Journal: bioRxiv

Article Title: HPV16 Utilizes Phospholipase C(s) for its Genome Egress

doi: 10.64898/2026.09.03.749318

Figure Lengend Snippet: HeLa:Cas9 cells in 10cm plates were transfected with overexpression constructs for HPV16L2, encoding a C-terminal HA tag, and either an overexpression construct for PLCδ4 or PLCγ1, both containing the sequence for both C-terminal FLAG tag and c-Myc tag, or pCMV6, an empty vector control. Transfected cells were harvested 24h post-transfection, lysed in buffer containing 1% NP40 and 0.1% SDS, and then (cleared) lysates were incubated with magnetic beads coupled to anti-HA antibodies to pull down L2. Co-immunoprecipitation (CoIP) of PLCδ4 in bead elutions (“Elutes”) was determined using Western blot. Western blotting against the HA and c-Myc tags was used to confirm the presence of L2, PLCδ4, and PLCγ1 in “elutes” and “cleared lysates”. Blots are representative of three independent experiments ( N =3).

Article Snippet: PLCβ4 and PLCδ4 gene edits in selected clonal HeLa cells were further confirmed via sequencing with Plasmidsaurus sequencing services using Oxford Nanopore Technology.

Techniques: Transfection, Over Expression, Construct, Sequencing, FLAG-tag, Plasmid Preparation, Control, Incubation, Magnetic Beads, Co-Immunoprecipitation Assay, Western Blot

Journal: bioRxiv

Article Title: Identification of soil microbes associated with real-time plastic degradation using in situ conductivity sensors

doi: 10.64898/2026.08.16.745074

Figure Lengend Snippet: Step 1 ) The sensors are printed with biodegradable PHBV, poly(3-hydroxybutuyrate- co -3-hydroxyvalerate), on one side and non-biodegradable PMMA, poly(methyl methacrylate) on the other side as a control. Step 2 ) UV-sterilized sensors are inserted into soil mesocosms and monitor PHBV degradation in real-time as an increase in resistance, which we normalize to the non-degraded PMMA. Step 3 ) At the end of the mesocosm incubation, the sensors are removed and we can summarise the PHBV degradation signal to identify which soils have the highest capacity to degrade PHBV. Step 4 ) PHBV and PMMA material from sensors incubated in the highest PHBV-degrading soils are scraped off and used as starting material for culture-independent ( Step 5 , left panel) and culture-dependent ( Step 6 , right panel) approaches to identify novel PHBV degraders. Step 5, left ) We used the PHBV and PMMA material from high degrading soils for 16S rRNA and ITS marker gene sequencing to identify microbes enriched on degraded PHBV compared to non-degraded PMMA from each in situ sensor. Step 5, right ) We also used the PHBV material from high degrading soils to isolate novel PHBV degraders on agar plates with a PHBV overlay (inset). Isolates that formed clearing zones on plates were cultured in liquid media to measure their continued capacity to degrade PHBV. We also measured the respiration rates of isolates growing with PHBV as their sole carbon source to confirm PHBV catabolism.

Article Snippet: DNA aliquots from two of our isolates, Streptomyces sp. [2CO Garden Isolate], Lysinibacillus sp. [2CO Garden Isolate] were sequenced by Plasmidsaurus using their bacterial genome sequencing service.

Techniques: Control, Incubation, Marker, Sequencing, In Situ, Cell Culture

Journal: Cancer Research

Article Title: GNAQ Induces Melanomagenesis in Mitfa-Independent Melanocyte Progenitors in a Zebrafish Model of Uveal Melanoma

