Journal: Micromachines

Article Title: A Novel 3D-Printed and Miniaturized Periodic Counter Current Chromatography System for Continuous Purification of Monoclonal Antibodies

doi: 10.3390/mi15030382

Figure Lengend Snippet: Chromatogram of a continuous membrane adsorber-based CEX run using three membrane adsorbers numbered MA1–MA3. A total of 4 cycles (55 min) and a cleansing post cycle using membrane adsorbers with a volume of 0.41 mL were performed. Small regular plateaus with peaks at the end emerge during loading.

Article Snippet: The consecutive polishing step was performed via cation exchange chromatography using membrane adsorbers (Sartobind ® S15, Sartorius Stedim Biotech, Göttingen, Germany).

Techniques: Membrane

Journal: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: ( A ) SARS-Cov-2 infection for 90 min (10 5 viral particles exposed apically using enteroid monolayers from 3 different donors) and studied at 48 h reduced mRNA for ACE2, NHE3, and DRA, but not CFTR, Results are means ±sem. *,n=4, p<0.05; ( B , C ) Virus alone reduced total NHE3 expression and the percent of total NHE3 in the apical domain. B , untreated control. C , monolayer infected with 10 5 virus particles for 90 min and studied 2 days later. Representative confocal XY images at level of the BB (i) and XZ cross sections (ii). NHE3 = green; F-actin = red, nuclei = blue. Scale bar = 20µm. SARS-CoV-2 infection decreased total NHE3 fluorescence intensity measured in projections of at least 100 XZ frames in each of two independent experiments from 8879.1 ±1852.8 in control monolayers to 3092.1 ± 1096.2 (p=0.036); SARS-CoV-2 infection decreased the BB NHE3 fluorescence also measured in at least100 XZ frames from 25410.5 ± 5466.7 in control monolayers to 4823.8 ± 4284.8 (p=0.038) in virus infection monolayers.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Infection, Virus, Expressing, Fluorescence

Journal: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: Human ileal enteroid monolayers were exposed apically to VLPs with either S-2PP or S-D614G forms of Spike protein of SARS-CoV-2 in the presence of IL-6/IL-8 (48h, 50 ng/ml). At 48 hours post exposure to VLPs at times 0 and 24 h, total RNA was isolated and mRNA expression of ACE2, NHE3, DRA, and CFTR was measured by qRT-PCR. Red dash = normalization to empty exosome controls. Results are mean ± sem. n=3.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Isolation, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: Modeling the molecular mechanisms of SARS-Cov-2 induced diarrhea using a functional interactome created from a small number of proteins that are known to be modulated by the viral infection of human intestinal epithelium ( https://string-db.org/ ). This analysis is based on the most inclusive gene ontology transport pathway (GO:0006810; false discovery rate (FDR) = 2.30e-07); magenta) and includes 21 functionally and physically interacting proteins that are potentially involved in virus-induced signaling that triggers the Isc response in human intestinal epithelium. This model indicates that NHE3 plays a central role in signal transduction between GO:0070102 - the interleukin-6-mediated signaling pathway (red; false discovery rate (FDR)= 4.08e-0) and GO:1902476 - chloride secretion (green, FDR = 5.96e-0). This cross-talk between interleukins and Cl secretion might be mediated by PAT-1 in a CFTR-independent or -dependent manner, leading to ANO1 activation. NHE3 is also crucial for the signal transduction from virus receptor ACE2 via intracellular calcium stores to a membrane Cl/bicarbonate transporter and basolateral voltage-gated potassium channel complex (yellow - CL:8927; FDR = 5.91e-07). The IL-6 functional network (red) together with IL-8 (CXCL8) influences the functions of the SARS-Cov-2 receptor complex and membrane transporters (magenta) which cause intestinal anion secretion via CaCC and via the Ca2+ signaling cascade (blue) that affects the activity of Ca 2+ - or cAMP-mediated basolateral K+ channels (yellow). Following string colors that connect the proteins represent the interactions based on: turquoise - from curated databases; magenta – experimentally determined; green – gene neighborhood; blue – gene co-occurrence; light green – text mining; black – co-expression; light purple – protein homology.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Functional Assay, Infection, Virus, Transduction, Activation Assay, Membrane, Activity Assay, Expressing