Journal: bioRxiv

Article Title: Metabolic imprinting drives epithelial memory during mucosal fungal infection

doi: 10.1101/2025.07.11.664387

Figure Lengend Snippet: A. Schematic of the C. albicans reinfection model and experimental timeline for assessment. Created with BioRender.com B . Oral fungal burden in immunocompetent wild-type mice at indicated time points during reinfection. N=6 ; Two-tailed Mann–Whitney Test. The y-axis represents the limit of detection (20 colony-forming units [CFUs] per gram of tissue). C. Heat map of chemokine and cytokine levels in tongue homogenates from immunocompetent wild-type mice after 11 days of primary infection and 8h post-reinfection . N=4-6 . D. Recruitment of immune cells to the tongue at 8h of reinfection. N=6 ; Two-tailed Mann–Whitney Test. E. Oral fungal burden in Rag1 −/− and Ccr2 −/− mice at 2 days post-reinfection. N=6 ; Two-tailed Mann–Whitney Test. F. Oral fungal burden in neutrophil-depleted mice 1 day post-reinfection. N=6 ; Two-tailed Mann–Whitney Test.

Article Snippet: C57BL/6J, Rag1 −/− (B6.129S7- Rag1 tm1Mom /J; Strain# 002216; non-leaky) and Ccr2 −/− ( B6.129S4-Ccr2 tm1Ifc /J; Strain#:004999) mice were purchased from The Jackson Laboratory.

Techniques: Two Tailed Test, MANN-WHITNEY, Infection

Journal: Nature Communications

Article Title: Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae

doi: 10.1038/s41467-025-61206-1

Figure Lengend Snippet: a RAG1 or RAG2 variants were tagged with eGFP on the C-terminus and integrated in BY4742-NAB2-mCherry yeast. Median eGFP fluorescence was then measured using flow cytometry after overnight culture in YPG. b Confocal microscopy images of eGFP-tagged RAG1 and RAG2 protein variants (green) in a NAB2-mCherry strain which natively highlights the nucleus (red). Scale bar represents 5 μm; all images are at the same scale. Gains have been adjusted from the original images to aid visual comparison. The brightfield channel for each image can be found in Supplementary Fig. . c Nuclear localization was estimated by calculating the Pearson correlation coefficient between the RAG-eGFP and NAB2-mCherry signals of the confocal images. d Confocal microscopy images of eGFP-tagged RAG1 protein variants (green) in a NOP56-mCherry strain which natively highlights the nucleolus (red). As in ( b ), scale bar represents 5 μm. The complete set of images can be found in Supplementary Fig. . e Nucleolar localization was estimated by calculating the Pearson correlation coefficient between the RAG-eGFP and NOP56-mCherry signals of the confocal images. RFU = relative fluorescence units, NLS = nuclear localization signal. In ( a , c , and e ) data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a one-way ANOVA and Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). From left to right, the highlighted p values are, in ( a ), p = 0.0014, <0.0001, 0.0012, and 0.0001; in ( c ), p = <0.0001, 0.0499, 0.0016, and 0.0194; and in ( e ), p = <0.0001 and <0.0001. Source data are provided in the Source Data file.

Article Snippet: To prepare recombination genes for integration, first yeast-codon-optimized, full-length RAG1 and RAG2 (human and mouse sequences for each), and human HMGB1 were synthesized as linear fragments by Twist Bioscience.

Techniques: Fluorescence, Flow Cytometry, Confocal Microscopy, Comparison

Journal: Nature Communications

Article Title: Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae

doi: 10.1038/s41467-025-61206-1

Figure Lengend Snippet: a Diagram of pY112-CJA-UP-H20 recombination target plasmid. Triangles represent RSSs. Cutting by RAG1/2 leads to homology-assisted DNA repair of the G418R gene that confers resistance to the antibiotic G418. b Yeast strains were engineered with both RAG1 and RAG2 genes (either full-length, core, or core NLS variants) and optionally HMGB1. After 4-d induction in SG-Leu media, cell cultures were plated on Leu, G418 plates and colonies were counted after outgrowth. c G418R recombination with yeast strains assessing the combination of full-length RAG2 with RAG1core. Cells were plated after a 4-d induction. d G418R recombination with alternate truncations of RAG1 that include more regions of the full-length protein than the initial RAG1core construct (which contains amino acids 383-1006). All strains include full-length RAG2 and HMGB1. Cells were plated after a 4-d induction. C = core protein, F = full-length protein, N-C = core protein with N-terminal nuclear localization signal, NLS = nuclear localization signal, CFU = colony forming unit. In ( b – d ) strains that appear in multiple plots are highlighted with a color, and data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a one-way ANOVA and Tukey test (ns = not significant, ** p < 0.01, and **** p < 0.0001). From left to right, the highlighted p values are, in ( b ), p = <0.0001 and 0.0042; in ( c ), p = <0.0001 and <0.0001; and in ( d ), p = <0.0001 and 0.0148. Source data are provided in the Source Data file.

