice. Shown are the relative expression levels of reconstituted dCas9-VPR (left) and transactivated Myo7b (right). Untreated organs served as reference. n = 3 organs/tissues. A two-tailed unpaired t test with Welch’s correction was used in ( d , g ). Scatter plots show mean ± SEM. All source data in this figure are provided as a Source Data file.

Article Snippet: Welch’s ANOVA with Dunnett T3 (SAS) or Kruskal-Wallis test with Dunnett T3 (SDS) was used. j Western blots of cells transfected with versions of v1 containing (+pA) or lacking (−pA) a polyadenylation signal and with versions of v2 containing (+prom) or lacking (−prom) a promoter. k Ratiometric quantification of reconstitution efficiencies relative to cis-ctrl. n = 5 (pA), n = 3 (prom).

Techniques: Plasmid Preparation, Expressing, Injection, Formulation, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

doi: 10.1038/s41467-023-42386-0

Figure Lengend Snippet: a Upper panel, Dual REVeRT AAV8(Y733F) vector cassettes expressing split Cas9-VPR and three sgRNAs targeting either exon 1 of Rho (g1) or the promoter region of Opn1mw (g2–g4). A lacZ -targeting sgRNA (gL) is used as a non-targeting control. Lower panel, The corresponding dual split intein AAV8(Y733F) vector cassettes used for side-by-side comparison to REVeRT shown in ( c ). b Experimental design and timeline of experiments shown in c-e. sgRNAs with different spacer lengths are used for a simultaneous knockout (20 nt, g1) of Rho and transcriptional activation (15 nt, g2-g4) of Opn1mw . g4 was added in ( d ) as indicated. c Relative expression of Rho and Opn1mw in retinas of subretinally injected WT mice. Saline-injected retinas served as a reference. n = 9 retinas (saline, REVeRT), n = 10 retinas (intein). d Relative expression of Rho and Opn1mw upon subretinal injection of Rho P23H/+ mice with dual REVeRT vectors expressing g1 and either two (REVeRT(3xg)) or three (REVeRT(4xg)) Opn1mw transactivating sgRNAs. Retinas injected with dual REVeRT vectors expressing a lacZ -targeting sgRNA served as a reference. The three retinas used for RNA-Seq analysis (Fig. S ) are shown in dark pink. n = 15 retinas (saline), n = 6 retinas (REVeRT(3xg)), n = 9 retinas (REVeRT(4xg)). Welch’s ANOVA with Dunnett T3 was used in c and d. e Immunostainings of retinal cryosections of Rho P23H/+ mice subretinally injected with REVeRT(3xg) (left). A control site distal from the injection site is shown as a reference (right). Magnification of areas marked by white rectangles are shown. Arrowheads indicate rod outer segments expressing M-opsin while lacking rhodopsin signal. Peanut agglutinin lectin (PNA) served as a cone outer segment marker. In the control site, M-opsin is only expressed in cones. Scale bar: 30 µm. Scatter plots show mean ± SEM. All source data are provided as a Source Data file.

Article Snippet: Welch’s ANOVA with Dunnett T3 (SAS) or Kruskal-Wallis test with Dunnett T3 (SDS) was used. j Western blots of cells transfected with versions of v1 containing (+pA) or lacking (−pA) a polyadenylation signal and with versions of v2 containing (+prom) or lacking (−prom) a promoter. k Ratiometric quantification of reconstitution efficiencies relative to cis-ctrl. n = 5 (pA), n = 3 (prom).

Techniques: Plasmid Preparation, Expressing, Control, Comparison, Knock-Out, Activation Assay, Injection, Saline, RNA Sequencing, Marker