Journal: PLOS Pathogens

Article Title: Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation

doi: 10.1371/journal.ppat.1011906

Figure Lengend Snippet: A) Representative images of HCT-8 cells infected with C . parvum and stained with V . villosa lectin, anti-DMC1 antibody and nuclear stain. Scale bars = 100 μM. Insets show enlarged image sections demonstrating the association of DMC1 staining with parasite vacuoles. B) Replicate experiments showing anti-DMC1 antibody staining and automated image analysis is sensitive and specific for screening purposes. HCT-8 cell monolayers grown in 384-well culture plates were infected with C . parvum , and then stained for the female gamont-specific marker DMC1 at 72 hpi. The DMC1 + parasite ratio was calculated for the 72 hpi timepoint, along with the z’ score for each replicate. N = 56 for each replicate, 28 each for wells stained with (red) or without (blue) inclusion of anti-DMC1 antibody. C) Combined assay strategy showing compound addition, imaging timepoints, and the different readouts. Each readout is normalized to the readout value of vehicle treated wells from the same plate to calculate percent inhibition. D) Scatter dot plot showing ReFRAME library screening results for the three assay readouts. Y axis denotes percent inhibition compared to DMSO control wells. Data for negative control (DMSO) wells and positive control (nitazoxanide) wells from all plates is also shown. The cutoff values in each readout (library mean ± 2 standard deviation) for hit identification are marked by straight lines in the corresponding plot. Hits selected for further validation shown in blue fell ≥ 2 standard deviations from the library mean for at least two of three assay readouts.

Article Snippet: The parasites were then stained with 1.33 μg/mL FITC-labeled V . villosa lectin (Vector Laboratories, FL-1231) in 1% BSA for 1 h at 37°C.

Techniques: Infection, Staining, Marker, Imaging, Inhibition, Library Screening, Control, Negative Control, Positive Control, Standard Deviation

Journal: PLOS Pathogens

Article Title: Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation

doi: 10.1371/journal.ppat.1011906

Figure Lengend Snippet: A) Categorization of confirmed inhibitors. B) Examples of dose response curves for compounds belonging to the indicated category of inhibitor. Each point is from a single biological replicate and each curve shows one of the three readouts: Black circles and lines (effect on asexual growth); red triangles and lines (effect on existing macrogamonts); pink diamonds and lines (effect on macrogamont differentiation). C) Macrogamont differentiation inducer S-1255. Representative images showing C . parvum 48 hpi of HCT-8 cell monolayers with or without S-1255 treatment (0.55 μM). V . villosa lectin was used to stain all parasites, and anti-DMC1 antibody to stain macrogamonts.

Article Snippet: The parasites were then stained with 1.33 μg/mL FITC-labeled V . villosa lectin (Vector Laboratories, FL-1231) in 1% BSA for 1 h at 37°C.

Techniques: Staining

Journal: PLOS Pathogens

Article Title: Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation

doi: 10.1371/journal.ppat.1011906

Figure Lengend Snippet: C . A) A summary of DAVID functional annotation results for significantly dysregulated C . parvum genes with different treatments, as well as female and asexual stage-specific gene set lists taken from the previously published dataset . Enrichments with an FDR of ≤ 0.01 from KEGG pathway are included only. The bars denote fold enrichment of the labeled pathway in the labeled gene set whereas points denotes the enrichment P-value. B) Heatmap of the average transcript per million (TPM) values of ribosomal protein genes of C . parvum at various timepoints (control) and in treatments with differentiation inhibitors (48 hpi). Each column represents a single gene encoding a ribosomal protein. TPM values are Z normalized across each column. Hierarchical clustering between the samples using the expression values of the ribosomal proteins was performed using the “complete” linkage algorithm and Euclidean distance metrics and is shown on the left of the heatmap. C) Nascent proteins in cultured C . parvum imaged using OP-puro. HCT-8 cells were infected with C . parvum and cultures were pulsed with the alkyne puromycin analog OP-puro 1 h prior to the indicated time points. Cells were then fixed, stained by CLICK-chemistry with Alexa 568-azide, and imaged by fluorescence microscopy. Treatment with the translation inhibitor cyclohexamide (CHX; 50 μM) served as a control for staining specificity. D) Quantified parasite protein translation vs. time of infection. The mean fluorescence intensity in the OP-puro channel of parasite vacuoles identified by V . villosa staining was measured. Note that out of focus light from ongoing host cell protein synthesis confounded the analysis. Data shown are the mean and SE for three independent experiments of the mean fluorescence intensity relative to the 36 h CHX control. E) Significant enrichment (p-value cutoff ≤ 0.5) of the two specific gene ontology (GO) terms during gene ontology enrichment analysis of apicomplexan genes that are differentially regulated between the listed life cycle stages. Dysregulated genes are defined as genes with a log2-fold difference of ≥ 1 at 10% false discovery rate (FDR).

