Rapid Evaluation of the Resistance of Citrus Germplasms Against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms ('Ichang' papeda and 'Huapi' kumquat) are resistant, whereas eight citrus germplasms ('Chongyi' wild mandarin, 'Mangshan' wild mandarin, 'Ledong' kumquat, 'Dali' citron, 'Yiliang' citron, 'Longyan' kumquat, 'Bawang' kumquat, and 'Daoxian' wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches.

Region-Wide Comprehensive Implementation of Roguing Infected Trees, Tree Replacement, and Insecticide Applications Successfully Controls Citrus Huanglongbing.

Huanglongbing (HLB) is a devastating citrus disease worldwide. A three-pronged approach to controlling HLB has been suggested, namely, removal of HLB-symptomatic trees, psyllid control, and replacement with HLB-free trees. However, such a strategy did not lead to successful HLB control in many citrus-producing regions, such as Florida. We hypothesize that this is because of the small-scale or incomprehensive implementation of the program; conversely, a comprehensive implementation of such a strategy at the regional level can successfully control HLB. To test our hypothesis, we investigated the effects of region-wide comprehensive implementation of this scheme to control HLB in Gannan region, China, with a total planted citrus acreage of over 110,000 ha from 2013 to 2019. With the region-wide implementation of comprehensive HLB management, the overall HLB incidence in Gannan decreased from 19.71% in 2014 to 3.86% in 2019. A partial implementation of such a program (without a comprehensive inoculum removal) at the regional level in Brazil resulted in HLB incidence increasing from 1.89% in 2010 to 19.02% in 2019. Using dynamic regression model analyses with data from both Brazil and China, we constructed a model to predict HLB incidence when all three components were applied at 100%. It was predicated that in a region-wide comprehensive implementation of such a program, HLB incidence would be controlled to a level of less than 1%. We conducted economic feasibility analyses and showed that average net profits were positive for groves that implemented the comprehensive strategy, but groves that did not implement it had negative net profits over a 10-year period. Overall, the key for the three-pronged program to successfully control HLB is the large scale (region-wide) and comprehensiveness in implementation. This study provides valuable information to control HLB and other economically important endemic diseases worldwide.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Chemical composition, antioxidant, antibacterial, and tyrosinase inhibition activity of extracts from Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) peel.

BACKGROUND: Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) is the major navel orange cultivar planted in China. Almost all Newhall navel orange peels produced in that country are currently discarded, which is not only harmful to the environment but also a waste of resources. It is therefore necessary to carry out research to explore the utilization potential of this resource. RESULTS: A 95% ethanol extract (EE) of Newhall orange peel was prepared and fractionated into three subextracts: petroleum ether extract (PEE), ethyl acetate extract (EtOAcE), and water extract (WE) by simple liquid / liquid extractions. These four extracts were then subjected to antioxidant, antibacterial, and tyrosinase inhibition assays. Interestingly, EtOAcE was significantly superior to all other three extracts, exhibiting the best biological effects. The total polyphenol content (TPC), total flavonoid content (TFC), and primary individual flavonoids of these four extracts were analyzed and compared. This was followed by principal component analysis (PCA) and the Pearson's correlation test. The result indicates that the primary bioactive compounds responsible for the biological effects of the EtOAcE are sinensetin, 4',5,6,7-tetramethoxyflavone, nobiletin, 3,3',4',5,6,7-hexamethoxyflavone, and narirutin. In view of its easy preparation and potent biological effects, EtOAcE might demonstrate excellent application potential in various industrial areas. CONCLUSIONS: This study successfully identified EtOAcE as a potent naturally occurring antioxidant, antibacterial, and tyrosinase inhibitory agent, which might add value to the utilization of Newhall navel orange peel in the food, cosmetic, and pharmaceutical industries. © 2020 Society of Chemical Industry.

Identification and functional analysis of an iron-binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus.

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.

Tomato histone H2B monoubiquitination enzymes SlHUB1 and SlHUB2 contribute to disease resistance against Botrytis cinerea through modulating the balance between SA- and JA/ET-mediated signaling pathways.

