Journal: bioRxiv

Article Title: Dopamine signaling drives skin invasion by human-infective nematodes

doi: 10.1101/2025.01.29.635547

Figure Lengend Snippet: A. Phylogenetic analysis shows the closest homologs of C. elegans CAT-2 (gray) in S. stercoralis (brown) and S. ratti (blue). Putative homologs in each genome were identified by performing TBLASTN searches of the C. elegans CAT-2A protein sequence (i.e ., the longest isoform) against either the S. stercoralis genome or the S. ratti genome in WormBase ParaSite WBPS18. The tree has the three members of the biopterin-dependent aromatic amino acid hydroxylases in C. elegans , of which CAT-2 is a member, and the predicted homologs in S. stercoralis and S. ratti . B. Co-expression of Ss-cat-2 and Ss-dat-1 in the putative dopaminergic (DA) neurons of S. stercoralis . Montage shows expression of the Ss-cat-2 transcriptional reporter in green, expression of the Ss-dat-1 transcriptional reporter in magenta, and co-expression of the two reporters and the relative positions of these neurons along the body of the iL3 in the differential interference contrast (DIC) overlay. The circles, asterisk, and arrow label the putative Ss- CEP, Ss- ADE, and Ss- PDE neurons, respectively. The iL3 is oriented with the dorsal side facing up and the ventral side facing down; the head is to the left. Scale bar = 100 µm. C-D. Violin plots show expression levels of Ss-cat-2 and Ss-dat-1 , expressed as log 2 counts per million (CPM), at the indicated life stages based on published RNA-seq datasets , , . **** p <0.0001; statistical tests for differential expression analysis were performed as previously described, with corrections for multiple comparisons . FLF = free-living female; iL3 = infective third-stage larva; ppL3 = post-parasitic third-stage larva; PF = parasitic female. Each dot indicates an independent replicate experiment. E. The cat-2 genes of C. elegans and S. stercoralis . Schematics show the gene models of Ce-cat-2 (isoform a), as annotated in WormBase WS292, and Ss-cat-2 . The gene model for Ss-cat-2 was initially derived from WBPS18 and then manually updated to include an additional exon ; the existence of this exon was supported by RNA-seq data , . Exons and introns are depicted as lavender boxes and black lines, respectively. The transcriptional start sites are indicated by black arrows. Ss-cat-2 has a single CRISPR/Cas9 target site, which is located in the second exon (depicted in red). Drawings are to scale and scale bar = 500 bp. F. Inactivation of Ss-cat-2 drastically alters skin-penetration behavior. Tracks show the skin-penetration behaviors of a representative wild-type iL3 that punctured and penetrated the skin and a representative Ss-cat-2 -/- iL3 that punctured but did not complete penetration. Representative worms were defined as in . The key details the behavioral motifs that were tracked. G. Inactivation of Ss-cat-2 severely impairs skin penetration. Bar graphs show the percentage of wild-type and Ss-cat-2 -/- iL3s that completed skin penetration. n = 21 iL3s per genotype. **** p <0.0001, Fisher’s exact test. H. Ss-cat-2 -/- iL3s pushed and punctured the skin for less time than control iL3s. Violin plot depicts the percentage of time on skin that control and Ss-cat-2 -/- iL3s spent engaging in pushes or punctures. n = 20-21 iL3s per genotype. **** p <0.0001, Mann-Whitney test. The iL3s that had initiated penetration by the time the recording started were excluded from this analysis. I. The pushing bouts of Ss-cat-2 -/- iL3s are shorter than those of control iL3s. For each worm, the duration of each individual pushing bout was averaged and then plotted. n = 20 iL3s per genotype. *** p <0.001, Mann-Whitney test. J. Inactivation of Ss-cat-2 inhibits punctures. Violin plot depicts the time taken by control vs. Ss-cat-2 -/- iL3s to puncture the skin for the first time since placement on skin. n = 21 iL3s per genotype. ** p <0.01, Mann-Whitney test. The dotted line at y = 5 indicates the time at which the assay ended; the dots above this line indicate animals that failed to puncture the skin by the end of the assay period. K. Ss-cat-2 -/- iL3s frequently reverse after a push or puncture event. Violin plot shows the percentage of pushes or punctures that were followed by backward locomotion that lasted at least 1 s for each genotype. n = 21 iL3s per genotype. **** p <0.0001, Mann-Whitney test. L. Ss-cat-2 -/- iL3s frequently abort penetration attempts. Violin plot depicts the percentage of penetration attempts, as defined by instances that the worm has punctured and partially entered the skin, that were aborted. n = 11-21 iL3s per genotype. ** p <0.01, Mann-Whitney test. For H-L, dots depict individual worms, dashed lines indicate medians, and dotted lines indicate interquartile ranges. Behavioral parameters plotted in G-L were obtained from 3 independent replicate experiments.