doi: 10.1158/0008-5472.CAN-25-5299

Figure Lengend Snippet: Single-cell profiling of uveal melanoma (UM) and the TME in adult zebrafish. A, UMAP representation of scRNA-seq from 3 dissociated WT eyes harboring UM. UM tumors were generated by TEAZ-Eye (sequencing replicates, n = 2). Control eyes represent pools of 2 tumor-free paired WT eyes and 4 sibling-matched WT eyes (sequencing replicates, n = 2). Annotated cell clusters as labeled. B, UMAP and feature plot showing eGFP and GNAQ Q209L expression in control eyes and UM tumor. Red dotted line highlights the melanocyte and UM clusters. C, Comparison of UM tumor marker genes with the markers of cell types within the control eye ME. Only genes with a log 2 FC > 1 and FDR < 0.01 were included in the analysis. D, Dot plot representing enriched genes (log 2 FC > 1; FDR < 0.01) in choroidal cell populations, UM tumors cells, immune cell populations, fibroblasts, Schwann cells, pericytes, and vascular endothelial cells as shown. Dot size indicates the percentage of cells expressing each gene; color intensity (gray to blue) reflects normalized average expression levels (low to high). E, UMAP clustering of cells in the choroidal ME. Annotated cell clusters as labeled. F, Comparison of relative cluster sizes between the 2 conditions, control and TEAZ-Eye, shown in E . Schwann cells serve as a control cluster, displaying no change in frequency between conditions. Gray = TEAZ-Eye; blue = control. G, Venn diagram representing the overlap of genes between UM tumors and choroidal melanocytes compared with the cells in their respective MEs. Enriched genes for each condition were identified by differential expression analysis comparing melanocytes with the ME or tumor cells to the TME using a threshold of log 2 FC > 1 and FDR < 0.01. Genes of interest have been labeled in their respective subsets; bolded genes represent genes associated with worse overall survival or disease-free survival in human UM TCGA datasets. H, Dot plot showing enriched genes (log 2 FC > 1; FDR < 0.01) in UM tumor cells, choroidal melanocytes, or the TME. Dot size indicates the percentage of cells expressing each gene; color intensity (gray to blue) reflects normalized average expression levels (low to high). I, HOMER promoter motif enrichment analysis of genes enriched in UM tumors vs the TME in G . The top ranked motif for “known” and “ de novo ” motifs are shown with their corresponding P values. J–L, Kaplan–Meier disease-free survival and overall survival curves generated using UM TCGA data. Patients stratified by relative expression levels of KIT , FABP3 , or LGALS2 . Gene expression thresholds were defined by median expression within each cohort. Log-rank P values as shown. Dotted lines represent 95% confidence interval. M, Dot plot showing enriched genes (log 2 FC > 1; FDR < 0.01) in CAFs, normal fibroblasts (NF), or the TME without CAFs. Dot size indicates the percentage of cells expressing each gene; color intensity (gray to blue) reflects normalized average expression levels (low to high). Genes have been grouped according to their labeled functions.

Article Snippet: Entire plasmid was sequenced using Plasmidsaurus sequencing service.

Techniques: Single Cell, Generated, Sequencing, Control, Labeling, Expressing, Comparison, Marker, Quantitative Proteomics, Gene Expression

Journal: Cancer Research

Article Title: GNAQ Induces Melanomagenesis in Mitfa-Independent Melanocyte Progenitors in a Zebrafish Model of Uveal Melanoma

doi: 10.1158/0008-5472.CAN-25-5299

Figure Lengend Snippet: Comparison of GNAQ-driven tumors and primary melanocytes in the skin and eyes of adult zebrafish. A, UMAP representation of GNAQ-positive tumors generated using TEAZ-Eye and TEAZ-Skin, along with their respective control tissues using scRNA-seq. Tumor samples include 3 dissociated WT eyes harboring uveal melanoma (UM) tumors generated via TEAZ-Eye and 2 dissociated WT skin UM tumors generated via TEAZ-Skin ( n = 2 sequencing replicates each). Control eye samples consist of a pool of 2 uninjected tumor paired WT eyes, 3 mock-injected sibling-matched WT eyes, and 4 uninjected sibling-matched WT eyes ( n = 3 sequencing replicates). Control skin samples consist of dissociated normal skin from 2 WT zebrafish ( n = 2 sequencing replicates). Cell clusters are annotated as tumor cells (red = eye; blue = skin), TME cells shared by both eye and skin samples (light gray), and TME cells specific to the eye samples (dark gray). B, Venn diagram showing overlapping genes between TEAZ-Eye, TEAZ-Skin, choroidal melanocytes, and skin melanocytes compared with the cells in their respective MEs. Enriched genes for each condition were identified by differential expression analysis comparing melanocytes with the ME or tumor cells to the tumor TME, using a threshold of log 2 FC > 0.5 and FDR < 0.01. Bar chart displaying overlaps between enriched gene sets from B . Percentages represented as the number of overlapping genes over the total unique genes in both gene sets. C, UMAP representation of primary melanocytes from both eye (salmon) and skin (teal). Volcano plot of DEGs between eye and skin primary melanocytes (log 2 FC > |0.5| and FDR < 0.01). D, GSEA enrichment plots of pathways that were enriched in skin melanocytes and of pathways enriched in eye melanocytes from C . P values and FDRs as labeled. E, UMAP representation of GNAQ Q209L -induced melanoma from both eye (salmon) and skin (teal). Volcano plot of DEGs between eye and skin tumors (log 2 FC > |0.5| and FDR < 0.01). F, GSEA enrichment plots of pathways that were enriched in skin melanoma and of pathways enriched in eye melanoma from E P values and FDRs as labeled.