BACKGROUND: Histone H2B monoubiquitination pathway has been shown to play critical roles in regulating growth/development and stress response in Arabidopsis. In the present study, we explored the involvement of the tomato histone H2B monoubiquitination pathway in defense response against Botrytis cinerea by functional analysis of SlHUB1 and SlHUB2, orthologues of the Arabidopsis AtHUB1/AtHUB2. METHODS: We used the TRV-based gene silencing system to knockdown the expression levels of SlHUB1 or SlHUB2 in tomato plants and compared the phenotype between the silenced and the control plants after infection with B. cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Biochemical and interaction properties of proteins were examined using in vitro histone monoubiquitination and yeast two-hybrid assays, respectively. The transcript levels of genes were analyzed by quantitative real time PCR (qRT-PCR). RESULTS: The tomato SlHUB1 and SlHUB2 had H2B monoubiquitination E3 ligases activity in vitro and expression of SlHUB1 and SlHUB2 was induced by infection of B. cinerea and Pst DC3000 and by treatment with salicylic acid (SA) and 1-amino cyclopropane-1-carboxylic acid (ACC). Silencing of either SlHUB1 or SlHUB2 in tomato plants showed increased susceptibility to B. cinerea, whereas silencing of SlHUB1 resulted in increased resistance against Pst DC3000. SlMED21, a Mediator complex subunit, interacted with SlHUB1 but silencing of SlMED21 did not affect the disease resistance to B. cinerea and Pst DC3000. The SlHUB1- and SlHUB2-silenced plants had thinner cell wall but increased accumulation of reactive oxygen species (ROS), increased callose deposition and exhibited altered expression of the genes involved in phenylpropanoid pathway and in ROS generation and scavenging system. Expression of genes in the SA-mediated signaling pathway was significantly upregulated, whereas expression of genes in the jasmonic acid (JA)/ethylene (ET)-mediated signaling pathway were markedly decreased in SlHUB1- and SlHUB2-silenced plants after infection of B. cinerea. CONCLUSION: VIGS-based functional analyses demonstrate that both SlHUB1 and SlHUB2 contribute to resistance against B. cinerea most likely through modulating the balance between the SA- and JA/ET-mediated signaling pathways.

The de novo biosynthesis of vitamin B6 is required for disease resistance against Botrytis cinerea in tomato.

Vitamin B6 (VB6), an essential cofactor for numerous metabolic enzymes, has recently been shown to act as a potent antioxidant and play important roles in developmental processes and stress responses. However, little is known about the possible function of VB6 in plant disease resistance response against pathogen infection. In the present study, we explored the possible involvement of VB6 in defense response against Botrytis cinerea through functional analysis of tomato VB6 biosynthetic genes. Three de novo VB6 biosynthetic genes (SlPDX1.2, SlPDX1.3, and SlPDX2) and one salvage pathway gene (SlSOS4) were identified and the SlPDX1.2, SlPDX1.3, and SlPDX2 genes were shown to encode functional enzymes involved in de novo biosynthesis of VB6, as revealed by complementation of the VB6 prototrophy in yeast snz1 and sno1 mutants. Expression of SlPDX1.2, SlPDX1.3, and SlSOS4 genes was induced by infection with B. cinerea. Virus-induced gene silencing-mediated knockdown of SlPDX1.2 or SlPDX1.3 but not SlPDX2 and SlSOS4 led to increased severity of disease caused by B. cinerea, indicating that the VB6 de novo biosynthetic pathway but not the salvage pathway is involved in tomato defense response against B. cinerea. Furthermore, the SlPDX1.2- and SlPDX1.3-silenced tomato plants exhibited reduced levels of VB6 contents and reactive oxygen species scavenging capability, increased levels of superoxide anion and H2O2 generation, and increased activity of superoxide dismutase after infection by B. cinerea. Our results suggest that VB6 and its de novo biosynthetic pathway play important roles in regulation of defense response against B. cinerea through modulating cellular antioxidant capacity.

Tomato SR/CAMTA transcription factors SlSR1 and SlSR3L negatively regulate disease resistance response and SlSR1L positively modulates drought stress tolerance.

BACKGROUND: The SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress. RESULTS: Expression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast. CONCLUSIONS: VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.

Tomato SlMKK2 and SlMKK4 contribute to disease resistance against Botrytis cinerea.

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that mediate the transduction of extracellular stimuli via receptors/sensors into intracellular responses and play key roles in plant immunity against pathogen attack. However, the function of tomato MAPK kinases, SlMKKs, in resistance against Botrytis cinerea remains unclear yet. RESULTS: A total of five SlMKK genes with one new member, SlMKK5, were identified in tomato. qRT-PCR analyses revealed that expression of SlMKK2 and SlMKK4 was strongly induced by B. cinerea and by jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual SlMKKs and disease assays identified that SlMKK2 and SlMKK4 but not other three SlMKKs (SlMKK1, SlMKK3 and SlMKK5) are involved in resistance against B. cinerea. Silencing of SlMKK2 or SlMKK4 resulted in reduced resistance to B. cinerea, increased accumulation of reactive oxygen species and attenuated expression of defense genes after infection of B. cinerea in tomato plants. Furthermore, transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK4DD in leaves of Nicotiana benthamiana plants led to enhanced resistance to B. cinerea and elevated expression of defense genes. CONCLUSIONS: VIGS-based knockdown of SlMKK2 and SlMKK4 expression in tomato and gain-of-function transient expression of constitutively active phosphomimicking forms SlMKK2DD and SlMKK2DD in N. benthamiana demonstrate that both SlMKK2 and SlMKK4 function as positive regulators of defense response against B. cinerea.

Synergy of enediyne antibiotic lidamycin and temozolomide in suppressing glioma growth with potentiated apoptosis induction.

The present work evaluated the synergistic efficacy of an enediyne antibiotic lidamycin (LDM) plus temozolomide (TMZ) against glioma in vitro and in vivo. LDM plus TMZ inhibited the proliferations of rat glioma C6 cells and human glioma U87 cells more efficiently than the single usage of LDM or TMZ. In addition, LDM also potentiated the apoptosis inductions by TMZ in rat C6 cells and human U87 cells. Meanwhile, the results of TdT-mediated dUTP Nick End Labeling assay for subcutaneous U87 tumor sections indicated an enhanced apoptosis induction in vivo by LDM plus TMZ, which confirmed the high potency of the combination for glioma therapy. As determined by Western blot, apoptosis signal pathways in C6 cells and U87 cells were markedly affected by the synergistic alteration of P53, bax, procaspase 3, and bcd-2 expression. In both subcutaneous U87 xenograft and C6 intracerebral orthotopic implant model, TMZ-induced glioma growth suppression was dramatically potentiated by LDM. As shown, the combination therapy efficiently reduced the tumor volumes and tumor weights of the human glioma U87 xenograft. Kaplan-Meier assay revealed that LDM plus TMZ dramatically prolonged the life span of C6 intracerebral tumor-bearing rats with decreased tumor size. This study indicates that the combination of LDM with TMZ might be a promising strategy for glioma therapy.

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response.

KEY MESSAGE: Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death. Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.

Identification of HSP70 genes in Diaphorina citri Kuwayama Reveals Their Involvement in Immunity and Development.

Huanglongbing (HLB), a global citrus threat, is transmitted by Diaphorina citri Kuwayama, a widespread insect pest. The disease's rapid spread and incurability necessitate efficient, sustainable control strategies. This study investigates heat shock protein 70 (HSP70) genes in D. citri, known to play a pivotal role in insect survival and stress response. The genome-wide identification, gene structure analysis, and conserved protein domain analysis of 22 HSP70 genes in D. citri were performed. Furthermore, the expression of these genes during HLB infection or developmental processes was gauged. Phylogenetic analysis revealed the functional categorization of the identified genes, while gene structure and conserved motifs offered insights into gene function. The expression analysis unveiled dynamic profiles in response to infection and across development stages, potentially aiding future targeted pest control strategies. These findings offer promising leads for the design of novel inhibitors or RNAi strategies targeting D. citri and HLB.

