Bio-Rad (Israel)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock‐out cell lines are cumbersome and labor intensive, comprising of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of droplet digital PCR (ddPCR) and high‐resolution melt analysis (HRM) to produce DNA sequence and western blot validated CRISPR/Cas9 knock‐out cell lines in under 5 weeks. Support or Funding Information Bio‐Rad, Inc Internal R&D support. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Objective: Several studies have found that protein concentrations of inflammatory cytokines are significantly increased in tissue and serum from patients with chronic venous insufficiency (CVI) relative to that of healthy controls. We sought to determine whether inflammatory protein concentrations differ between patients with moderate clinical disease, classified as clinical, etiology, anatomy, pathophysiology (CEAP) Classes 2 and 3, and more severe clinical disease, classified as CEAP Class 4. Methods: Twenty patients with abnormal venous function were included in the study. Blood from a competent leg vein and from an incompetent superficial vein was collected in addition to incompetent vein tissue extracted through a phlebectomy procedure. Cytokine levels of venous tissue lysate and serum samples were determined using a multiplex assay. Results: Thirteen patients (65%) were classified as clinical CEAP Class 2 or 3, with seven patients (35%) falling into the more severe Class 4 category. Twenty-seven cytokines were measured. Serum isolated from normal veins had significantly higher levels of IFN-gamma in patients with Classes 2 and 3 than Class 4 disease (95.17 pg/mL vs. 71.97 pg/mL; p=0.036). In serum from incompetent veins, IFN-gamma concentrations averaged 95.47 pg/mL in Class 2 and 3 patients and 76.97 pg/mL in Class 4 patients (p=0.048). Eotaxin levels from diseased vein tissue averaged 3.37 pg/mL in Classes 2 and 3 patients, and 1.57 pg/mL in Class 4 patients (p=0.037). IP-10 levels in diseased vein tissue was also significantly less in Class 4 patients at 74.20 pg/mL in Class 2 and 3 patients versus 31.06 pg/mL in Class 4 patients (p=0.004). Conclusion: Despite several studies documenting increased inflammatory cytokines in patients with CVI, our study shows that those patients with more severe disease have significantly lower levels of several inflammatory cytokines. Thus, particular inflammatory cytokines may function in a reparative capacity following tissue injury or as a control mechanism to inhibit further tissue destruction.

Inspired by biology where pathways are triggered and suppressed by specific binding of two molecules, we realize a functional interface between electronics and biology by replacing one of the pair molecules with a two-state "electronic antigen" device comprising a hydroquinone monolayer assembled on gold, and choosing for the pair molecule an antibody that discriminates between the two electrically selected redox states of the monolayer. Application of an oxidative +0.6 V pulse to the antigen switches it to its benzoquinone state where antibodies bind the layer. A subsequent -0.6 V pulse reduces the monolayer back to the unbinding hydroquinone state, releases the specifically bound antibody molecules, and prevents further binding.