Journal: Biomédica

Article Title: Validación clínica de la prueba RT-LAMP para el diagnóstico rápido del SARS-CoV-2

doi: 10.7705/biomedica.6523

Figure Lengend Snippet: Detección colorimétrica de SARS-CoV-2 por RT-LAMP; empleando dos sets de cebadores, Broughton (A) y Lalli (B). Se recolectaron 117 muestras de ARN purificado a partir de hisopado nasofaríngeo de individuos sintomáticos para COVID-19. La reacción fue ejecutada a una temperatura de 65 °C durante 30 minutos, usando el estuche comercial Warmstart colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB) en 20 μΙ de volumen final. La reacción de RT-LAMP tenía como diana el gen N del SARS-CoV-2. Las muestras negativas para SARS-CoV-2 se muestran en color rosa y las muestras positivas se visualizan por un cambio de color de rosa a amarillo. Los amplicones producto de la reacción de amplificación fueron separados en un gel de agarosa al 2 % y teñidos con SYBR Safe (Invitrogen). El patrón de tipo escalera observado en las muestras positivas, es coherente con una amplificación positiva y el subsecuente viraje de color en las muestras con presencia del SARS-CoV-2, en tanto que los productos de amplificación están ausentes en las muestras negativas. CP: fragmento sintético del gen N del SASR-CoV-2. CN: reacciones incubadas con agua de grado para biología molecular en lugar de ARN.

Article Snippet: Como control positivo, en las reacciones de RT-LAMP se incluyó un plásmido que tiene clonado un fragmento del gen N del SARS-CoV-2, adquirido de la casa comercial New England Biolabs (NEB, Germany).

Techniques:

Journal: Biomédica

Article Title: Validación clínica de la prueba RT-LAMP para el diagnóstico rápido del SARS-CoV-2

doi: 10.7705/biomedica.6523

Figure Lengend Snippet: Detección colorimétrica de SARS-CoV-2 por RT-LAMP en muestras de hisopado nasofaríngeo, sin previa extracción de ARN. Se precalentaron 36 muestras de hisopado nasofaríngeo durante 5 minutos a 95 °C; posteriormente, se agregaron 3 μl de muestra a la reacción RT-LAMP usando el estuche comercial Warmstart colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB) en 20 μl de volumen final. La RT-LAMP tenía como diana el gen N del SASR-CoV-2. Las muestras negativas para este virus se muestran en color rosa y las muestras positivas se visualizan por un cambio de color de rosa a amarillo. Los amplicones producto de la reacción de amplificación fueron resueltos en un gel de agarosa al 2 % y teñidos con SYBR Safe (Invitrogen). Se muestra el patrón típico de amplificación de la técnica RT- LAMP en muestras positivas y el cambio de color en la reacción; en muestras negativas, la mezcla reactiva permanece de color rosa y no se observan bandas en el gel electroforético. CP: fragmento sintético del gen N del SASR-CoV-2. CN: reacciones incubadas con agua de grado para biología molecular en lugar de muestras de hisopado.

Article Snippet: Como control positivo, en las reacciones de RT-LAMP se incluyó un plásmido que tiene clonado un fragmento del gen N del SARS-CoV-2, adquirido de la casa comercial New England Biolabs (NEB, Germany).