A Transcriptome Analysis of Poncirus trifoliata, an Aurantioideae Species Tolerant to Asian Citrus Psyllid, Has Identified Potential Genes and Events Associated with Psyllid Resistance.

Citrus huanglongbing (HLB) is a devastating disease for citrus production, largely caused by the Asian citrus psyllid (ACP). Poncirus trifoliata exhibits high resistance to ACP; however, this resistance is weakened when C. sinensis is co-cultivated. This study aimed to identify the differentially expressed genes (DEGs) during ACP feeding and to uncover potential ACP resistance genes in P. trifoliata. In comparison to independent cultivation, 1247 and 205 DEGs were identified in P. trifoliata when co-cultivated with C. sinensis after 7 and 14 days, respectively. Analysis of enriched Gene Ontology categories revealed that DEGs were significantly associated with the cell wall, glucometabolic activities, and secondary metabolites. Additionally, these genes were found to be involved in phytohormone signaling, cell wall metabolism, redox state homeostasis, and secondary metabolites, as well as a number of transcription factor genes (TFs). Furthermore, we examined the impact of the ACP feeding factor on the gene expression patterns in P. trifoliata. Results showed an increase in the JA signaling pathway and various TFs. The RNA-seq results were verified using reverse transcription quantitative PCR. Our findings shed light on the molecular basis of ACP resistance in P. trifoliata and identified potential genes associated with this resistance.

Evaluation of antibacterial activity of compounds isolated from the peel of Newhall navel orange.

Fifteen compounds including one flavanone hesperitin (1), two cinnamic acid derivatives as (E)-4-hydroxycinnamic acid (2) and (E)-ferulic acid (3), eight polymethoxyflavones (PMFs) (4-11), and four hydroxylated PMFs (12-15) isolated from orange peel were subjected to antibacterial evaluation. Compounds 1-3 exhibited wide-spectrum antibacterial effects against four test strains involving Bacillus subtilis, Staphylococcus aureus, Xanthomonas citri subsp. citri (Xcc), and Meticillin-resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentrations (MICs) ranging from 0.0312 to 0.125 mg/mL. In contrast, all PMFs showed weak antibacterial activity against these four strains with MICs being equal to or more than 0.125 mg/mL. Hydroxylated PMFs demonstrated better antibacterial effect against Xcc relative to PMFs. In addition, the synergistic effect against Xcc was obtained when compounds 1 and 3 were combined. Furthermore, the scanning electron microscopy (SEM) results of Xcc treated with both compounds 2 and 3 showed shrunken and rough surface morphologies, indicative of the cell membrane damage.

Erratum: Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

[This corrects the article DOI: 10.1016/j.isci.2023.106024.].

Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

Citrus HLB caused by Candidatus Liberibacter asiaticus is a pathogen-triggered immune disease. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. We first developed a phased diploid assembly of Citrus sinensis 'Newhall' genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 accessions for genome-wide association mapping of the HLB response. Genes determinants for HLB pathogenicity were associated with host immune response, ROS production, and antioxidants. Overall, this study has provided a significant resource of citrus genomic data and identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity.

Pomelo seed oil: Natural insecticide against cowpea aphid.

Cowpea aphid (Aphis craccivora Koch) is a plant pest that causes serious damage to vegetable crops. Extensive use of synthetic chemical pesticides causes deleterious effects on consumers as well as the environment. Hence, the search for environmentally friendly insecticides in the management of cowpea aphids is required. The present work aims to investigate the aphicidal activity of pomelo seed oil (PSO) on cowpea aphids, the possible insecticidal mechanisms, its chemical constituent profile, as well as the toxicity of its primary compounds. The results of the toxicity assay showed that PSO had significant insecticidal activity against aphids with a 72-hour LC50 value of 0.09 µg/aphid and 3.96 mg/mL in the contact and residual toxicity assay, respectively. The enzymatic activity of both glutathione S-transferase (GST) and acetyl cholinesterase (AChE) significantly decreased, as well as the total protein content, after PSO treatment, which suggested that the reduction of AChE, GST, and the total protein content in aphids treated with PSO might be responsible for the mortality of A. craccivora. The GC-MS analysis revealed that PSO contained limonene (22.86%), (9Z,12Z)-9,12-octadecadienoic acid (20.21%), n-hexadecanoic acid (15.79%), (2E,4E)-2,4-decadienal (12.40%), and (2E,4Z)-2,4-decadienal (7.77%) as its five major compounds. Furthermore, (9Z,12Z)-9,12-octadecadienoic acid showed higher toxicity to aphids than both PSO and thiamethoxam (positive control). This study emphasized the potential of PSO as a natural plant-derived insecticide in controlling cowpea aphids and also provided a novel approach for the value-added utilization of pomelo seed.