Article Snippet: Entire plasmid was sequenced using Plasmidsaurus sequencing service.

Techniques: Comparison, Generated, Control, Sequencing, Injection, Quantitative Proteomics, Labeling

Journal: Cancer Research

Article Title: GNAQ Induces Melanomagenesis in Mitfa-Independent Melanocyte Progenitors in a Zebrafish Model of Uveal Melanoma

doi: 10.1158/0008-5472.CAN-25-5299

Figure Lengend Snippet: mitfa -independent melanocyte progenitor cells are expanded in mitfa -deficient zebrafish and are susceptible to GNAQ Q209L transformation. A, H&E analysis representation of the retina and choroid in WT (left), nacre (middle), and casper (right) zebrafish (20× magnification). Note the loss of melanocytes (red arrows) in the choroid of nacre and casper eyes. Scale bar, 50 μm. B, Immunofluorescent analysis of transgenic Tg ( mitfa : GFP ); mitfa w2/w2 ( nacre ) zebrafish using an anti-GFP antibody. Sections were counterstained with DAPI to visualize nuclei. Representative images showing GFP-positive cells in the ciliary body and choroid are shown by yellow arrowheads. Secondary-only antibody was used as background control. Scale bar, 20 μm. C, UMAP representation of scRNA-seq data from 3 paired dissociated casper eyes and from 3 casper eyes harboring uveal melanoma (UM) tumors induced using strategy B via TEAZ-Eye ( n = 1, sequencing replicate). Annotated cell clusters are labeled. Progenitor cells and UM tumor clusters are outlined with dashed red lines. Representative brightfield images of a normal eye and a UM tumor in casper zebrafish are shown in the top left corner of the UMAP. Scale bar, 250 μm. D, Reclustered UMAP representing progenitor cells from control eyes and UM tumor cells from TEAZ-Eye–injected casper zebrafish. Heterogeneous cell populations as labeled. E, Reclustered UMAP from D showing melanocyte progenitor cells from control eyes in red and UM tumor cells from TEAZ-Eye in blue. Feature plots for pax3a , foxd3 , and sox10 expression are shown; dashed line marks the progenitor cell population. F, Dot plot illustrating DEGs (log 2 FC > 0.5; FDR < 0.05) between heterogeneous cell populations in D . Dot size indicates the percentage of cells expressing each gene; color intensity (blue to red) reflects normalized average expression levels (low to high). G, Kaplan–Meier curves comparing tumor-free survival in WT and casper zebrafish following TEAZ-Eye injection using strategy B plasmids for mitfa conditional KO. WT zebrafish were injected with either strategy B plasmids plus nontargeting gRNAs ( n = 9) or strategy B plasmids plus mitfa -targeting gRNAs ( n = 9). Casper zebrafish were injected with strategy B plasmids and mitfa -targeting gRNAs ( n = 9). H, Representative brightfield and GFP-overlay images of tumors that formed in casper (9 of 9) and WT zebrafish injected with mitfa -targeting gRNAs (8 of 9). Metastatic cells are indicated by red arrows. Scale bar, 0.5 cm. I, UMAP obtained after clustering GFP -positive cells sorted from Tg ( mitfa : GFP ) and Tg ( mitfa : GFP ); mitfa w2/w2 ( nacre ) zebrafish embryos at 28 hpf. Annotated cell clusters as labeled. J, Comparison of relative cluster size for the 2 genotypes; Tg ( mitfa : GFP ) and Tg ( mitfa : GFP ); mitfa w2/w2 ( nacre ) in I . Scale bar, 0.5 mm. K, Dot plot illustrating DEGs (log 2 FC > 0.5; FDR < 0.05) in melanophores and progenitor cells in mitfa w2/w2 and sibling embryos at 28 hpf. Dot size indicates the percentage of cells expressing each gene; color intensity (gray to red) reflects normalized average expression levels (low to high). CH, choroid; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; POS, photoreceptor outer segment; VIT, vitreous. **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Entire plasmid was sequenced using Plasmidsaurus sequencing service.

Techniques: Transformation Assay, Transgenic Assay, Control, Sequencing, Labeling, Injection, Expressing, Eye Injection, Comparison