We report to use the main protease (Mpro) of SARS-Cov-2 to screen plant flavan-3-ols and proanthocyanidins. Twelve compounds, (–)-afzelechin (AF), (–)-epiafzelechin (EAF), (+)-catechin (CA), (–)-epicatechin (EC), (+)-gallocatechin (GC), (–)-epigallocatechin (EGC), (+)-catechin-3-O-gallate (CAG), (–)-epicatechin-3-O-gallate (ECG), (–)-gallocatechin-3-O-gallate (GCG), (–)-epigallocatechin-3-O-gallate (EGCG), procyanidin A2 (PA2), and procyanidin B2 (PB2), were selected for docking simulation. The resulting data predicted that all 12 metabolites could bind to Mpro. The affinity scores of PA2 and PB2 were predicted to be −9.2, followed by ECG, GCG, EGCG, and CAG, −8.3 to −8.7, and then six flavan-3-ol aglycones, −7.0 to −7.7. Docking characterization predicted that these compounds bound to three or four subsites (S1, S1′, S2, and S4) in the binding pocket of Mpro via different spatial ways and various formation of one to four hydrogen bonds. In vitro analysis with 10 available compounds showed that CAG, ECG, GCG, EGCG, and PB2 inhibited the Mpro activity with an IC50 value, 2.98 ± 0.21, 5.21 ± 0.5, 6.38 ± 0.5, 7.51 ± 0.21, and 75.3 ± 1.29 μM, respectively, while CA, EC, EGC, GC, and PA2 did not have inhibitory activities. To further substantiate the inhibitory activities, extracts prepared from green tea (GT), two muscadine grapes (MG), cacao, and dark chocolate (DC), which are rich in CAG, ECG, GAG, EGCG, or/and PB2, were used for inhibitory assay. The resulting data showed that GT, two MG, cacao, and DC extracts inhibited the Mpro activity with an IC50 value, 2.84 ± 0.25, 29.54 ± 0.41, 29.93 ± 0.83, 153.3 ± 47.3, and 256.39 ± 66.3 μg/ml, respectively. These findings indicate that on the one hand, the structural features of flavan-3-ols are closely associated with the affinity scores; on the other hand, the galloylation and oligomeric types of flavan-3-ols are critical in creating the inhibitory activity against the Mpro activity.

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Gossypium barbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G . barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G . barbadense . In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G . barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G . barbadense . The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G . barbadense , and help to contribute desirable traits for breeding of G . barbadense and G. hirsutum.

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LiNi 1 − x Fe x O 2 (0 ≤ x ≤ 0.3) and Li y Ni 0.8 Fe 0.2 O 2 (0.8 ≤ y ≤ 1.2) catalysts for the oxygen evolution reaction (OER) were systematically investigated to discover the influence of the composition and layered structure on electrochemical activity. LiNi 0.8 Fe 0.2 O 2 exhibits OER activity that is better than that of LiNiO 2 and other Fesubstituted LiNiO 2 catalysts, while Li 1.2 Ni 0.8 Fe 0.2 O 2 shows OER activity that is much higher than that of LiNi 0.8 Fe 0.2 O 2 and Li 0.8 Ni 0.8 Fe 0.2 O 2 . The best OER activity is achieved on Li 1.2 Ni 0.8 Fe 0.2 O 2 with a Tafel slope of 59 mV dec −1 and a current density of 10 mA cm −2 at an overpotential of 302 mV, better than that for the benchmark IrO 2 catalyst. Combined with the density functional theory calculations, the enhanced OER activity is mainly attributed to the unique electronic structure derived from the interaction of Li, Ni, and Fe in the materials and the layered structure which plays an important role in stabilizing the high valence states of Ni and Fe during the OER.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., et al. Phase-plate cryo-EM structure of a class B GPCR-G protein complex. Nature 2017, 546, 118−123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Insulin-like peptide 5 (INSL5), a member of the insulin/relaxin superfamily, can activate the G-protein-coupled receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), but its precise biological functions are largely unknown. Recent studies suggest that INSL5/RXFP4 is involved in the control of food intake and glucose homoeostasis. We report in the present study that RXFP4 is present in the mouse insulinoma cell line MIN6 and INSL5 augments glucose-stimulated insulin secretion (GSIS) both in vitro and in vivo. RXFP4 is also expressed in the mouse intestinal L-cell line GLUTag and INSL5 is capable of potentiating glucose-dependent glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. We propose that the insulinotrophic effect of INSL5 is probably mediated through stimulation of insulin/GLP-1 secretion and the INSL5/RXFP4 system may be a potential therapeutic target for Type 2 diabetes.