Journal: medRxiv

Article Title: Resident microbes shape the vaginal epithelial glycan landscape

doi: 10.1101/2022.02.23.22271417

Figure Lengend Snippet: ( A ) Representative confocal images of VECs stained with MAL-II lectin (Green) recognizing α2-3-linked sialic acids from individual women without BV (n=3) and with BV (n=12). Specimens were compared across varying levels of endogenous sialidase activity. Nuclei and bacterial cells (Blue) are stained with DAPI. Scale bars = 50 μm. ( B ) Quantification of endogenous sialidase activity in the vaginal swab eluates using the fluorogenic substrate Neu5Ac-4-methyl umbelliferone (4MU-sialic acid). Data shown is the rate of 4MU hydrolysis and the points represent values for individual women (n=16 without BV and n=20 with BV). Data in A and B is combined from 3 independent experiments. Images shown in A are from a subset of specimens used in B . ( C and D ) Fluorimetric quantification of 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labelled sialic acid (Neu5Ac) in isolated VEC N - and O -glycans, using reversed-phase chromatography after mild acid hydrolysis. Graph shows sialic acid quantification on glycans derived from protein extracts of pools of VECs from women with BV (Nugent scores 7-10, n=7 pools from a total N=45 specimens, with 5 or 10 specimens in each pool) and without BV (Nugent scores 0-3, No BV, n=8 pools from a total of N=55 specimens, with 5 or 10 specimens in each pool). Glycans derived from No BV VEC pools pretreated with A.u. sialidase were included as a control (n=2 pools with 10 specimens in each pool). Data in C and D are from same pools of VECs and was combined from 2 independent experiments. Error bars show standard deviation for each group, Mann–Whitney U test was used. ***P < 0.001, ****P<0.0001. A.u. sialidase = sialidase from Arthrobacter ureafaciens. A total of N=151 specimens were used to generate these data. A subset of VEC pools from C and D were also used for studies reported in , and . See methods for pooling rationale.

Article Snippet: To measure sialidase activity, 50 µL of vaginal swab eluate was diluted 3-fold into 100 mM sodium acetate, pH 5.5, containing 200 µM 4-methylumbelliferyl-Neu5Ac (4-MU-Sialic acid) (Goldbio) in a 96 well round bottom black polypropylene plates and sealed with an optical clear film.

Techniques: Staining, Activity Assay, Isolation, Reversed-phase Chromatography, Derivative Assay, Standard Deviation, MANN-WHITNEY

Journal: medRxiv

Article Title: Resident microbes shape the vaginal epithelial glycan landscape

doi: 10.1101/2022.02.23.22271417

Figure Lengend Snippet: ( A-D ) Cells from the same pool of VECs, from women with or without BV, were treated with (i) vector control (Control) or (ii) commercial Arthrobacter ureafaciens sialidase ( A.u. ) or (iii) recombinant Gardnerella NanH2 sialidase (NanH2) or (iv) recombinant Gardnerella NanH3 sialidase (NanH3) as indicated in the labels. (A) Fluorimetric quantification of sialic acid (Neu5Ac) released from No BV VECs treated with either vector control or sialidase enzymes. ( B, C ) Flow cytometric analysis of PNA binding to VECs. Galactose exposure was assessed by comparing PNA binding to A.u. sialidase treated cells from the same pool. ( B ) Galactose exposure on No BV VECs. ( C ) Galactose exposure on BV VECs (n=3 pools, with 2-3 specimens in each pool). (D) Representative confocal images of VECs from women with and without BV stained with PNA (Red) lectin under each condition. Nuclei and bacterial cells (Blue) are stained with DAPI. Each row shows analysis of one pooled specimen. Scale bars are 50 µm. Data in A - D combined from 2 independent experiments. For No BV groups treated with vector control or A.u. sialidase or G.v. NanH2 sialidase - n=10 pools, with 2 – 5 specimens in each pool; for the No BV group treated with G.v. NanH3 sialidase - n=8 pools,, with 2 – 5 specimens in each pool. **p<0.01 (Wilcoxon signed rank test). A total of N=38 specimens from individual women were used to generate the data in A - D . Data in 7A , 7B , and 7D is from the same No BV VEC pools. Data in 7C and 7D is from the same BV VEC pools. See methods for pooling rationale.

Article Snippet: To measure sialidase activity, 50 µL of vaginal swab eluate was diluted 3-fold into 100 mM sodium acetate, pH 5.5, containing 200 µM 4-methylumbelliferyl-Neu5Ac (4-MU-Sialic acid) (Goldbio) in a 96 well round bottom black polypropylene plates and sealed with an optical clear film.

Techniques: Plasmid Preparation, Recombinant, Binding Assay, Staining

Journal: Journal of renal nutrition : the official journal of the Council on Renal Nutrition of the National Kidney Foundation

Article Title: Variation in Sodium Intake and Intra-Individual Change in Blood Pressure in Chronic Kidney Disease

doi: 10.1053/j.jrn.2017.07.002

Figure Lengend Snippet: Relationships between urine sodium/urine creatinine and mean arterial pressure among 119 trial participants with CKD stage 3–4. The black lines denote regression lines (calculated from 3 or 4 data points) for each individual. The red line shows the mean of the slopes of regression lines for all the patients.

Article Snippet: A maximum of four 24-hour urine samples were collected from each patient, and urine sodium (Na) and creatinine (Cr) were measured (Litholink Corp [Chicago, IL]).

Techniques: