Evaluation of the inhibitory effects and the mechanism of terpenoids on Toxoplasma gondii tachyzoites.

BACKGROUND: Currently, symptomatic toxoplasmosis is treated with a combination of sulfadiazine and pyrimethamine. However, significant adverse effects and drug resistance have been reported. Terpenoids are widely found in nature, with numerous studies demonstrating that they have effective inhibitory effects on a variety of parasites. METHODS: In this study, we employed intermediates or derivatives of lindenane sesquiterpenoids to evaluate their intracellular and extracellular inhibitory effects on Toxoplasma gondii tachyzoites and their cytotoxicity on macrophages. RESULTS: We demonstrated that two of these target terpenoids could effectively reduce the number of extracellular tachyzoites, probably by inducing tachyzoite apoptosis through altering tachyzoites mitochondrial membrane potential and calcium homeostasis. In addition, the two target terpenoids were able to promote intracellular tachyzoites elimination, possibly by enhancing macrophage activities. CONCLUSIONS: Two derivatives of lindenane sesquiterpenoids with low cytotoxicity to macrophages demonstrated direct and indirect antitoxoplasma effects.

Design, synthesis and properties of hydrogen peroxide fluorescent probe based on benzothiazole.

Abnormal intracellular hydrogen peroxide (H2O2) concentration is related to cancer, cardiovascular, cerebrovascular diseases and other diseases. A novel H2O2 fluorescent probe (BBS) was designed and synthesized. The fluorescence intensity shows good linear relationship with the concentration of H2O2 (0-40 µM), and the detection limit is 132 nM. The response mechanism of probe BBS was verified and analyzed by UV-Vis spectra, titration spectra and density functional theory (DFT) on Gaussian 09 programme. BBS can be used for bioimaging in living cells.

Chloroplast Genome Sequences and Comparative Analyses of Combretaceae Mangroves with Related Species.

In the Combretaceae family, only two species of Lumnitzera and one species of Laguncularia belong to mangroves. Among them, Lumnitzera littorea (Jack) Voigt. is an endangered mangrove plant in China for the limited occurrence and seed abortion. In contrast, Lumnitzera racemosa Willd. is known as the most widespread mangrove plant in China. Laguncularia racemosa C. F. Gaertn., an exotic mangrove in China, has the fast growth and high adaptation ability. To better understand the phylogenetic positions of these mangroves in Combretaceae and in Myrtales and to provide information for studies on evolutionary adaptation for intertidal habitat, the complete chloroplast (cp) genomes of Lu. racemosa and La. racemosa were sequenced. Furthermore, we present here the results from the assembly and annotation of the two cp genomes, which were further subjected to the comparative analysis with Lu. littorea cp genomes we published before and other eleven closely related species within Myrtales. The chloroplast genomes of the three Combretaceae mangrove species: Lu. littorea, Lu. racemosa, and La. racemosa are 159,687 bp, 159,473 bp, and 158,311 bp in size. All three cp genomes host 130 genes including 85 protein-coding genes, 37 tRNAs, and 4 rRNAs. A comparative analysis of those three genomes revealed the high similarity of genes in coding-regions and conserved gene order in the IR and LSC/SSC regions. The differences between Lumnitzera and Laguncularia cp genomes are the locations of rps19 and rpl2 genes in the IR/SC boundary regions. Investigating the effects of selection events on shared protein-coding genes showed a relaxed selection had acted on the ycf2, ycf1, and matK genes of Combretaceae mangroves compared to the nonmangrove species Eucalyptus aromaphloia. The phylogenetic analysis based on the whole chloroplast genome sequence with one outgroup species strongly supported three Combretaceae mangroves together with other two Combretaceae species formed a cluster in Combretaceae. This study is the first report on the comparative analysis of three Combretaceae mangrove chloroplast genomes, which will provide the significant information for understanding photosynthesis and evolution in Combretaceae mangrove plants.

CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya.

