Journal: G3: Genes|Genomes|Genetics

Article Title: Tissue-Specific Split sfGFP System for Streamlined Expression of GFP Tagged Proteins in the Caenorhabditis elegans Germline

doi: 10.1534/g3.119.400162

Figure Lengend Snippet: Sequences used for CRISPR/Cas9, MosSCI, cloning and diagnosis. Table of the DNA/RNA sequences used for construction of the strains in this manuscript

Article Snippet: Ultramer oligonucleotides, tracrRNA, and crRNAs were obtained from IDT and mixed in the following concentrations: 14.35 µM Cas9-NLS (Berkeley MacroLab), 17.6 µM tracrRNA (IDT), 1.5 µM dpy10 crRNA (IDT), 5 µM dpy10 ssODN (IDT), 16.2 µM of target crRNA (IDT), and 6 µM of target ssODN (IDT) (see ).

Techniques: CRISPR, Clone Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Effects of Extracellular DNA on Plasminogen Activation and Fibrinolysis *

doi: 10.1074/jbc.M111.301218

Figure Lengend Snippet: Effects of DNA in solution and DNA incorporated into a FITC-fibrin film on fibrinolysis. A, changes in the fluorescence emission at 520 nm (excitation 493 nm) during the incubation of FITC-fibrin film with 100 μl of a mixture of 100 nm Glu-Plg with 0.25 nm tctPA without DNA (●) and with 1.0 (○) and 25 (□) μg/ml dsDNA in 0.05 m Hepes/NaOH buffer (pH 7.4) (with 20 mm NaCl and BSA 1 mg/ml). Inset, dependences of rates of fibrinolysis of FITC-fibrin film by 20 nm PL (Δ), mixtures of 100 nm Glu-Plg with 0.25 nm tctPA (●), or 0.25 nm tcuPA (○), 100 nm Plg/tctPA with 15 nm α2AP (■) and 100 nm Plg/tcuPA with 15 nm α2AP (□) on the concentration of dsDNA in the solution. The rates of fibrinolysis were calculated as slopes of a linear (r2 >0.9) increase in the fluorescein fluorescence emission with time (PL) or the square of time (tctPA/Plg and tcuPA/Plg) and expressed as the % of the control reaction (no DNA added). B, effects of dsDNA incorporated into a FITC-fibrin film on the rates of fibrinolysis by 20 nm PL (Δ) and a mixture of 100 nm Glu-Plg with 0.25 nm tctPA (●) or 0.25 nm tcuPA (○). The indicated amounts of dsDNA (μg/well) were added to 20 μg of FITC-Fbg before its polymerization. The rates of fibrinolysis were calculated as slopes of a linear (r2 >0.9) increase in the fluorescein fluorescence emission with time (PL) or the square of time (tctPA/Plg) and expressed as % of the control reaction (no DNA added). C, effects of oligo(dT)65 incorporated into FITC-fibrin film on the rates of fibrinolysis by 20 nm PL (Δ) and a mixture of 100 nm Glu-Plg with 0.25 nm tctPA (●) or 0.25 nm tcuPA (○). The indicated amounts of nanomoles of oligo(dT)65 per well were added to 20 μg of FITC-Fbg before its polymerization. Rates of fibrinolysis were calculated as slopes of a linear (r2 >0.9) increase in the fluorescein fluorescence emission with time (PL) or the square of time (tctPA/Plg) and expressed as % of the control reaction (no oligo(dT)65 added). D, effects of α2AP (15 nm) on the rates of fibrinolysis of FITC-fibrin films with incorporated dsDNA (circles) or oligo(dT)65 (squares) by 100 nm Plg/tctPA (filled symbols) and 100 nm Plg/tcuPA (open symbols). The indicated amounts of dsDNA (μg/well) or oligo(dT)65 (nanomoles/well) were added to 20 μg of FITC-Fbg before its polymerization. The rates of fibrinolysis were calculated as slopes of a linear (r2 >0.9) increase in the fluorescein fluorescence emission with the square of time and expressed as % of the control reaction (no dsDNA or oligo(dT)65 added).

