Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle.

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

Evolution and Function of the Populus SABATH Family Reveal That a Single Amino Acid Change Results in a Substrate Switch.

Evolutionary mechanisms of substrate specificities of enzyme families remain poorly understood. Plant SABATH methyltransferases catalyze methylation of the carboxyl group of various low molecular weight metabolites. Investigation of the functional diversification of the SABATH family in plants could shed light on the evolution of substrate specificities in this enzyme family. Previous studies identified 28 SABATH genes from the Populus trichocarpa genome. In this study, we re-annotated the Populus SABATH gene family, and performed molecular evolution, gene expression and biochemical analyses of this large gene family. Twenty-eight Populus SABATH genes were divided into three classes with distinct divergences in their gene structure, expression responses to abiotic stressors and enzymatic properties of encoded proteins. Populus class I SABATH proteins converted IAA to methyl-IAA, class II SABATH proteins converted benzoic acid (BA) and salicylic acid (SA) to methyl-BA and methyl-SA, while class III SABATH proteins converted farnesoic acid (FA) to methyl-FA. For Populus class II SABATH proteins, both forward and reverse mutagenesis studies showed that a single amino acid switch between PtSABATH4 and PtSABATH24 resulted in substrate switch. Our findings provide new insights into the evolution of substrate specificities of enzyme families.

Transoral laser microsurgery versus radiotherapy for T1 glottic carcinoma: a systematic review and meta-analysis.

Transoral laser microsurgery (TLM) and radiotherapy (RT) are both accepted treatment modalities for glottic cancer. The objective of the study was to assess the oncologic outcomes and life quality of TLM in comparison with RT for T1 glottic carcinoma. We searched Medline/PubMed, Web of knowledge, EMBASE, the Cochrane Library, the Wiley online library, Springer, Google, China National Knowledge Infrastructure (CNKI), etc. We screened the literature, assessed the quality of the studies, and extracted the relevant data through the establishment of inclusion and exclusion criteria. Meta-analysis was done using the Cochrane collaboration' s RevMan 5.0 for data analysis. A total of 11 studies were included in this meta-analysis. The laryngeal preservation for patients undergoing TLM was significantly better than that for RT (P < 0.00). The laser surgery significantly improved the overall survival of patients with T1 glottic carcinoma (P = 0.04). No statistically significant differences were found between TLM and RT regarding the local control (P = 0.91). The funnel plot demonstrates no apparent publication bias in the overall survival and laryngeal preservation comparison. Our meta-analysis suggested that laser surgery was a preferred method than radiotherapy with respect to significantly better overall survival and laryngeal preservation. But the local control was not significant different. Further prospective randomized controlled studies will be needed.

Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates.

Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.

Molecular evolution and expression divergence of the Populus polygalacturonase supergene family shed light on the evolution of increasingly complex organs in plants.

Plant polygalacturonases (PGs) are involved in cell separation processes during many stages of plant development. Investigation into the diversification of this large gene family in land plants could shed light on the evolution of structural development. We conducted whole-genome annotation, molecular evolution and gene expression analyses of PG genes in five species of land plant: Populus, Arabidopsis, rice, Selaginella and Physcomitrella. We identified 75, 44, 16 and 11 PG genes from Populus, rice, Selaginella and Physcomitrella genomes, respectively, which were divided into three classes. We inferred rapid expansion of class I PG genes in Populus, Arabidopsis and rice, while copy numbers of classes II and III PG genes were relatively conserved in all five species. Populus, Arabidopsis and rice class I PG genes were under more relaxed selection constraints than class II PG genes, while this selective pressure divergence was not observed in Selaginella and Physcomitrella PG families. In addition, class I PG genes underwent marked expression divergence in Populus, rice and Selaginella. Our results suggest that PG gene expansion occurred after the divergence of the lycophytes and euphyllophytes, and this expansion was likely paralleled by the evolution of increasingly complex organs in land plants.

Different myrosinases activate sequestered glucosinolates in larvae and adults of the horseradish flea beetle.

ß-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous ß-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

Association of Interleukin-6 Genetic Polymorphisms (rs1800795, -174C > G and rs1800796, -572G > C) With Risk of Essential Hypertension in the Chinese Population.

