Search

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

In this research, a coated graphite polyvinyl chloride (PVC) membrane electrode was fabricated for the potentiometric determination of lithium based on 13-(3-nitrophenyl)-12H,14H-benzo[g]chromeno[2,3-b]chromene-7,12,14(13H)-trione (L) as the ionophore. The best Nernstian response was observed from the membrane composition of 31% PVC, 7% L, 2% NaTPB, and 60% dioctyl phthalate (DOP) as the plasticizer. The designed sensor showed a wide linearity over the concentration range of 1 x 10-8-1 x 10-3 M with a slope of 60.2 ± 0.3 mV decade-1, and the limit of detection (LOD) of 7.5 x 10-9 M. The selectivity of the designed sensor was investigated by the matched potential method and no serious interference was observed. The response time and life span of the electrode were 5 s and 16 weeks, respectively. The potential response of the electrode was independent of solution pH in the pH range of 3.0-12.0. The influence of the presence of organic solvents on the potential response of the electrode was also scrutinized, and the results showed that the designed sensor keeps its Nernstian behavior in solutions with 20% non-aqueous content. In the end, the electrode was successfully applied to determine lithium carbonate in tablet, urine, and serum samples with recovery% values of 96.0 to 106.33 and RSD% 1.99-4.45. [ABSTRACT FROM AUTHOR]

In this report, natural dyes extracted from three different, black-colored fruits were used as photosensitizers for the construction of dye-sensitized solar cells (DSSCs). The natural dyes were extracted from the dark-colored peels of jamun (also known as Indian black plum), black plum, and blackberry fruit. These natural dyes contain polyphenolic compounds—most prominently anthocyanins—which interact strongly with titanium dioxide (TiO2) semiconductors and accordingly enhance the efficiency of DSSCs. The natural dyes extracted from the various fruits were characterized utilizing UV-Vis and fluorescence spectroscopy. The interaction between the dyes and TiO2 was monitored with FTIR and Raman spectroscopy. The fabricated DSSCs were characterized via current–voltage measurements and electrochemical impedance analysis. DSSCs fabricated with jamun produced the highest efficiency of 1.09% with a short-circuit current of 7.84 mA/cm2, an open-circuit voltage of 0.45 V, and a fill factor of 0.31. The efficiencies of the DSSCs from black plum and blackberry were 0.55% and 0.38%, respectively. The flow of charge occurring at the interfaces between the natural dye and the TiO2 layers were investigated using electrochemical impedance spectroscopy (EIS). To the best of our knowledge, this study is the first to directly compare three distinct types of black DSSCs. Computation analysis was also carried out utilizing SCAPS-1D software (version 3.3.07), which revealed how the type of defects in the devices impacts their performance. [ABSTRACT FROM AUTHOR]

Background and purpose: Enzyme engineering is the process of raising enzyme efficiency and activity by altering amino acid sequences. Kallikrein 6 (KLK6) enzyme is a secreted serine protease involved in a variety of physiological and pathological activities. The increased expression of KLK6 plays a key role in various diseases. Instability and spontaneous activation and deactivation are major challenges in the study of this enzyme. This study aimed to create a stable pro-KLK6 enzyme by enzyme engineering, designing a specific cleavage site for enterokinase, and using Pichia pastoris GS115 as a host cell. Then, recombinant pro-KLK6 was used to introduce a novel inhibitor for it. Experimental approach: An engineered pro-KLK6 gene was cloned into the pPICZα A expression vector. Then, it was expressed in P. pastoris GS115 and purified by Ni-NTA chromatography. An inactive engineered pro-KLK6 gene was cleaved by enterokinase and converted to an active KLK6. The KLK6 enzyme activity and its kinetic parameters were measured using N-benzoyl-L-arginine ethyl ester (BAEE) substrates. Findings/Results: The secretory form of the pro-KLK6 was expressed at about 11 mg/L in P. pastoris (GS115). Before activation with enterokinase, pro-KLK6 was inactive and did not activate spontaneously. The kinetic parameters, including Km and Vmax, were estimated at 113.59 µM and 0.432 µM/s, respectively. Conclusion and implications: A stable pro-KLK6 enzyme was produced using P. pastoris (GS115) as the host cell and a specific cleavage site for enterokinase. Additionally, this study assessed the kinetic parameters of the KLK6 enzyme using the BAEE substrate for the first time. [ABSTRACT FROM AUTHOR]

Background and purpose: Properties of Alzheimer's disease, can be caused by several reasons and there is no definite treatment for it. We aimed to study the effect of the hydroalcoholic extract, methanolic and n-hexane fractions of brown algae Sargassum angustifolium on memory impairment in mice and rats. Experimental approach: Hydroalcoholic extract (25, 50, 100, 200 mg/kg), methanolic (20 and 40 mg/kg) and n-hexane (40 and 60 mg/kg) fractions of S. angustifolium were administered for 21 days intraperitoneally before scopolamine injection (2 mg/kg) on day 21. Rivastigmine was administered for 3 weeks intraperitoneally as well. Then, cognitive function was evaluated by three behavioral tests: passive avoidance, object recognition, and the Morris Water Maze test. Findings/Results: Scopolamine induced memory impairment and rivastigmine significantly reversed the memory dysfunction in all three tests. Hydroalcoholic extract and methanolic fraction significantly reversed scopolamine-induced memory impairment in passive avoidance by 64% and 55% and enhanced the recognition index in the object recognition test. In the Morris water maze test probe trial and training session, on days 3 and 4, the hydroalcoholic extract showed a significant decrease in time spent in the target quadrant and path length, respectively. Also, hydroalcoholic extract and methanolic fraction decreased escape latency time in training sessions on days 3 and 4, by 50% and 31% in comparison to scopolamine. N-hexane fractions had no significant effect on scopolamine-induced cognitive impairment. Conclusion and implications: Although the n-hexane fraction wasn't effective, the administration of hydroalcoholic extract and the methanolic fraction of S. angustifolium enhanced scopolamine-induced memory impairment. [ABSTRACT FROM AUTHOR]