Papaya (Carica papaya L.) is a commercially important fruit crop. Various phytohormones, particularly ethylene and auxin, control papaya fruit ripening. However, little is known about the interaction between auxin and ethylene signaling during the fruit ripening process. In the present study, we determined that the interaction between the CpARF2 and CpEIL1 mediates the interaction between auxin and ethylene signaling to regulate fruit ripening in papaya. We identified the ethylene-induced auxin response factor CpARF2 and demonstrated that it is essential for fruit ripening in papaya. CpARF2 interacts with an important ethylene signal transcription factor CpEIL1, thus increasing the CpEIL1-mediated transcription of the fruit ripening-associated genes CpACS1, CpACO1, CpXTH12 and CpPE51. Moreover, CpEIL1 is ubiquitinated by CpEBF1 and is degraded through the 26S proteasome pathway. However, CpARF2 weakens the CpEBF1-CpEIL1 interaction and interferes with CpEBF1-mediated degradation of CpEIL1, promoting fruit ripening. Therefore, CpARF2 functions as an integrator in the auxin-ethylene interaction and regulates fruit ripening by stabilizing CpEIL1 protein and promoting the transcriptional activity of CpEIL1. To our knowledge, we have revealed a novel module of CpARF2/CpEIL1/CpEBF1 that fine-tune fruit ripening in papaya. Manipulating this mechanism could help growers tightly control papaya fruit ripening and prolong shelf life.

A novel fluorescent sensor for specific recognition of GSH based on the copper complex and its bioimaging in living cells.

Given the important role of biothiols in various physiological processes, there is a need to develop novel fluorescent sensors for detecting them. Herein, a novel "on-off-on" fluorescent sensor (E)-N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-6-((quinolin-8-yloxy)methyl)picolinohydrazide (PQC) was synthesized and its absorbance and fluorescence properties were characterized. The sensor PQC could form a stable complex and showed a significant fluorescence quenching response to Cu2+ with a quenching efficiency of approximately 100%, and the PQC-Cu2+ complex showed a fluorescence enhancement response to GSH with a higher recovery rate of above 80% in a CH3OH/HEPES (9:1 v/v, pH = 7.23) buffer system. Its detection limits were determined to be 0.17 µM for Cu2+ and 0.20 µM for GSH, and the binding stoichiometry of PQC-Cu2+ was determined to be 1: 1 by Job's plot method. Importantly, the sensor PQC can be used for filter paper strip tests and bioimaging in living cells.

A novel fluorescent chemosensor based on coumarin and quinolinyl-benzothiazole for sequential recognition of Cu2+ and PPi and its applicability in live cell imaging.

In this study, a highly selective fluorescent sensor (E)-2-((2-(benzo[d]thiazol-2-yl)quinolin-8-yl)oxy)-N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)acetohydrazide (TQC) was synthesized from 2-methylquinolin-8-ol and 4-(diethylamino)-2-hydroxybenzaldehyde and its structure was characterized by 1H NMR, 13C NMR, ESI-HR-MS and density functional theory (DFT) calculation. Sensor TQC showed an obvious "on-off-on" fluorescence response to Cu2+ and PPi in a DMSO/HEPES (3:2 v/v, pH = 7.4) buffer system. The detection limits of sensor TQC were 0.06 µM to Cu2+ and 0.01 µM to PPi. In addition, sensor TQC showed a 1:1 binding stoichiometry to Cu2+ and TQC-Cu2+ complex showed a 2:1 binding stoichiometry to PPi. The optimum pH range of sensor TQC and TQC-Cu2+ was 3-8. Further studies demonstrated that sensor TQC could be made into test paper strips for the qualitative of Cu2+ and PPi and showed sequentially "on-off-on" fluorescent bio-imaging of Cu2+ and PPi in HeLa cells.

A simple fluorescent probe for detection of Ag+ and Cd2+ and its Cd2+ complex for sequential recognition of S2.

In this study, we designed and synthesized a simple probe 2-(8-((8-methoxyquinolin-2-yl)methoxy)quinolin-2-yl)benzo[d]thiazole (DQT) for detection of Ag+ and Cd2+ in a CH3OH/HEPES (9 : 1 v/v, pH = 7.30) buffer system. Its structure was characterized by NMR, ESI-HR-MS and DFT calculations, and its fluorescence performance was also investigated. Probe DQT showed fluorescence quenching in response to Ag+ and Cd2+ with low detection limits of 0.42 µM and 0.26 µM, respectively. Importantly, the complexation of the probe with Cd2+ resulted in a red shift from blue to green, making it possible to detect Ag+ and Cd2+ by the naked eye under an ultraviolet lamp. The DQT-Cd2+ complex could be used for sequential recognition of S2-. The recovery response could be repeated 3 times by alternate addition of Cd2+ and S2-. A filter paper strip test further demonstrated the potential of probe DQT as a convenient and rapid assay.

