Activity

Retinopathy development (OR 1.05 [95%CI 1.02-1.07] per mmol/mol increase) and progression (OR 1.05 [1.04-1.06]) at seven years was associated with higher HbA

atdiabetesdiagnosis. Obesity (OR 0.88 [0.79-0.98]) and high socioeconomic status (OR 0.63 [0.53-0.74]) were negatively associated with retinopathy development at seven years.

Baseline retinopathy prevalence has declined since UKPDS. Additionally, HbA

at diabetes diagnosis remains important for retinopathy development and progression.

Baseline retinopathy prevalence has declined since UKPDS. PEG300 Additionally, HbA1c at diabetes diagnosis remains important for retinopathy development and progression.In this study, we developed and evaluated a luciferase immunosorbent assay (LISA) for quantitative detection of IgG antibody against SARS-CoV-2 nucleoprotein (NP). Anti-SARS-CoV-2 NP antibody in serum or plasma samples was captured by protein G-coated microtiter plate and detected using the crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with SARS-CoV-2 NP. After the addition of furimazine substrate, the levels of anti-SARS-CoV-2 NP IgG antibody were quantitatively measured as luciferase light units. As expected, SARS-CoV-2 NP showed cross-reactivity with the monoclonal antibodies against SARS-CoV NP, but not MERS-CoV NP-specific monoclonal antibodies or the monoclonal antibodies against SARS-CoV Spike protein. LISA for detecting murine monoclonal antibody against SARS-CoV NP showed a low limit of detection of 0.4 pg/μl and linear detection range from 0.4 pg/μl to 75 pg/μl. Furthermore, LISA had a sensitivity of 71 % when testing COVID-19 patients at the second week post onset and a specificity of 100 % when testing healthy blood donors.Molecular chaperones maintain proteostasis by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to numerous neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s disease. Hsp110 is related to the canonical Hsp70 class of protein folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF). In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has implicated the sole Hsp110 gene (Hsc70cb) in proteinopathic neurodegeneration. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function as a protective protein “holdase”, preventing the aggregation of unfolded polypeptides via the SBD-b subdomain. We demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138 amino-acid region of Hsp110 carboxyl-terminal to both SBD-b and SBD-α (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105α, inhibits the formation of amyloid Aβ-42 and α-synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings establish capacity for metazoan Hsp110 chaperones to suppress both general protein aggregation and amyloidogenesis, raising the possibility of exploitation of this IDR for therapeutic benefit.It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5-P450 interactions has been the limited solution assay methods. We constructed a fluorescently-labeled variant of human b5 that can be used in titrations. The labeled b5 bound to wild-type P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b5P450 17A1 complex resulted in a concentration-dependent, partial restoration of b5 fluorescence, indicative of a ternary P450b5POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.The enzyme NUDT15 efficiently hydrolyses the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and pre-emptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants due to their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared to TH1760, while showing no cytotoxicity on its own.