Article Snippet: Custom oligonucleotides (oligo(dT) 20 ; oligo(dT) 65 , oligo(dAT) 10 (a 20-mer containing 10 AT repeats); oligo(dAT) 33 (a 66-mer containing 33 AT repeats); and TEX615-oligo(dAT) 33 , (an oligonucleotide labeled with red wavelength dye TEX615 at the 5′ end) were synthesized by Integrated DNA Technologies Inc. (Iowa City, IA).

Techniques: Fluorescence, Incubation, Concentration Assay

Journal: Experimental Biology and Medicine

Article Title: Absence of antibody responses to SARS-CoV-2 N protein in COVID-19 vaccine breakthrough cases

doi: 10.1177/15353702221134097

Figure Lengend Snippet: Study design and sampling time points. Participants included naïve and convalescent individuals given either two or three doses of Pfizer vaccine; a convalescent individual given the J&J vaccine; and unvaccinated convalescent individuals. Viral RNA, antibodies against S or N proteins, and inhibition capacities against the viral receptor-binding domain (RBD) of SARS-CoV-2 were evaluated at T1 (2 weeks post the first dose) and T2 (2 weeks post the second dose) in individual participants. At T3 (24 weeks post the first dose or ~5 months postvaccination) and T4 (36 weeks post the first dose or ~8 months postvaccination), viral RNA, antibodies against S or N proteins of SARS-CoV-2, and neutralizing antibodies were evaluated by live-virus-based microneutralization assay. Since the Delta variant emerged (around T3), we evaluated neutralization capacities against the basal B.1 virus (original SARS-CoV-2) and the Delta variant. The Delta variant became the dominant variant ( ⩾ 99%) as most of our participants were reaching T4, thus, we mainly focused on the Delta variant at this time point. Participants gradually received BNT162b2 booster shots after T3 until the endpoint of our study.

Article Snippet: Viral RNA was detected by (qRT-PCR using FDA-approved Luminex SARS-CoV-2 panel 2 (IDT, Cat# 10006941).

Techniques: Sampling, Inhibition, Binding Assay, Microneutralization Assay, Variant Assay, Neutralization

Journal: Experimental Biology and Medicine

Article Title: Absence of antibody responses to SARS-CoV-2 N protein in COVID-19 vaccine breakthrough cases

doi: 10.1177/15353702221134097

Figure Lengend Snippet: Reduced neutralizing capacity against SARS-CoV-2 Delta variant at 5 months postvaccination and in unvaccinated convalescent individuals. (a) Correlation between neutralization capacities (presented as EC50: half-maximal effective concentration) against B.1-isolate and Delta variant as evidenced by the Spearman correlation coefficient. (b) Neutralization capacities against B.1-isolate (top panels) and Delta variant (bottom panels) in older Vac_naive individuals as compared to younger Vac_naive individuals. Significance was determined using two-sided Mann–Whitney U tests. (c) Age-adjusted neutralization capacities against Delta (filled circles) compared to those against B.1-isolate (open circles). #: Significance was determined using the two-sided Kruskal–Wallis test followed by a two-stage-step-up Benjamini, Krieger, and Yekutieli false discovery rate method for multiple comparison correction. &: The rank ANCOVA was used to evaluate neutralization capacities across study groups, while controlling for the confounding effect of age. The rank ANCOVA results were presented at F -distribution with p -value. The rank ANCOVA was followed by two-sided Dunnett’s post hoc tests to correct multiple comparisons.

Article Snippet: Viral RNA was detected by (qRT-PCR using FDA-approved Luminex SARS-CoV-2 panel 2 (IDT, Cat# 10006941).

Techniques: Variant Assay, Neutralization, Concentration Assay, MANN-WHITNEY