Heterobifunctional Cross-Linker with Dinitroimidazole and N-Hydroxysuccinimide Ester Motifs for Protein Functionalization and Cysteine-Lysine Peptide Stapling.

A heterobifunctional cross-linker with one sulfhydryl-reactive dinitroimidazole end and another amine-reactive N-hydroxysuccinimide (NHS) ester end was designed and synthesized. The two motifs of this cross-linker, dinitroimidazole and NHS ester, proved to react with thiol and amine, respectively, in an orthogonal way. The cross-linker was further applied to construct stapled peptides of different sizes and mono- and dual functionalization (including biotinylation, PEGylation, and fluorescence labeling) of protein.

The miRNA-mRNA Regulatory Modules of Pinus massoniana Lamb. in Response to Drought Stress.

Masson pine (Pinus massoniana Lamb.) is a major fast-growing woody tree species and pioneer species for afforestation in barren sites in southern China. However, the regulatory mechanism of gene expression in P. massoniana under drought remains unclear. To uncover candidate microRNAs, their expression profiles, and microRNA-mRNA interactions, small RNA-seq was used to investigate the transcriptome from seedling roots under drought and rewatering in P. massoniana. A total of 421 plant microRNAs were identified. Pairwise differential expression analysis between treatment and control groups unveiled 134, 156, and 96 differential expressed microRNAs at three stages. These constitute 248 unique microRNAs, which were subsequently categorized into six clusters based on their expression profiles. Degradome sequencing revealed that these 248 differentially expressed microRNAs targeted 2069 genes. Gene Ontology enrichment analysis suggested that these target genes were related to translational and posttranslational regulation, cell wall modification, and reactive oxygen species scavenging. miRNAs such as miR482, miR398, miR11571, miR396, miR166, miRN88, and miRN74, along with their target genes annotated as F-box/kelch-repeat protein, 60S ribosomal protein, copper-zinc superoxide dismutase, luminal-binding protein, S-adenosylmethionine synthase, and Early Responsive to Dehydration Stress may play critical roles in drought response. This study provides insights into microRNA responsive to drought and rewatering in Masson pine and advances the understanding of drought tolerance mechanisms in Pinus.

Effects of drought and rehydration on root gene expression in seedlings of Pinus massoniana Lamb.

The mechanisms underlying plant response to drought involve the expression of numerous functional and regulatory genes. Transcriptome sequencing based on the second- and/or third-generation high-throughput sequencing platforms has proven to be powerful for investigating the transcriptional landscape under drought stress. However, the full-length transcriptomes related to drought responses in the important conifer genus Pinus L. remained to be delineated using the third-generation sequencing technology. With the objectives of identifying the candidate genes responsible for drought and/or rehydration and clarifying the expression profile of key genes involved in drought regulation, we combined the third- and second-generation sequencing techniques to perform transcriptome analysis on seedling roots under drought stress and rewatering in the drought-tolerant conifer Pinus massoniana Lamb. A sum of 294,114 unique full-length transcripts were produced with a mean length of 3217 bp and N50 estimate of 5075 bp, including 279,560 and 124,438 unique full-length transcripts being functionally annotated and Gene Ontology enriched, respectively. A total of 4076, 6295 and 18,093 differentially expressed genes (DEGs) were identified in three pair-wise comparisons of drought-treatment versus control transcriptomes, including 2703, 3576 and 8273 upregulated and 1373, 2719 and 9820 downregulated DEGs, respectively. Moreover, 157, 196 and 691 DEGs were identified as transcription factors in the three transcriptome comparisons and grouped into 26, 34 and 44 transcription factor families, respectively. Gene Ontology enrichment analysis revealed that a remarkable number of DEGs were enriched in soluble sugar-related and cell wall-related processes. A subset of 75, 68 and 97 DEGs were annotated to be associated with starch, sucrose and raffinose metabolism, respectively, while 32 and 70 DEGs were associated with suberin and lignin biosynthesis, respectively. Weighted gene co-expression network analysis revealed modules and hub genes closely related to drought and rehydration. This study provides novel insights into root transcriptomic changes in response to drought dynamics in Masson pine and serves as a fundamental work for further molecular investigation on drought tolerance in conifers.

Combination Therapies against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus disease 2019 (COVID-19), which was announced as a pandemic leading to devastating economic and medical burden worldwide. The virus attacks the organ system across the body by binding to its receptor (for example, angiotensin converting enzyme 2) on the surface of the host cell of various organs. The patients present with a variety of pathological symptoms ranging from fever, cough and cytokine storm to acute respiratory distress syndrome (ARDS). Many combination therapies have been developed to combat the disease, via blocking one or more processes of the viral life cycle and/or relieving host complications simultaneously. In this review, the progress of those combination therapies containing at least one small molecule is updated. We believe it'll provide significant inspiration for further development of treatment strategy against SARS-CoV-2, especially its mutant variants.

Physiological and molecular mechanisms of the response of roots of Pinus massoniana Lamb. to low-temperature stress.

Pinus massoniana Lamb. is the timber species with the widest distribution and the largest afforestation area in China, providing a large amount of timber, turpentine and ecological products. but low temperature limits its growth and geographical distribution. Physiological and molecular studies can well explain the mechanism of P. massoniana response to low temperature. In this study, physiological and biochemical indexes, cell morphology, lignin content, gene regulatory networks, and gene expression patterns of different P. massoniana varieties (cold-tolerant and cold-sensitive) were studied from physiological, biochemical, and molecular perspectives. The results indicated that under low-temperature stress, the cold-tolerant cultivar maintained high contents of osmoregulatory substances, and the root morphology and structure remained intact. In the initial stage of low-temperature stress, the number of differentially expressed genes was 7148, and with the extension of stress time, the number of differentially expressed genes decreased to 1991. P. massoniana might direct its responses to low temperature by regulating phenylpropane metabolism, starch and sucrose metabolism, hormone signaling pathways, and transcription factors. BAM, 4CL, CCoAOMT, PRX5, WRKYs, and hormone synthesis related genes play important roles. P. massoniana cultivars may vary in response mechanisms. In this study, physiological and analytical techniques were used to study the root tip response mechanism of Masson's pine to low temperature stress. The results of this study lay a foundation for in-depth research on the molecular functions of P. massoniana under low-temperature stress conditions.