Techniques: Virus

Journal: Cell reports. Medicine

Article Title: Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

doi: 10.1016/j.xcrm.2023.101134

Figure Lengend Snippet: Figure 3. SARS-CoV-2 IgG and neutralizing antibody responses (A and B) Serum IgG titers against wild-type SARS- CoV-2 RBD protein (A) and pseudovirus neutralizing antibody titers (ID50) against SARS-CoV-2 variants (B) for non-elderly (left side) and elderly (right side) participants (n = 10 per dose group, and n = 6 for the placebo group). Participants received one injection of VLPCOV-01 (0.3, 1.0, or 3.0 mg), 30 mg BNT162b2, or placebo on day 1 (week 0). Logarithmic values are reported as geometric mean titers for serum IgG and neutralizing antibody against pseudovirus. Bars indicate 95% CIs. (C) The correlation between serum neutralizing antibody titers against pseudovirus Wuhan (wild type) and IgG antibody titers against SARS-CoV-2 RBD protein following booster vaccination. Pearson’s product-moment correlation coefficient and p value were calculated following log trans- formation of source data (r = 0.950, p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human CD154-FITC (clone: TRAP1) BD Biosciences cat# 555699; RRID: AB_396049 Mouse anti-human CD3-BUV615 (clone: SP34-2) BD Biosciences cat# 751249; RRID: AB_2875266 Mouse anti-human CD4-PE-Cy5.5 (clone: S3.5) Thermo Ficher Scientific cat# MHCD0418; RRID: AB_10376013 Mouse anti-human CD8-BUV563 (clone: RPA-T8) BD Biosciences cat# 612914; RRID: AB_2870200 Mouse anti-human CD27-PE-Cy5 (clone: 1A4CD27) Beckman coulter cat# 6607107 Mouse anti-human CD45RO-BUV805 (clone: UCHL1) BD Biosciences cat# 748367; RRID: AB_2872786 Mouse anti-human IFN-g-BV786 (clone: 4S.B3) BioLegend cat# 502542; RRID: AB_2563882 Mouse anti-human TNF-BV650 (clone: MAb11) BioLegend cat# 502938; RRID: AB_2562741 Rat anti-human IL-13-BV421 (clone: JES10-5A2) BD Biosciences cat# 563580; RRID: AB_2738290 Mouse anti-human IL-21-Ax647 (clone: 3A3-N21) BD Biosciences cat# 560493; RRID: AB_1645421 Mouse anti-human IL-4-PE-Cy7 (clone:8D4-8) BD Biosciences cat# 560672; RRID: AB_1727547 Mouse anti-human IL-17A-BV605 (clone: BL168) BioLegend cat# 512326; RRID: AB_2563887 Rat anti-human IL-2-BUV737 (clone: MQ117H12) BD Biosciences cat# 612836 Mouse anti-human CD107A-BV711 (clone: H4A3) BioLegend cat# 328640; RRID: AB_2565840 Mouse anti-human MIP1b-Alexa700 (clone: D21-1351) BD Biosciences cat# 561278; RRID: AB_10612008 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1 (AS35) ACROBiosystems cat# SAD-S35 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG2 (AS35) ACROBiosystems cat# SAD-S66 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG3 (AS35) ACROBiosystems cat# SAD-S67 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG4 (AS35) ACROBiosystems cat# SAD-S68 Mouse anti-Human IgG1 Fc Secondary Antibody, HRP Thermo Fisher Scientific cat# MH1715; RRID: AB_2539710 Mouse Anti-Human IgG2 Fc-BIOT HP6002 SouthernBiotech cat# 9070-08; RRID: AB_2796638 Mouse Anti-Human IgG3 Hinge-BIOT HP6050 SouthernBiotech cat# 9210-08; RRID: AB_2796700 Mouse Anti-Human IgG4 Fc-BIOT HP6025 SouthernBiotech cat# 9200-08; RRID: AB_2796692 Biological samples Human PBMCs This study This study Chemicals, peptides, and recombinant proteins Benzonase Nuclease, Purity >90% MERCK Millipore cat# 70746 (Continued on next page) Cell Reports Medicine 4, 101134, August 15, 2023 e1

