Development of FRET Assay into Quantitative and High-throughput Screening Technology Platforms for Protein–Protein Interactions

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein–protein interactions. Here we report the development of dissociation constant (Kd) determination for protein–protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction Kd determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The Kd determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both Kd determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications

Overlap of Genetic Risk between Interstitial Lung Abnormalities and Idiopathic Pulmonary Fibrosis

Rationale: Interstitial lung abnormalities (ILAs) are associated with the highest genetic risk locus for idiopathic pulmonary fibrosis (IPF); however, the extent to which there are unique associations among individuals with ILAs or additional overlap with IPF is not known.Objectives: To perform a genome-wide association study (GWAS) of ILAs.Methods: ILAs and a subpleural-predominant subtype were assessed on chest computed tomography (CT) scans in the AGES (Age Gene/Environment Susceptibility), COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease [COPD]), Framingham Heart, ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points), MESA (Multi-Ethnic Study of Atherosclerosis), and SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study) studies. We performed a GWAS of ILAs in each cohort and combined the results using a meta-analysis. We assessed for overlapping associations in independent GWASs of IPF.Measurements and Main Results: Genome-wide genotyping data were available for 1,699 individuals with ILAs and 10,274 control subjects. The MUC5B (mucin 5B) promoter variant rs35705950 was significantly associated with both ILAs (P = 2.6 × 10-27) and subpleural ILAs (P = 1.6 × 10-29). We discovered novel genome-wide associations near IPO11 (rs6886640, P = 3.8 × 10-8) and FCF1P3 (rs73199442, P = 4.8 × 10-8) with ILAs, and near HTRE1 (rs7744971, P = 4.2 × 10-8) with subpleural-predominant ILAs. These novel associations were not associated with IPF. Among 12 previously reported IPF GWAS loci, five (DPP9, DSP, FAM13A, IVD, and MUC5B) were significantly associated (P < 0.05/12) with ILAs.Conclusions: In a GWAS of ILAs in six studies, we confirmed the association with a MUC5B promoter variant and found strong evidence for an effect of previously described IPF loci; however, novel ILA associations were not associated with IPF. These findings highlight common genetically driven biologic pathways between ILAs and IPF, and also suggest distinct ones

Age and associated outcomes among patients receiving venoarterial extracorporeal membrane oxygenation-analysis of the Extracorporeal Life Support Organization registry

PURPOSE: Venoarterial extracorporeal membrane oxygenation (V-A ECMO) can be used to support severely ill patients with cardiogenic shock. While age is commonly used in patient selection, little is known regarding its association with outcomes in this population. We sought to evaluate the association between increasing age and outcomes following V-A ECMO. METHODS: We used individual-level patient data from 440 centers in the international Extracorporeal Life Support Organization registry. We included all adult patients receiving V-A ECMO from 2017 to 2019. The primary outcome was hospital mortality. Secondary outcomes included a composite of complications following initiation of V-A ECMO. We conducted Bayesian analyses of the relationship between increasing age and outcomes of interest. RESULTS: We included 15,172 patients receiving V-A ECMO. Of these, 8172 (53.9%) died in hospital. For the analysis conducted using weakly informed priors, and as compared to the reference category of age 18-29, the age bracket of 30-39 (odds ratio [OR] 0.94, 95% credible interval [CrI] 0.79-1.10) was not associated with hospital mortality, but age brackets 40-49 (odds ratio [OR] 1.26, 95% CrI: 1.08-1.47), 50-59 (OR 1.78, 95% CrI: 1.55-2.06), 60-69 (OR 2.24, 95% CrI: 1.94-2.59), 70-79 (OR 2.90, 95% CrI: 2.49-3.39) and?=?80 (OR 4.02, 95% CrI: 3.13-5.20) were independently associated with increasing hospital mortality. Similar results were found in the analysis conducted with an informative prior, as well as between increasing age and post-ECMO complications. CONCLUSIONS: Among patients receiving V-A ECMO for cardiogenic shock, increasing age is strongly associated with increasing odds of death and complications, and this association emerges as early as 40 years of age