Article Snippet: To prepare recombination genes for integration, first yeast-codon-optimized, full-length RAG1 and RAG2 (human and mouse sequences for each), and human HMGB1 were synthesized as linear fragments by Twist Bioscience.

Techniques: Plasmid Preparation, Construct

Journal: Nature Communications

Article Title: Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae

doi: 10.1038/s41467-025-61206-1

Figure Lengend Snippet: a G418R recombination assay using substrates with mutated RSSs that cannot be recognized by the RAG proteins (Mut12 and Mut23) or nonstandard pairings (12 + 12 and 23 + 23). Cells were cultured for 4 d in SG-Leu prior to plating. b G418R recombination assay showing the effect of adjusting homology length between the two halves of the G418R gene in the recombination target plasmid. Cells were plated after an 8-d induction. c Diagram of the original intersignal region for “UP” plasmid design and the shorter variant sequences between the RSSs that were tested. For all alternatives, 20 bp of homology between the two halves of G418R was used. d G418R recombination assay varying the intersignal region between RSSs. See ( c ) for region descriptions. Cells were plated after a 4-d induction. e Similar to ( d ), G418R recombination assay testing additional intersignal regions between RSSs. Cells were plated after a 4-d induction. f G418R recombination assay using enhanced pY112-CJA-U-H20 plasmid with additional recombination strains. Recombination strains contained RAG2, HMGB1, and the specified variant of RAG1. Cells were plated after 4-d induction. In all figures, BY4742 are wild-type cells that do not contain V(D)J recombination genes. CFU = colony forming unit. In ( a , b , d – f ) data are presented as mean values ± SD; n = 3 biological replicates. In ( a , d , and e ), statistical significance was calculated using a two-way ANOVA and Tukey test. In ( b ), significance was calculated using multiple two-sided t-tests with a Holm-Sidak correction for multiple comparisons. In ( f ), statistical significance was calculated using a one-way ANOVA and Tukey test (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). From left to right, the highlighted p values are, in a, p = 0.9811, 0.9916, 0.9916, 0.7759, 0.9602, and <0.0001; in ( b ), p = 0.2606, 0.0078, 0.0028, and 0.0004; in ( d ) p = 0.0329, < 0.0001; in ( e ) p = 0.0017 and <0.0001; and in ( f ) p = 0.0001 and 0.1147. Source data are provided in the Source Data file.

Article Snippet: To prepare recombination genes for integration, first yeast-codon-optimized, full-length RAG1 and RAG2 (human and mouse sequences for each), and human HMGB1 were synthesized as linear fragments by Twist Bioscience.

Techniques: Recombination Assay, Cell Culture, Plasmid Preparation, Variant Assay

Journal: Nature Communications

Article Title: Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae

doi: 10.1038/s41467-025-61206-1

Figure Lengend Snippet: a Diagram of the integrated SJUE target in chromosome XV (Chr XV). Cutting by RAG1/2 can lead to NHEJ repair which fuses the RSSs together and forms a signal joint. Such an event permanently removes eGFP and URA3 , leading to cells that do not fluoresce and are 5-FOA-resistant. b The SJUE target was integrated in both a recombination-competent (AC518: NLS-RAG1core, RAG2, and HMGB1) and recombination-incompetent (BY4742) strain. After an 8-d induction in YPG media, cells were plated on SD, 5-FOA plates. Both eGFP-positive and eGFP-negative colonies were separately counted. CFU = colony forming unit. In ( b ), data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated using a two-way ANOVA with repeated measures across fluorescence types and Fisher LSD test. Source data are provided in the Source Data file.

Article Snippet: To prepare recombination genes for integration, first yeast-codon-optimized, full-length RAG1 and RAG2 (human and mouse sequences for each), and human HMGB1 were synthesized as linear fragments by Twist Bioscience.

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae

doi: 10.1038/s41467-025-61206-1

Figure Lengend Snippet: a Genetic map of human RAG1 showing the core region and the location of five mutations which have been shown to cause varying degrees of immunodeficiency. Certain domains of RAG1 and their function are highlighted, including the zinc dimerization domain (ZDD) and nonamer-binding domain (NBD). b Table of human RAG1 mutants and their corresponding mutation in mouse RAG1. Additionally, the previously reported activity of the mutants in mammalian cells is given along with the activity measured in yeast in this work. WT = wild-type RAG1. c G418R recombination with pY112-CJA-U-H20 plasmid. BY4742 are the base strain of yeast. All other strains contain mouse RAG1core with a mutation (or without in the case of wild type, WT), RAG2, and HMGB1. Cells were plated after a 4-d induction. CFU = colony forming unit. In ( c ), data are presented as mean values ± SD; n = 3 biological replicates. Source data are provided in the Source Data file.

Article Snippet: To prepare recombination genes for integration, first yeast-codon-optimized, full-length RAG1 and RAG2 (human and mouse sequences for each), and human HMGB1 were synthesized as linear fragments by Twist Bioscience.

Techniques: Binding Assay, Mutagenesis, Activity Assay, Plasmid Preparation