Identification of WRKY transcription factor family genes in Pinus massoniana Lamb. and their expression patterns and functions in response to drought stress.

BACKGROUND: Pinus massoniana Lamb. is the timber species with the widest distribution and the largest afforestation area in China, providing a large amount of timber, turpentine and ecological products. Seasonal drought caused by climate warming severely constrains the quality and growth of P. massoniana forests. WRKY transcription factors play an important role in plant responses to abiotic stress. In this study, the molecular mechanisms by which P. massoniana responds to drought stress were analysed based on the P. massoniana WRKY (PmWRKY) family of genes. RESULTS: Forty-three PmWRKYs are divided into three major families, 7 sub-families, and the conserved motifs are essentially the same. Among these 43 PmWRKYs express under drought stress but with different expression patterns in response to stress. PmWRKYs respond to drought stress induced by exogenous hormones of SA, ABA, and MeJA. The expression of PmWRKY6, PmWRKY10, and PmWRKY30 up-regulate in different families and tissues under drought stress, while PmWRKY22 down-regulate. Transgenetic tobaccos of PmWRKY31 are with lower malondialdehyde (MDA) content and higher proline (Pro) content than wild type (WT) tobaccos. In transgenic tobaccos of PmWRKY31, expression levels of related genes significantly improve, and drought tolerance enhance. CONCLUSIONS: This study analysed the molecular biological characteristics of PmWRKYs and investigated the expression patterns and functions of PmWRKYs in response to drought stress in P. massoniana. The results of this study provide a basis for in-depth research of the molecular functions of PmWRKYs in response to drought stress.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

High-throughput sequencing-based assembly of chloroplast genomes of five pine tree species.

Pinus plants are the largest existing group of gymnosperms and one of the most highly differentiated taxa. Due to its huge ecological, economic, and scientific value, the genetic diversity and the relationship between the intraspecific evolution of Pinus plants have gained wide attention. In this study, the chloroplast genomes of several common pine trees in southwest and south China, including P. massoniana (masson pine), P. yunnanensis (yunnan pine), P. latteri (south asia pine), P. crassicorticea (la ya pine), and P. elliottii (slash pine), and entire cpDNA sequences were obtained. Characteristics including the structure, repeated sequence, and codon bias of the cpDNA for these five pine tree species were analyzed.

Dichloroacetophenone Derivatives: A Class of Bioconjugation Reagents for Disulfide Bridging.

A mild and biocompatible method for the construction of disulfide bridging in peptides using dichloroacetophenone derivatives is developed. This method is highly selective (chemo, diastereo, regio, etc.) and atom economic and works under biocompatible reaction conditions (metal-free, water, pH 7, rt, etc.).

Dinitroimidazoles as bifunctional bioconjugation reagents for protein functionalization and peptide macrocyclization.

Efficient and site-specific chemical modification of proteins under physiological conditions remains a challenge. Here we report that 1,4-dinitroimidazoles are highly efficient bifunctional bioconjugation reagents for protein functionalization and peptide macrocyclization. Under acidic to neutral aqueous conditions, 1,4-dinitroimidazoles react specifically with cysteines via a cine-substitution mechanism, providing rapid, stable and chemoselective protein bioconjugation. On the other hand, although unreactive towards amine groups under neutral aqueous conditions, 1,4-dinitroimidazoles react with lysines in organic solvents in the presence of base through a ring-opening & ring-close mechanism. The resulting cysteine- and lysine-(4-nitroimidazole) linkages exhibit stability superior to that of commonly employed maleimide-thiol conjugates. We demonstrate that 1,4-dinitroimidazoles can be applied in site-specific protein bioconjugation with functionalities such as fluorophores and bioactive peptides. Furthermore, a bisfunctional 1,4-dinitroimidazole derivative provides facile access to peptide macrocycles by crosslinking a pair of cysteine or lysine residues, including bicyclic peptides of complex architectures through highly controlled consecutive peptide macrocyclization.

De novo transcriptome analysis of Liriodendron chinense petals and leaves by Illumina sequencing.

Liriodendron chinense (Hemsl.) Sarg is an endangered species and occupies a pivotal position in phylogenetic studies of flowering plants, while its genomic resources are limited. In this study, we performed transcriptome sequencing for L. chinense petals and leaves using the Illumina paired-end sequencing technique. Approximately 17.02-Gb clean reads were obtained, and de novo assembly generated 87,841 unigenes, with an average length of 778 bp. Of these, there were 65,535 (74.61%) unigenes with significant similarity to publically available plant protein sequences. There were 3386 genes identified as significant differentially expressed between petals and leaves, among them 2969 (87.68%) were up-regulated and 417 (12.31%) down-regulated in petals. Metabolic pathway analysis revealed that 25 unigenes were predicted to be responsible for the biosynthesis of carotenoids, with 7 genes differentially expressed between these two tissues. This report is the first to identify genes associated with carotenoid biosynthesis in Liriodendron and represents a valuable resource for future genomic studies on the endangered species L. chinense.