Synthesis of Four Illudalane Sesquiterpenes Utilizing a One-Pot Diels-Alder/Oxidative Aromatization Sequence.

The concise, divergent total syntheses of four illudalane sesquiterpenes using an indanone as the key intermediate are reported. The key elements in these total syntheses, which involve only four to six operational steps, consist of a Suzuki cross-coupling and a one-pot Diels-Alder/oxidative aromatization reaction.

Comparative proteomic analysis provides novel insights into the regulation mechanism underlying papaya (Carica papaya L.) exocarp during fruit ripening process.

BACKGROUND: Papaya (Carica papaya L.) is a popular climacteric fruit, undergoing various physico-chemical changes during ripening. Although papaya is widely cultivated and consumed, few studies on the changes in metabolism during its ripening process at the proteasome level have been performed. Using a newly developed TMT-LCMS analysis, proteomes of papaya fruit at different ripening stages were investigated. RESULTS: In total, 3220 proteins were identified, of which 2818 proteins were quantified. The differential accumulated proteins (DAPs) exhibited various biological functions and diverse subcellular localizations. The KEGG enrichment analysis showed that various metabolic pathways were significantly altered, particularly in flavonoid and fatty acid metabolisms. The up-regulation of several flavonoid biosynthesis-related proteins may provide more raw materials for pigment biosynthesis, accelerating the color variation of papaya fruit. Variations in the fatty acid metabolism- and cell wall degradation-related proteins were investigated during the ripening process. Furthermore, the contents of several important fatty acids were determined, and increased unsaturated fatty acids may be associated with papaya fruit volatile formation. CONCLUSIONS: Our data may give an intrinsic explanation of the variations in metabolism during the ripening process of papaya fruit.

Total synthesis, structural revision and biological evaluation of γ-elemene-type sesquiterpenes.

Total synthesis and absolute configuration confirmation of γ-elemene-type sesquiterpenes, which possess vast potential for biological activities, was investigated based on a convergent synthetic strategy. A key intermediate with all functional groups of this family of natural products was accessed by an intermolecular aldol reaction and then an acetylation of a known ketone (12) derived from commercially available verbenone. The versatile intermediate can be easily transformed into structurally different γ-elemene-type sesquiterpenes based on control of base-promoted cyclization manipulation in different solvents. The utility of this robust approach is illustrated by the first syntheses of elema-1,3,7(11),8-tetraen-8,12-lactam (4') and 8ß-methoxy-isogermafurenolide (6a), as well as the syntheses of elem-1,3,7,8-tetraen-8,12-olide (3) and hydroxyisogermafurenolide (5) in only 6 or 7 steps. In addition, the structure of the reported 5ßH-elem-1,3,7,8-tetraen-8,12-olide (1) was revised as elem-1,3,7,8-tetraen-8,12-olide (3) by comparison of their identified datum, and the absolute configuration of elema-1,3,7(11),8-tetraen-8,12-lactam was confirmed as 4'. Furthermore, the inhibitory effect of all synthesized natural compounds and their natural analogues on cancer cell proliferation was evaluated. Among them compounds 3, 4 and 4' were found to possess potent inhibitory activity against Kasumi-1 and Pfeiffer. Meanwhile, preliminary structure-activity relationships for these compounds are discussed.

A qualitative proteome-wide lysine crotonylation profiling of papaya (Carica papaya L.).

Lysine crotonylation of histone proteins is a recently-identified post-translational modification with multiple cellular functions. However, no information about lysine crotonylation of non-histone proteins in fruit cells is available. Using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity antibody analysis, a global crotonylation proteome analysis of papaya fruit (Carica papaya L.) was performed. In total, 2,120 proteins with 5,995 lysine crotonylation sites were discovered, among which eight conserved motifs were identified. Bioinformatic analysis linked crotonylated proteins to multiple metabolic pathways, including biosynthesis of antibiotics, carbon metabolism, biosynthesis of amino acids, and glycolysis. particularly, 40 crotonylated enzymes involved in various pathways of amino acid metabolism were identified, suggesting a potential conserved function for crotonylation in the regulation of amino acid metabolism. Numerous crotonylation sites were identified in proteins involved in the hormone signaling and cell wall-related pathways. Our comprehensive crotonylation proteome indicated diverse functions for lysine crotonylation in papaya.