Techniques: Injection

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 2 Methyl-β cyclodextrin (MβCD)-induced reductions in ACE2 binding of Wuhan-Hu-1 strain of SARS-CoV-2 (WT) and Delta SARS- CoV-2 spike receptor-binding domains (RBDs) negatively correlate with the applied RBD concentrations. HEK/ACE2 + TMPRSS2 cells were treated with different concentrations of MβCD ranging between 0.25 and 10 mM. Fluorescence intensities of cells were measured using flow cytometry for 5 min continuously starting immediately after the addition of GFP-conjugated WT (a) or Delta (b) SARS-CoV-2 spike RBDs applied at various concentrations between 0.1 and 5 μgml1, or 0.05 and 2.5 μgml1, respectively. Moving averages of time-correlated fluorescence intensities of at least 50,000 cells of normal morphology were determined with a window size of 10 s and subsequently normalized to the average of intensities obtained in the last time window of untreated control samples. Mean ± SD values were calculated from data of six independent samples per treatment condition and plotted as a function of time (starting 20 s after RBD addition). The extents of inhibition of WT (a) and Delta (b) RBD binding at the end of the measurement time in response to various MβCD concentrations were calculated and plotted as mean ± SD as a function of the applied RBD concentration in the right panels. Asterisks indicate significant differences of samples treated with the lowest versus highest RBD concentrations at each applied CD concentration (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S2.

Article Snippet: When using Omicron spike trimer conjugated with Alexa Fluor 488 (R&D Systems; AFG11060), experiments were repeated after SBECD, Veklury® P or Veklury® S treatments in HEK/ACE2 + TMPRSS2 and Calu-3 cells using the trimer at 0.5 and 2.5 μg ml 1, respectively.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Control, Inhibition, Concentration Assay

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 6 Veklury® (remdesivir) formulations effectively decrease ACE2 binding of Omicron SARS-CoV-2 spike receptor-binding domain (RBD) and the cellular uptake of Omicron SARS-CoV-2 spike trimer due to their SBECD content. (a) HEK/ACE2 + TMPRSS2 (left panel) or Calu-3 cells (right panel) were treated with SBECD, Veklury® P (powder) or Veklury® S (solution) at CD concentrations of 1, 5 and 10 mM. Then, HEK/ ACE2 + TMPRSS2 cells were incubated in the presence of 0.2-μgml1 Omicron SARS-CoV-2 spike RBD–GFP, whereas Calu-3 cells were labelled with 1-μgml1 Omicron RBD–GFP for 4 min. The emitted fluorescence intensities of individual cells were subsequently measured using flow cytometry, and the mean intensity was calculated from data of at least 10,000 cells of normal morphology per sample. The average values of six independent measurements (±SD) were calculated, normalized to the mean value determined in untreated control samples and plotted as a function of the applied concentrations of CD. Asterisks indicate significant differences of samples treated with the highest applied concentrations compared to the control samples (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S6. (b) HEK/ACE2 + TMPRSS2 (left panel) or Calu-3 cells (right panel) treated with SBECD, Veklury® P or Veklury® S at CD concentrations of 1 and 5 mM for 1 h were subsequently incubated for 4 h in the presence of Omicron SARS-CoV-2 spike trimers conjugated with Alexa Fluor 488. The average fluorescence intensity values emitted by Alexa Fluor 488-labelled trimers were calculated using data of intracellular pixels identified using the membrane marker N-[3-(40-dihexylamino-3-hydroxy-flavonyl-6-oxy)-propyl]N,N-dimethyl-N-(3-sulfopropyl)-ammonium inner salt (F66) as described in Figure 5 for individual cells, which were subsequently normalized to the median value determined in untreated control samples. The number of cells obtained from five independent experiments and involved in the analysis was from left to right: 382, 348, 373, 396, 388, 402 and 393 for Omicron trimer uptake in HEK/ACE2 + TMPRSS2 and 308, 250, 256, 223, 222, 232 and 296 for Omicron trimer uptake in Calu-3. Data points indicated in the figure were obtained from individual cells and plotted along with median values with quartiles. Grey shaded areas show ranges between 0.5 and 1. Asterisks indicate significant differences compared to the control samples (*P < 0.05; one-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S6.

Article Snippet: When using Omicron spike trimer conjugated with Alexa Fluor 488 (R&D Systems; AFG11060), experiments were repeated after SBECD, Veklury® P or Veklury® S treatments in HEK/ACE2 + TMPRSS2 and Calu-3 cells using the trimer at 0.5 and 2.5 μg ml 1, respectively.

Techniques: Binding Assay, Incubation, Fluorescence, Flow Cytometry, Control, Membrane, Marker