Background Interleukin-6 (IL-6) plays a critical role in essential hypertension (EH) and cardiovascular disease. Evidence suggests two hotspot single nucleotide polymorphisms (SNPs) of the IL-6 gene (rs1800795, -174C > G and rs1800796, -572G > C) might be associated with the susceptibility of EH. However, no consensus has yet been established. Thus, we aimed to investigate the potential association between IL-6 gene polymorphisms and the risk of EH based on a case-control study in a Chinese population. Materials and methods A total of 479 subjects (272 healthy controls and 207 EH patients) were randomly enrolled in our study. After extracting the genomic DNA, two SNPs of the IL-6 gene (rs1800795, -174C > G and rs1800796, -572G > C) were genotyped to analyze the potential association between these genetic variants and EH risk. Multiple genetic models were performed to investigate the strength of association by calculating the odds ratio (OR) and 95% confidence interval (95% CI). The potential effect of SNPs on gene expression was evaluated using expression quantitative trait loci (eQTL) analysis. Results The genotyping findings of IL-6 rs1800795, -174C > G polymorphism showed three study participants with CG genotype and 204 with GG genotype in the EH patients. The IL-6 -174C > G polymorphism was significantly associated with EH risk (P = 0.046) and conferred a reduced risk of EH development (OR = 0.99, 95%CI = 0.97-1.00). Conversely, no substantial association between IL-6 rs1800796, -572G > C polymorphism and the risk of EH was found in all genetic models (P > 0.05). Moreover, the eQTL analysis indicated that the -174C > G polymorphism was significantly associated with gene expression of IL-6 (P = 0.006), and the G allele corresponded to a reduced IL-6 gene expression (Beta = -0.397). Compared with -174C > G, the -572G > C polymorphism was not found to be significantly associated with IL-6 gene expression (Beta = -0.120, P = 0.560). Conclusions Our findings provide evidence that the rs1800795, -174C > G polymorphism can affect the expression levels of IL-6, and the risk of EH occurrence. However, the rs1800796, -572G > C polymorphism does not regulate the IL-6 gene expression levels and the susceptibility of EH.

Extensive functional diversification of the Populus glutathione S-transferase supergene family.

Identifying how genes and their functions evolve after duplication is central to understanding gene family radiation. In this study, we systematically examined the functional diversification of the glutathione S-transferase (GST) gene family in Populus trichocarpa by integrating phylogeny, expression, substrate specificity, and enzyme kinetic data. GSTs are ubiquitous proteins in plants that play important roles in stress tolerance and detoxification metabolism. Genome annotation identified 81 GST genes in Populus that were divided into eight classes with distinct divergence in their evolutionary rate, gene structure, expression responses to abiotic stressors, and enzymatic properties of encoded proteins. In addition, when all the functional parameters were examined, clear divergence was observed within tandem clusters and between paralogous gene pairs, suggesting that subfunctionalization has taken place among duplicate genes. The two domains of GST proteins appear to have evolved under differential selective pressures. The C-terminal domain seems to have been subject to more relaxed functional constraints or divergent directional selection, which may have allowed rapid changes in substrate specificity, affinity, and activity, while maintaining the primary function of the enzyme. Our findings shed light on mechanisms that facilitate the retention of duplicate genes, which can result in a large gene family with a broad substrate spectrum and a wide range of reactivity toward different substrates.

Molecular evolution and functional characterization of chitinase gene family in Populus trichocarpa.

Chitinases, the chitin-degrading enzymes, have been shown to play important role in defense against the chitin-containing fungal pathogens. In this study, we identified 48 chitinase-coding genes from the woody model plant Populus trichocarpa. Based on phylogenetic analysis, the Populus chitinases were classified into seven groups. Different gene structures and protein domain architectures were found among the seven Populus chitinase groups. Selection pressure analysis indicated that all the seven groups are under purifying selection. Phylogenetic analysis combined with chromosome location analysis showed that Populus chitinase gene family mainly expanded through tandem duplication. The Populus chitinase gene family underwent marked expression divergence and is inducibly expressed in response to treatments, such as chitosan, chitin, salicylic acid and methyl jasmonate. Protein enzymatic activity analysis showed that Populus chitinases had activity towards both chitin and chitosan. By integrating sequence characteristic, phylogenetic, selection pressure, gene expression and protein activity analysis, this study shed light on the evolution and function of chitinase family in poplar.