Comparative transcriptomic analyses of normal and malformed flowers in sugar apple (Annona squamosa L.) to identify the differential expressed genes between normal and malformed flowers.

BACKGROUND: Sugar apple (Annona squamosa L.), a popular fruit with high medicinal and nutritional properties, is widely cultivated in tropical South Asia and America. The malformed flower is a major cause for a reduction in production of sugar apple. However, little information is available on the differences between normal and malformed flowers of sugar apple. RESULTS: To gain a comprehensive perspective on the differences between normal and malformed flowers of sugar apple, cDNA libraries from normal and malformation flowers were prepared independently for Illumina sequencing. The data generated a total of 70,189,896 reads that were integrated and assembled into 55,097 unigenes with a mean length of 783 bp. A large number of differentially expressed genes (DEGs) were identified. Among these DEGs, 701 flower development-associated transcript factor encoding genes were included. Furthermore, a large number of flowering- and hormone-related DEGs were also identified, and most of these genes were down-regulated expressed in the malformation flowers. The expression levels of 15 selected genes were validated using quantitative-PCR. The contents of several endogenous hormones were measured. The malformed flowers displayed lower endogenous hormone levels compared to the normal flowers. CONCLUSIONS: The expression data as well as hormone levels in our study will serve as a comprehensive resource for investigating the regulation mechanism involved in floral organ development in sugar apple.

Genome-wide analysis and characterization of Aux/IAA family genes related to fruit ripening in papaya (Carica papaya L.).

BACKGROUND: Auxin/indole-3-acetic acid (Aux/IAA) family genes encode short-lived nuclear proteins that mediate the responses of auxin-related genes and are involved in several plant developmental and growth processes. However, how Aux/IAA genes function in the fruit development and ripening of papaya (Carica papaya L.) is largely unknown. RESULTS: In this study, a comprehensive identification and a distinctive expression analysis of 18 C. papaya Aux/IAA (CpIAA) genes were performed using newly updated papaya reference genome data. The Aux/IAA gene family in papaya is slightly smaller than that in Arabidopsis, but all of the phylogenetic subfamilies are represented. Most of the CpIAA genes are responsive to various phytohormones and expressed in a tissues-specific manner. To understand the putative biological functions of the CpIAA genes involved in fruit development and ripening, quantitative real-time PCR was used to test the expression profiling of CpIAA genes at different stages. Furthermore, an IAA treatment significantly delayed the ripening process in papaya fruit at the early stages. The expression changes of CpIAA genes in ACC and 1-MCP treatments suggested a crosstalk between auxin and ethylene during the fruit ripening process of papaya. CONCLUSIONS: Our study provided comprehensive information on the Aux/IAA family in papaya, including gene structures, phylogenetic relationships and expression profiles. The involvement of CpIAA gene expression changes in fruit development and ripening gives us an opportunity to understand the roles of auxin signaling in the maturation of papaya reproductive organs.

Total Syntheses of Sarcandrolide†J and Shizukaol†D: Lindenane Sesquiterpenoid [4+2] Dimers.

The syntheses of members of a family of lindenane sesquiterpenoid [4+2] dimers led to the total syntheses of sarcandrolide J and shizukaol D. Inspired by a modified biosynthetic pathway, a cascade featuring furan formation/alkene isomerization/Diels-Alder cycloaddition was devised to construct the congested polycyclic architecture of the target molecules with the correct stereochemistry. This study presents a pioneering synthetic entry to this family of natural products and paves the way for fully exploring their biological functions.

Transcriptome Analysis and Identification of Genes Associated with Floral Transition and Flower Development in Sugar Apple (Annona squamosa L.).

