fresh frozen normal colon tissue  (BioIVT Inc)

Journal: Nature Communications

Article Title: NXPE1 alters the sialoglycome by acetylating sialic acids in the human colon

doi: 10.1038/s41467-025-59671-9

Figure Lengend Snippet: A Schematic showing samples and NGS strategy employed to identify regions associated with colon mPAS staining. Negative staining samples with a rare positive crypt were presumed to be of heterozygous genotype, where a spontaneous loss of heterozygosity in a stem cell caused positive staining of a crypt (right). Images shown are 10x. B Bar plot showing the number of SNPs correlated with the mPAS phenotype by chromosome as evaluated by WGS (green shading). Only SNPs on chromosome 11 contained perfect matches between genotype and phenotype in all samples (orange shading). C Region on chromosome 11q23.2 identified by whole genome sequencing as highly associated with mPAS staining. Samples that largely stain negative but contain rare positive crypts are suspected of having a heterozygous genotype and are listed separately (blue shading). Homozygous reference, heterozygous and homozygous alternate genotypes are noted by yellow, blue and red shading respectively. SNPs are shown on the y -axis based on their location on chromosome 11. Formal linkage disequilibrium analysis of this region is shown in figure . D Independent validation of WGS results. 3 × 3 table showing genotype for SNP rs661946 (located in the promoter of NXPE1 ) and mPAS staining phenotype on a set of 91 normal colon tissue samples. Allele frequencies are consistent with Hardy-Weinberg equilibrium (Haldane Exact = 0.45). A two-sided Fisher’s Exact Test for the genotype-phenotype relationship yields a p < 2.2e-16.

Article Snippet: Fresh frozen normal colon tissue was obtained from BioIVT (Westbury, NY).

Techniques: Staining, Negative Staining, Sequencing, Biomarker Discovery

Journal: Nature Communications

Article Title: NXPE1 alters the sialoglycome by acetylating sialic acids in the human colon

doi: 10.1038/s41467-025-59671-9

Figure Lengend Snippet: A mPAS, NXPE1 IHC and NXPE4 IHC on adjacent sections from normal colon FFPE tissue with the indicated genotypes for SNP rs661946. Heterozygous samples were primarily negative by mPAS, and positive for NXPE1 and NXPE4, but the images chosen show rare spontaneously mPAS positive crypts. Images are representative examples from more than 20 unique patients, and are shown at 20x. B Adjacent sections of normal colon FFPE tissue heterozygous for rs661946 containing spontaneous mPAS positive crypts stained by IHC for NXPE1, NXPE4 or known sialic acid O-acetyltransferase CASD1. Circles highlight the same crypt in each sample. Note that the crypt in patient 1 denoted with a * is positive only in the bottom half of the crypt. NXPE1 protein appears only in the portion of the crypt that is negative for mPAS. Images are representative examples of 10 patients with similar results and are shown at 20x. C Adjacent sections from the same samples in B now stained with SIGLEC-15 and detected by immunofluorescence and DAPI. SIGLEC-15 staining matches the mPAS staining pattern. All images are shown at 20x. D Adjacent sections stained with mPAS or immunofluorescence with SIGLEC-15 on normal FFPE colon tissue heterozygous for the NXPE1 promoter SNP rs661946. mPAS and SIGLEC-15 stain the same cells/crypts. All images are 20x and representative of experiments repeated in at least 10 unique patients.

Article Snippet: Fresh frozen normal colon tissue was obtained from BioIVT (Westbury, NY).

Techniques: Staining, Immunofluorescence

Journal: Nature Communications

Article Title: NXPE1 alters the sialoglycome by acetylating sialic acids in the human colon

doi: 10.1038/s41467-025-59671-9

Figure Lengend Snippet: A Flow cytometry with biotinylated SIGLEC-15 conjugated to APC on a pooled population of Jurkat cells infected with lentiviral clones containing a NXPE1 expression cassette. B Sialyl-Tn IHC staining on Jurkat wildtype and a pooled population of Jurkat cells overexpressing NXPE1. Images shown are representative of 2 unique pools of cells. Images are shown at 2x and 20x. C Fraction of total RNA expression from the allele in linkage with the T allele of rs661946 in normal colon tissue. Data is from targeted deep RNA-sequencing comparing the transcript levels of each allele using coding region heterozygous SNPs in NXPE1 in perfect genetic linkage with the promoter SNP rs661946 in all samples tested. More specifically, the C and A variants of rs524911 and rs10891692, respectively, are tightly linked with the T allele of rs661946, which lacked detectable NXPE1 expression above (Fig. ). The coding region SNP from the gene NXPE4 is shown as a control. Boxplots show median value, box indicates 75 th and 25 th quartile and whiskers extend to the farthest value (largest and smallest). n = 4 biological replicates for each boxplot. D Cartoon describing CRISPR-Cas9 knock in approach to change rs661946 in LS180 cells from T/T (homozygous VAR) to C/T (heterozygous) or C/C (homozygous REF). Red letter indicates base changed for rs661946. E mPAS and NXPE1 IHC staining on LS180 cells with the indicated genotypes for rs661946, with heterozygous (C/T) and homozygous REF (C/C) being created by CRISPR-Cas9 knock in. Cells positive for mPAS staining are circled. F Counts of the number of mPAS positive LS180 cells with the three possible genotypes for rs661946 as created by knock-in. Quantitation was performed using ImageJ on three different random images from each cell plug at 10x. Error bars indicate 95% confidence intervals, bar tops indicate mean. P values are as indicated by one-sided Student T-Test.

Article Snippet: Fresh frozen normal colon tissue was obtained from BioIVT (Westbury, NY).

Techniques: Flow Cytometry, Infection, Clone Assay, Expressing, Immunohistochemistry, RNA Expression, RNA Sequencing, Control, CRISPR, Knock-In, Staining, Quantitation Assay