Rapid and Selective Absorption of Plant Defense Compounds From the Gut of a Sequestering Insect.

Many herbivorous insects exploit defense compounds produced by their host plants for protection against predators. Ingested plant defense compounds are absorbed via the gut epithelium and stored in the body, a physiological process that is currently not well understood. Here, we investigated the absorption of plant defense compounds from the gut in the horseradish flea beetle, Phyllotreta armoraciae, a specialist herbivore known to selectively sequester glucosinolates from its brassicaceous host plants. Feeding experiments using a mixture of glucosinolates and other glucosides not found in the host plants showed a rapid and selective uptake of glucosinolates in adult beetles. In addition, we provide evidence that this uptake mainly takes place in the foregut, whereas the endodermal midgut is the normal region of absorption. Absorption via the foregut epithelium is surprising as the apical membrane is covered by a chitinous intima. However, we could show that this cuticular layer differs in its structure and overall thickness between P. armoraciae and a non-sequestering leaf beetle. In P. armoraciae, we observed a thinner cuticle with a less dense chitinous matrix, which might facilitate glucosinolate absorption. Our results show that a selective and rapid uptake of glucosinolates from the anterior region of the gut contributes to the selective sequestration of glucosinolates in P. armoraciae.

Sugar transporters enable a leaf beetle to accumulate plant defense compounds.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

Adverse reactions of vancomycin in humans: A protocol for meta-analysis.

BACKGROUND: Vancomycin is effective against Gram-positive bacteria and considered as a last resort in the case of ineffective use of other antigens. While due to the occurrence of adverse reactions, the application of vancomycin is strictly limited. We will conduct a meta-analysis to summarize adverse reactions of vancomycin in humans. METHODS: To collect comprehensive randomized controlled trials (RCTs), the following electronic databases will be searched: PubMed, Embase, Web of Science, Cochrane Library, the China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and China Science and Technology Journal Database. The range of publication time will be from the inception of the database to August 2020 without language limitation. Two reviewers will independently conduct selection of studies, data extraction and management, and assessment of risk of bias. Any disagreement will be resolved by discussion with the third reviewer. Review Manager 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration) will be used for meta-analysis. The Cochrane risk of bias tool will be used to assess the risk of bias. RESULTS: This study will synthesize the data from the present eligible high quality RCTs to explore the incidence of adverse reactions such as hypersensitivity reactions, nephrotoxicity, ototoxicity, phlebitis, and agranulocytosis. CONCLUSION: This meta-analysis will provide systematic evidence for adverse reactions of vancomycin in humans. STUDY REGISTRATION NUMBER: INPLASY202080094.

Identification and biochemical characterization of the glutathione reductase family from Populus trichocarpa.

Glutathione reductase (GR; EC 1.6.4.2) is a key NADPH-dependent flavo-protein oxidoreductase which can catalyze the oxidized glutathione (GSSG) to reduced glutathione (GSH) to protect plant cells from oxidative damage induced by Reactive oxygen species (ROS) burst. To investigate the biochemical characteristics and functional divergence of Populus GR family, three GR genes (PtGR1.1/1.2/2) were cloned from Populus trichocarpa and their biochemical characteristics were analyzed in this study. All the three genes were expressed in root, stem, leaf and bud, and the expression of PtGR genes were general upregulated under salicylic acid and alamethicin treatment. PtGR1.1 and PtGR1.2 were localized in cytoplasm, while PtGR2 was in chloroplast. The three PtGR proteins showed different enzymatic activities, apparent kinetic characteristic and thermal stability profiles. However, they have similar bivalent metal ions (Cu2+, Cd2+, Zn2+ and Pb2+) sensitivity and optimum pH profiles. Our study sheds light on a comprehensive information of glutathione reductase family in P. trichocarpa, and proved PtGR genes play critical roles when suffering different stresses.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

Two cases of corona virus disease 2019 (COVID-19) treated with the combination of acupuncture and medication in bedridden patients2.

