Chikungunya E2 protein produced in E. coli and HEK293-T cells-comparison of their performances in ELISA

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)-a prokaryotic system unable to produce post-translational modifications-or in HEK-293T mammalian cells-a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays

MntC-bound plasminogen is converted to functionally active plasmin.

<p>Recombinant proteins or BSA (10 µg/mL), immobilized on microtiter plate wells, were incubated with plasminogen (20 µg/mL). After washing, uPA (3 U) and the chromogenic substrate D-valyl-leucyl-lysine-ρ-nitroanilide dihydrochloride (25 µg/well) were added. Data represent the mean absorbance value at 405 nm ± the standard deviation of two independent experiments, each performed in duplicate. For this analysis, a Student's t-test was used (* <i>p</i><0.05). PAI-1 (plasminogen activator inhibitor 1).</p

Purification and circular dichroism of recombinant MntC.

<p>(A): 12% SDS-PAGE showing purified recombinant protein MntC (1) and after removal of N-terminal polyhistidine tag (2) by enterokinase (asterisk); molecular mass marker (M). (B) MntC circular dichroism spectrum: predominant α-helical secondary structure is shown. Far-UV CD spectrum is presented as an average of five scans recorded from 182 to 262 nm; , molar ellipticity.</p

Degradation of human fibrinogen by plasmin(ogen) bound to immobilized MntC.

<p>Plasminogen (20 µg/mL) was added to immobilized recombinant proteins (10 µg/mL). After washing, fibrinogen (10 µg or 500 ng) and uPA (3 U) were added, and incubation proceeded for the indicated time points. Samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and further stained with Coomassie Blue (A) or transferred to a nitrocellulose membrane, and probed with a mouse monoclonal antibody recognizing the fibrinogen α-chain followed by the corresponding secondary HRP-conjugated antibodies (B). BSA was also included as negative control. Controls omitting uPA and/or plasminogen were included.</p

Binding of MntC to ECM components as a function of protein concentration.

<p>(A) Collagen type IV (CIV), (B) laminin (LAM), (C) cellular fibronectin (CF), (D) plasma fibronectin (PF), (E) plasminogen (PLG), (F) fibrinogen (FIB). LigBC and LIC10301 were included as positive and negative controls, respectively. Recombinant protein concentrations ranged from 0 to 2 µM. Each point represents the mean absorbance value at 492 nm ± the standard error of two independent experiments, each performed in duplicate. MntC binding to each ECM component was compared to LIC10301 binding to these molecules by the two-tailed t test (* <i>p</i><0.05).</p

Analysis toxicity by different methods and anxiolytic effect of the aqueous extract Lippia sidoides Cham.

Abstract Lippia sidoides Cham. (Verbenaceae) is a species often mentioned in traditional medicine due to the medicinal properties attributed to its leaves, which include antibacterial, antifungal, acaricidal and antioxidant. Several of these actions have been scientifically proven, according to reports in the literature; however, little is known about toxicological aspects of this plant. This work included studies to determine the chemical composition and toxicity tests, using several methods aiming to evaluate the safety for use of the aqueous extract of L. sidoides leaves, in addition, the anxiolytic effect on adult zebrafish was investigated, thus contributing to the pharmacological knowledge and traditional medicine concerning the specie under study. The chemical profile was determined by liquid chromatography coupled to mass spectrometry-HPLC/MS with electrospray ionization. Toxicity was evaluated by zebrafish, Drosophila melanogaster, blood cells, and Artemia salina models. 12 compounds belonging to the flavonoid class were identified. In the toxicity assays, the observed results showed low toxicity of the aqueous extract in all tests performed. In the analysis with zebrafish, the highest doses of the extract were anxiolytic, neuromodulating the GABAa receptor. The obtained results support the safe use of the aqueous extract of L. sidoides leaves for the development of new drugs and for the use by populations in traditional medicine

MELD 3.0 adequately predicts mortality and renal replacement therapy requirements in patients with alcohol-associated hepatitis

Model for End-Stage Liver Disease (MELD) score better predicts mortality in alcohol-associated hepatitis (AH) but could underestimate severity in women and malnourished patients. Using a global cohort, we assessed the ability of the MELD 3.0 score to predict short-term mortality in AH. This was a retrospective cohort study of patients admitted to hospital with AH from 2009 to 2019. The main outcome was all-cause 30-day mortality. We compared the AUC using DeLong's method and also performed a time-dependent AUC with competing risks analysis. A total of 2,124 patients were included from 28 centres from 10 countries on three continents (median age 47.2 ± 11.2 years, 29.9% women, 71.3% with underlying cirrhosis). The median MELD 3.0 score at admission was 25 (20-33), with an estimated survival of 73.7% at 30 days. The MELD 3.0 score had a better performance in predicting 30-day mortality (AUC:0.761, 95%CI:0.732-0.791) compared with MELD sodium (MELD-Na; AUC: 0.744, 95% CI: 0.713-0.775; p = 0.042) and Maddrey's discriminant function (mDF) (AUC: 0.724, 95% CI: 0.691-0.757; p = 0.013). However, MELD 3.0 did not perform better than traditional MELD (AUC: 0.753, 95% CI: 0.723-0.783; p = 0.300) and Age-Bilirubin-International Normalised Ratio-Creatinine (ABIC) (AUC:0.757, 95% CI: 0.727-0.788; p = 0.765). These results were consistent in competing-risk analysis, where MELD 3.0 (AUC: 0.757, 95% CI: 0.724-0.790) predicted better 30-day mortality compared with MELD-Na (AUC: 0.739, 95% CI: 0.708-0.770; p = 0.028) and mDF (AUC:0.717, 95% CI: 0.687-0.748; p = 0.042). The MELD 3.0 score was significantly better in predicting renal replacement therapy requirements during admission compared with the other scores (AUC: 0.844, 95% CI: 0.805-0.883). MELD 3.0 demonstrated better performance compared with MELD-Na and mDF in predicting 30-day and 90-day mortality, and was the best predictor of renal replacement therapy requirements during admission for AH. However, further prospective studies are needed to validate its extensive use in AH. Severe AH has high short-term mortality. The establishment of treatments and liver transplantation depends on mortality prediction. We evaluated the performance of the new MELD 3.0 score to predict short-term mortality in AH in a large global cohort. MELD 3.0 performed better in predicting 30- and 90-day mortality compared with MELD-Na and mDF, but was similar to MELD and ABIC scores. MELD 3.0 was the best predictor of renal replacement therapy requirements. Thus, further prospective studies are needed to support the wide use of MELD 3.0 in AH