Article Snippet: This guide RNA sequence was synthesized and fused with the promoter of the S. ratti U6 gene, the sequence of the sgRNA scaffold, and the S. ratti U6 3′ UTR by GenScript, as previously described .

Techniques: Sequencing, Expressing, RNA Sequencing Assay, Derivative Assay, CRISPR, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Dopamine signaling drives skin invasion by human-infective nematodes

doi: 10.1101/2025.01.29.635547

Figure Lengend Snippet: A. Phylogenetic analysis shows the closest homologs of C. elegans TRP-4 (gray) in S. stercoralis (brown). Putative homologs in each genome were identified by performing TBLASTN searches of the C. elegans TRP-4 protein sequence against the S. stercoralis genome in WBPS18. The tree has all known TRP family members in C. elegans - and the predicted homologs in S. stercoralis . B. Co-expression of Ss-trp-4 and Ss-dat-1 in the putative dopaminergic (DA) neurons of S. stercoralis . Montage shows expression of the Ss-trp-4 transcriptional reporter in green, expression of the Ss-dat-1 transcriptional reporter in magenta, and co-expression of the two reporters and the relative positions of these neurons along the body of the iL3 in the DIC overlay. The circles, asterisk, and arrow label the putative Ss- CEP, Ss- ADE, and Ss- PDE neurons, respectively; we never observed expression of the Ss-trp-4 reporter in Ss- PDE. The iL3 is oriented with the dorsal side facing up and head to the left. Scale bar = 100 µm. C. Violin plot shows expression levels of Ss-trp-4 , expressed as log 2 counts per million (CPM), in the indicated life stages based on published RNA-seq datasets , , . **** p <0.0001; statistical tests for differential expression analysis were performed as previously described, with corrections for multiple comparisons . FLF = free-living female; iL3 = infective third-stage larva; ppL3 = post-parasitic third-stage larva; PF = parasitic female. Each dot indicates an independent replicate experiment. D. The trp-4 genes of C. elegans and S. stercoralis . Schematics show the gene models of Ce-trp-4 and Ss-trp-4 , which were derived from annotations in WS292 and WBPS18, respectively. Notably, the Ss-trp-4 gene model has a 5′ UTR that is not annotated in WBPS18 but is supported by published RNA-seq data , . Exons, introns, and UTRs are depicted as pink boxes, black lines, and gray boxes, respectively. The transcriptional start sites are indicated by black arrows. Ss-trp-4 has two CRISPR/Cas9 target sites in the first exon, which are depicted in red; we used two distinct sgRNAs, one targeting each CRISPR site, to inactivate Ss-trp-4 and generate a mutant stable line. Drawings are to scale and scale bar = 1000 bp. E. Ss-trp-4 mutants have reduced skin-penetration drive. Tracks show the skin-penetration behaviors of a representative wild-type iL3 that punctured and completed penetration and a representative Ss-trp-4 -/- iL3 that neither punctured nor completed penetration. Representative worms were defined as in . The key details the behavioral motifs that were tracked. F. Inactivation of Ss-trp-4 severely inhibits skin penetration. Bar graph shows the percentage of wild-type and Ss-trp-4 -/- iL3s that completed skin penetration. n = 24 iL3s per genotype. **** p <0.0001, Fisher’s exact test. G. Ss-trp-4 -/- iL3s pushed and punctured the skin for less time than control worms. Violin plot depicts the percentage of time on skin that control and Ss-trp-4 -/- iL3s spent engaging in pushes or punctures. n = 23-24 iL3s per genotype. **** p <0.0001, Mann-Whitney test. The iL3s that had initiated penetration by the time the recording started were excluded from this analysis. H. The pushing bouts of Ss-trp-4 -/- iL3s are shorter than those of control iL3s. For each worm, the duration of each individual pushing bout was averaged and then plotted. n = 23 iL3s per genotype. ** p <0.01, Mann-Whitney test. I. Inactivation of Ss-trp-4 inhibits punctures. Violin plot depicts the time taken by control vs. Ss-trp-4 -/- iL3s to puncture the skin for the first time since placement on skin. n = 24 iL3s per genotype. **** p <0.0001, Mann-Whitney test. The dotted line at y = 5 indicates the time at which the assay ended; the dots above this line indicate animals that failed to puncture the skin by the end of the assay. J. Ss-trp-4 -/- iL3s frequently reverse after a push or puncture event. Violin plot shows the percentage of pushes or punctures that were followed by backward locomotion that lasted at least 1 s for each genotype. n = 23-24 iL3s per genotype. *** p <0.001, Mann-Whitney test. K. Ss-trp-4 -/- iL3s frequently abort penetration attempts. Violin plot depicts the percentage of penetration attempts, as defined by instances that the worm has punctured and partially entered the skin, that were aborted. n = 9-24 iL3s per genotype. *** p <0.001, Mann-Whitney test. For G-K, dots depict individual worms, dashed lines indicate the median, and dotted lines indicate the interquartile range. Behavioral parameters plotted in F-K were obtained from 3 independent replicate experiments.

