An integrative drug repositioning framework discovered a potential therapeutic agent targeting COVID-19.

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires an urgent need to find effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). In this study, we developed an integrative drug repositioning framework, which fully takes advantage of machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 can interact with the nucleocapsid (N) protein of SARS-CoV-2 and is able to suppress the LPS-induced production of several inflammatory cytokines that are highly relevant to the prevention of immunopathology induced by SARS-CoV-2 infection.

Drought-deciduous behavior reduces nutrient losses from temperate deciduous trees under severe drought.

Nutrient resorption from senescing leaves is an important mechanism of nutrient conservation in temperate deciduous forests. Resorption, however, may be curtailed by climatic events that cause rapid leaf death, such as severe drought, which has been projected to double by the year 2100 in the eastern United States. During a record drought in the southeastern US, we studied 18 common temperate winter-deciduous trees and shrubs to understand how extreme drought affects nutrient resorption of the macronutrients N, P, K, and Ca. Four species exhibited drought-induced leaf senescence and maintained higher leaf water potentials than the remaining 14 species (here called drought-evergreen species). This strategy prevented extensive leaf desiccation during the drought and successfully averted large nutrient losses caused by leaf desiccation. These four drought-deciduous species were also able to resorb N, P, and K from drought-senesced leaves, whereas drought-evergreen species did not resorb any nutrients from leaves lost to desiccation during the drought. For Oxydendrum arboreum, the species most severely affected by the drought, our results indicate that trees lost 50% more N and P due to desiccation than would have been lost from fall senescence alone. For all drought-deciduous species, resorption of N and P in fall-senesced leaves was highly proficient, whereas resorption was incomplete for drought-evergreen species. The lower seasonal nutrient losses of drought-deciduous species may give them a competitive advantage over drought-evergreen species in the years following the drought, thereby impacting species composition in temperate deciduous forests in the future.

Molecular cloning and expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from Lonicera japonica.

In jasmonate biosynthetic pathway, allene oxide synthase (AOS, EC 4.2.1.92), which is a cytochrome P450 (CYP74A), catalyzes the first committed step. We herein cloned a novel cDNA from Lonicera japonica Thunb., named LjAOS (GenBank accession: DQ303120), which was homologous to other AOSs. Southern blot analysis revealed that it was a multi-copy gene. Real-time quantitative PCR analysis showed that LjAOS mRNA accumulated most abundantly in alabastrums, in which the content of chlorogenic acid (CA, the major important active ingredient indicator) was previously proven to be the highest.

Molecular cloning, characterization and expression of a jasmonate biosynthetic pathway gene encoding allene oxide cyclase from Camptotheca acuminata.

AOC (allene oxide cyclase; EC 5.3.99.6), an essential enzyme in jasmonic acid and its methyl ester biosynthesis, was cloned from Camptotheca acuminata (named as CaAOC), a native medicinal plant species in China. CaAOC had significant similarity at the amino-acid level with AOCs from other plant species. Comparison between the sequences of the full-length cDNA and genomic DNA of CaAOC revealed that the genomic DNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern-blot analysis indicated that CaAOC was a multiple-copy gene, and real-time quantitative PCR analysis showed that CaAOC was expressed constitutively in all organs tested, with the highest expression level in leaves. The results from treatment experiments using different signalling components, including methyl jasmonate, abscisic acid, salicylic acid and H(2)O(2), revealed that expression of CaAOC had a prominent diversity. Heavy metal (copper) significantly enhanced CaAOC expression, whereas wounding (induced by UV-B) was not so effective.

Development of tobacco callus cultures over expressing Arabidopsis PAP1/MYB75 transcription factor and characterization of anthocyanin biosynthesis.

The Arabidopsis PAP1 gene (At1g56650) encodes the MYB75 transcription factor, which has been demonstrated to essentially regulate the biosynthesis of anthocyanins. Our previous study showed that ectopic expression of the PAP1 gene led to high pigmentation of anthocyanins in all tissues of transgenic tobacco plants. In order to understand the mechanisms of how PAP1 regulates anthocyanin biosynthesis and what can regulate the function of PAP1, we have established PAP1 transgenic tobacco callus cultures. Phenotypically different calli including anthocyanin-producing red and anthocyanin-free white calli lines were differentially induced from the same genotype of PAP1 transgenic plants. RT-PCR analysis showed that the expression of the PAP1 transgene was similar in the two types of calli, indicating that the transgenic red and white calli had differential responses to the regulation of PAP1. The growth of transgenic red calli followed a "sigmoid-like" curve in a 25-day callus culture period, during which the time course obviously impacted the profiles and the average levels of anthocyanins even though the expression of the PAP1 transgene was constitutive. A HPLC-UV-ESI-mass spectrum-based profiling characterized nine anthocyanin molecules (e.g., 595, 579 and 609 m/z) in the transgenic red calli over the course of the culture period. Cyanidin, pelargonidin, and peonidin were the major anthocyanidins identified by HPLC-mass spectrum analysis. We have demonstrated that dark, nitrogen nutrients, and auxin apparently affect the anthocyanin profiles in PAP1 transgenic callus cultures; and suggest that these cell cultures are an appropriate system to study the regulatory function of PAP1 on the anthocyanin biosynthesis at post-transcriptional level in vivo.

Molecular cloning and characterization of a mannose-binding lectin gene from Pinellia cordata.

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.

Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene from Ginkgo biloba.

Ginkgo biloba contains terpene triclactones of high pharmaceutical value such as ginkgolides. 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) reductase (HDR) is proved to be the terminal-acting enzyme in the plastid MEP pathway which provides isoprenoid precursors for the biosynthesis of ginkgolides. The full-length cDNA encoding HDR, designated as GbHDR (Genbank Accession Number DQ364231), was isolated for the first time from G. biloba by RACE method. GbHDR contained a 1,422-bp open reading frame encoding 474 amino acids. The deduced GbHDR protein, showing high identity to HDRs of other plant species, was predicted to possess a chloroplast transit peptide at the N-terminal and four conserved cysteine residues. Two-dimensional structural analysis showed that GbHDR had a similar secondary structure with HDR from Arabidopsis thaliana. Southern blot analysis indicated that GbHDR belonged to a small gene family. Transcription pattern analysis revealed that GbHDR had high transcription in roots, and low in leaves and stems. The cloning of GbHDR gene will enable us to further understand the role of GbHDR involved in terpene triclatones biosynthetic pathway in G. biloba at molecular level.

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata.

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.

Genomic cloning and characterization of a Pto-like gene SsPto-2 from Solanum surattense.

A Pto-like gene (designated as SsPto-2) was isolated from Solanum surattense by using genomic walker technology which encoded a cytoplasmically localized serine-threonine protein kinase. Analysis of the 2365 bp segment revealed a gene including a 905 bp 5' flanking region, a 924 bp open reading frame (ORF) and a 536 bp 3' flanking region. The deduced amino acid sequence of the SsPto-2 gene shared high homology with other known Ptos. The deduced SsPto-2 protein contained no signal peptide with a calculated molecular weight of 34.61 kDa. The analysis of SsPto-2 promoter region and terminator region was also presented. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that SsPto-2 transcripts were up-regulated by defense-related factors such as gibberellic acid (GA(3)), salicylic acid (SA) and down-regulated by darkness. The cloning of the SsPto-2 gene will allow us to further study its potential role in disease resistance.