The paper reports the experiences in treatment of two cases of corona virus disease 2019 (COVID-19) with the combination of acupuncture and medication in bedridden patients confirmed in C7 Inpatient Ward, Wuhan Leishenshan Hospital, China. The combined treatment of acupuncture with the oral administration of "Shanghai leishen No.1 formula" was given every day. The prescription was modified weekly according the symptoms of the patients. Besides, the antivirus, anti-infectious and symptomatic treatment of western medicine was combined. Both of the two cases were improved and discharged. It is anticipated that the treatment experiences in these two cases may provide the instruction and enlightenment for the prevention and treatment of COVID-19.

Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.

Abstract Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coli following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84 micromol/min per mg) and high affinity (a K(m) of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 degrees C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

Identification and evolution of glucosinolate sulfatases in a specialist flea beetle.

Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. Several insect herbivores, including the cabbage stem flea beetle (Psylliodes chrysocephala), prevent glucosinolate activation by forming desulfo-glucosinolates. Here we investigated the molecular basis of glucosinolate desulfation in P. chrysocephala, an important pest of oilseed rape. Enzyme activity assays with crude beetle protein extracts revealed that glucosinolate sulfatase (GSS) activity is associated with the gut membrane and has narrow substrate specificity towards the benzenic glucosinolate sinalbin. In agreement with GSS activity localization in vivo, we identified six genes encoding arylsulfatase-like enzymes with a predicted C-terminal transmembrane domain, of which five showed GSS activity upon heterologous expression in insect cells. PcGSS1 and PcGSS2 used sinalbin and indol-3-ylmethyl glucosinolate as substrates, respectively, whereas PcGSS3, PcGSS4, and PcGSS5 showed weak activity in enzyme assays. RNAi-mediated knock-down of PcGSS1 and PcGSS2 expression in adult beetles confirmed their function in vivo. In a phylogenetic analysis of coleopteran and lepidopteran arylsulfatases, the P. chrysocephala GSSs formed a cluster within a coleopteran-specific sulfatase clade distant from the previously identified GSSs of the diamondback moth, Plutella xylostella, suggesting an independent evolution of GSS activity in ermine moths and flea beetles.

A cytochrome P450 from the mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis.

Larvae of the leaf beetle Phaedon cochleariae synthesize the iridoid chysomelidial via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to (2E,6E)-2,6-dimethylocta-2,6-diene-1,8-diol (ω-OH-geraniol). Here we identify and characterize the P450 monooxygenase CYP6BH5 as the geraniol hydroxylase using integrated transcriptomics, proteomics and RNA interference (RNAi). In the fat body, 73 cytochrome P450s were identified, and CYP6BH5 was among those that were expressed specifically in fat body. Double stranded RNA mediated knockdown of CYP6BH5 led to a significant reduction of ω-hydroxygeraniol glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to ω-OH-geraniol. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α,ω-dihydroxy compounds.

Genome-wide profiling of expression and biochemical functions of the Medicago glutathione S-transferase gene family.

Glutathione S-transferases are ubiquitous enzyme in plants, playing vital roles in several physiological and developmental processes. In this study we identified 73 GST genes from the genome of Medicago truncatula. The Medicago GSTs were divided to eight classes with tau and phi being the most numerous. Six clusters were found on four Medicago chromosomes. The local gene duplication mainly contributed to the expansion of this large gene family. Functional divergence was found in their gene structures, gene expression patterns, and enzyme properties. A genomic comparative analysis revealed lineage-specific loss/gain events between Medicago and Glycine. This study offered new insights into the evolution of gene family between closely related species.

[Studies on the chemical constituents from Asarum Chinese Franch. f. fargesii (Franch.) C. Y. Cheng (II)].

Three compounds were isolated from the roots and stems of Asarum chinese Franch. f. fargesii (Franch.) C. Y. Cheng with column chromatography methods. Their structures were identified as kakuol (I), 2-methoxy-4,5-methylenedioxypropiophenone (II) and pluviatilol (III) on the basis of physicochemical and spectroscopic analysis. The compound II and III were isolated from the genus of Asarum for the first time.