Article Snippet: This guide RNA sequence was synthesized and fused with the promoter of the S. ratti U6 gene, the sequence of the sgRNA scaffold, and the S. ratti U6 3′ UTR by GenScript, as previously described .

Techniques: Sequencing, Expressing, RNA Sequencing Assay, Derivative Assay, CRISPR, Mutagenesis, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer

doi: 10.1101/2023.03.19.533333

Figure Lengend Snippet: ( A ) Oxygen tension (y-axis) as determined by an OxyLite probe in tumors from KPC mice breathing ambient air or pure oxygen (x-axis). Dark blue circles represent averages per tumor and boxplots illustrate their distribution. Light blue circles represent repeat measurments per tumor. ( B ) HIF target genes (orange) controlled directly or indirectly by BMAL2 (yellow). Indirect control involves both BMAL2’s negative influence on first (dark blue) or second tier (light blue) RP repressing HIF target genes, and positive influence on first (dark red) and second (light red) tier RP activating HIF target genes. ( C ) Fold change in cell growth relative to cells expressing non-targeting sgRNA in normoxic (21% O2) and hypoxic (1% O2) conditions ( D ) Number of migrated cells for cells expressing non-targeting or BMAL2-directed sgRNA in the indicated oxygen environment. P-values are derived from testing the indicated coefficients from a linear regression model. Significant interaction term suggests synergistic effects of BMAL2 pertubation and hypoxia on cell migration. ( E ) Media pH levels after 72 hours incubation. P-values derived from Welch’s t-test ( F ) Fold change in lactate levels in the indicated oxygen environment expressing either non-targeting or BMAL2-directed sgRNA ( G ) Western Blot for HIF1a, HIF2a,

Article Snippet: The sgRNA (small-guide RNA) to knocking-out BMAL2 as well as non-targeting (NT) sequence were purchased from GenScript (Piscataway, NJ) using pLentiGuide-Puro vector as a backbone.

Techniques: Control, Expressing, Derivative Assay, Migration, Incubation, Western Blot