Article Snippet: The parasites were then stained with 1.33 μg/mL FITC-labeled V . villosa lectin (Vector Laboratories, FL-1231) in 1% BSA for 1 h at 37°C.

Techniques: Functional Assay, Labeling, Control, Expressing, Cell Culture, Infection, Staining, Fluorescence, Microscopy

Journal: Theranostics

Article Title: Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion

doi: 10.7150/thno.67409

Figure Lengend Snippet: CD44-Tn and STn glycoproteoforms present high cancer specificity. A) Tn and STn antigens are not expressed in the healthy urothelium, are elevated in cancer, and increased in invasive tumors. The STn antigen, defined by affinity for the anti-tag-72 antibody [B72.3+CC49], is not expressed in the healthy urothelium and is elevated in cancer, being significantly overexpressed in muscle invasive bladder cancer (MIBC). The Tn antigen, based on VVA lectin immunoaffinity, is also not expressed in the healthy urothelium and is elevated in a subset of patients, specially at more advanced stages. B) CD44s colocalizes with Tn and STn antigens in MIBC. CD44 was diffusively expressed across the tumor section without a defined pattern. The STn antigen was observed both in superficial and invasive layers and the Tn antigen was found in scattered niches without a defined expression pattern. Immunohistochemistry also showed the co-localization of CD44 with Tn and STn positive areas in CD44s high tumors, exhibiting low amounts of other isoforms (according to RT-PCR, data not shown). The healthy urothelium expressed high amounts of CD44 and the Tn antigen is present in the cytoplasm of upper stratum umbrella cells while the STn antigen was not detected. C) In situ proximity ligation assays (PLA) supports CD44s-STn glycoproteoforms in MIBC and its presence within tumor invasive fronts. In situ proximity ligation assay showed close spatial proximity between CD44 and STn in the same cells, strongly supporting CD44-STn glycoproteoforms in tumors. This phenotype was mostly observed in invasive fronts of CD44s high tumors and was not detected in the healthy urothelium, suggesting cancer-specificity. D) Double staining immunofluorescence supports CD44s-Tn glycoproteoforms in bladder cancer. CD44s high tumors presented niches of cells co-expressing CD44 and Tn antigen, strongly suggesting CD44-Tn glycoproteoforms. This was not observed in the healthy urothelium. E) Glycosylation with Tn and STn antigens provides cancer specificity to CD44. CD44 was abundantly expressed in all studied healthy tissues. STn expression was either absent or low in secretions and cells facing the lumen of the respiratory, gastrointestinal, and colorectal tracts. Immunohistochemistry suggested STn and CD44 co-localization in the stomach, appendix, small intestine, colon, gallbladder, and white blood cells in MALT. PLA did not confirm these hypotheses, suggesting cancer specificity of CD44-STn glycoproteoforms. In healthy tissues, the Tn was restricted to the cytoplasm of goblet cells in the intestinal tract, Leydig cells in testicular tissue, pancreatic acini, hepatocytes, mucinous cells of the gastric epithelium, alveolar macrophages, and gallbladder epithelium. CD44 and Tn antigen co-expression was suggested in pancreatic tissue, testicle, and gallbladder, which was not confirmed by double staining immunofluorescence.

Article Snippet: FFPE bladder tumors and healthy tissue sections were screened by immunohistochemistry for CD44 and Tn and STn antigens, as previously described by Peixoto, A et al . . Tn antigen expression was evaluated using the biotinylated VVA lectin (Vector Laboratories, 40 mg/mL, 1 hour at 37 ºC; ) and the detection of STn and CD44 antigens were performed using the anti-tag-72 (B72.3 + CC49; Abcam, 0.5 mg/mL, overnight at 4 ºC; ) and anti-CD44 (1:5000, ab157107, Abcam; ) antibodies, respectively.

Techniques: Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, In Situ, Ligation, Proximity Ligation Assay, Double Immunofluorescence Staining