Sugar apple (Annona squamosa L.) is a semi-deciduous subtropical tree that progressively sheds its leaves in the spring. However, little information is available on the mechanism involved in flower developmental pattern. To gain a global perspective on the floral transition and flower development of sugar apple, cDNA libraries were prepared independently from inflorescent meristem and three flowering stages. Illumina sequencing generated 107,197,488 high quality reads that were assembled into 71,948 unigenes, with an average sequence length of 825.40 bp. Among the unigenes, various transcription factor families involved in floral transition and flower development were elucidated. Furthermore, a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that unigenes exhibiting differential expressions were involved in various phytohormone signal transduction events and circadian rhythms. In addition, 147 unigenes exhibiting sequence similarities to known flowering-related genes from other plants were differentially expressed during flower development. The expression patterns of 20 selected genes were validated using quantitative-PCR. The expression data presented in our study is the most comprehensive dataset available for sugar apple so far and will serve as a resource for investigating the genetics of the flowering process in sugar apple and other Annona species.

Identification, Expression and IAA-Amide Synthetase Activity Analysis of Gretchen Hagen 3 in Papaya Fruit (Carica papaya L.) during Postharvest Process.

Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya.

Genome-wide identification and characterization of auxin response factor (ARF) family genes related to flower and fruit development in papaya (Carica papaya L.).

BACKGROUND: Auxin and auxin signaling are involved in a series of developmental processes in plants. Auxin Response Factors (ARFs) is reported to modulate the expression of target genes by binding to auxin response elements (AuxREs) and influence the transcriptional activation of down-stream target genes. However, how ARF genes function in flower development and fruit ripening of papaya (Carica papaya L.) is largely unknown. In this study, a comprehensive characterization and expression profiling analysis of 11 C. papaya ARF (CpARF) genes was performed using the newly updated papaya reference genome data. RESULTS: We analyzed CpARF expression patterns at different developmental stages. CpARF1, CpARF2, CpARF4, CpARF5, and CpARF10 showed the highest expression at the initial stage of flower development, but decreased during the following developmental stages. CpARF6 expression increased during the developmental process and reached its peak level at the final stage of flower development. The expression of CpARF1 increased significantly during the fruit ripening stages. Many AuxREs were included in the promoters of two ethylene signaling genes (CpETR1 and CpETR2) and three ethylene-synthesis-related genes (CpACS1, CpACS2, and CpACO1), suggesting that CpARFs might be involved in fruit ripening via the regulation of ethylene signaling. CONCLUSIONS: Our study provided comprehensive information on ARF family in papaya, including gene structures, chromosome locations, phylogenetic relationships, and expression patterns. The involvement of CpARF gene expression changes in flower and fruit development allowed us to understand the role of ARF-mediated auxin signaling in the maturation of reproductive organs in papaya.

An entry to vinylcyclopropane through palladium-catalyzed intramolecular cyclopropanation of alkenes with unstabilized allylic tosylhydrazones.

A diastereoselective Pd-catalyzed intramolecular cyclopropanation of alkenes with unstabilized allylic tosylhydrazones was developed. This methodology provides an efficient entry toward synthesis of the bicyclo[3.1.0] hexane system with an exo-double bond, and sets the basis for future elaboration of more complex polycyclic motifs.

Asymmetric total synthesis of onoseriolide, bolivianine, and isobolivianine.

In this article, we describe our efforts on the total synthesis of bolivianine (1) and isobolivianine (2), involving the synthesis of onoseriolide (3). The first generation synthesis of bolivianine was completed in 21 steps by following a chiral resolution strategy. Based on the potential biogenetic relationship between bolivianine (1), onoseriolide (3), and ß-(E)-ocimene (8), the second generation synthesis of bolivianine was biomimetically achieved from commercially available (+)-verbenone in 14 steps. The improved total synthesis features an unprecedented palladium-catalyzed intramolecular cyclopropanation through an allylic metal carbene, for the construction of the ABC tricyclic system, and a Diels-Alder/intramolecular hetero-Diels-Alder (DA/IMHDA) cascade for installation of the EFG tricyclic skeleton with the correct stereochemistry. Transformation from bolivianine to isobolivianine was facilitated in the presence of acid. The biosynthetic mechanism and the excellent regio- and endo selectivities in the cascade are well supported by theoretical chemistry based on the DFT calculations.