A Fluorescent Reporter-Based Evaluation Assay for Antibacterial Components Against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the agent of citrus bacterial canker (CBC) disease, which has significantly reduced citrus quantity and quality in many producing areas worldwide. Copper-based bactericides are the primary products for CBC control and management, but the problems derived from copper-resistant and environmental contamination have become issues of anxiety. Thus, there is a need to find alternative antibacterial products instead of relying on a single type of agent. This study developed a method to evaluate the inhibition of antibacterial agents using the fluorescence-labeled recombinant Xcc strain (Xcc-eYFP). The optimization of timelines and parameters for the evaluation of antibacterial agents involved the use of a Spark™ multimode microplate reader. This evaluation and screening method can be applied to bactericides, cocktail-mixture formulations, antagonistic bacteria, and derived metabolites. The results showed that the minimum inhibitory concentration (MIC) of commercial bactericides determined by fluorescence agrees with the MIC values determined by the conventional method. A screened cocktail-mixture bactericide presents more activity than the individual agents during the protective effects. Notably, this method has been further developed in the screening of Xcc-antagonistic bacterial strains. In summary, we provide a validated strategy for screening and evaluation of different antibacterial components for inhibition against Xcc for CBC control and management.

[Inhibitory effect of lidamycin upon vasculogenic mimicry and its induction of apoptosis in glioma cells].

OBJECTIVE: To evaluate the in vitro effects of lidamycin upon vasculogenic mimicry and apoptosis induction in glioma cells. METHODS: Tube formation assay was performed to estimate the inhibitory effects of lidamycin upon vasculogenic mimicry in C6 and U87 glioma cells. The vasculogenic mimicry of glioma cells was photographed and enumerated. Annexin V-FITC/PI was used for determination of glioma cell apoptosis with flow cytometry. RESULTS: Vasculogenic mimicry assay indicated that 0.1 nmol/L, 0.5 nmol/L and 1 nmol/L lidamycin showed significant inhibition of vasculogenic mimicry in C6 and U87 cells. Comparing with C6 control group (14.7 +/- 1.2), 0.1 nmol/L (12.7 +/- 0.6), 0.5 nmol/L (9.0 +/- 1.7) and 1 nmol/L (4.7 +/-0.6) lidamycin inhibited vasculogenic mimicry in C6 cells with statistical significances (P = 0.013, P = 0.005 and P = 0.0002 respectively). Comparing with U87 control group (14.7 +/- 1.2), the vasculogenic mimicry of 0.1 nmol/L (10.0 +/- 2.0), 0.5 nmol/L (8.3 +/- 1.5) and 1 nmol/L lidamycin (4.3 +/- 0.6) treated U87 cells showed statistical significances (P = 0.025, P = 0.00 and P = 0.0009 respectively). The apoptotic ratios of same dosa ges lidamycin treated C6 cells and U87cells showed a similar tendency as vasculogenic mimicry inhibition (P < 0.001). Lidamycin was more potent than neocarzinostatin in vasculogenic mimicry inhibition and apoptosis induction of C6 cells and U87 cells. Conclusion Lidamycin can inhibit vasculogenic mimicry and promote apoptosis of glioma cells. Thus it is a promising drug in glioma treatment. Further researches on the therapeutic efficacy of enediyne antibiotics in glioma are needed.

Mitochondrial genome of Tamarixia radiata (Hymenoptera: Chalcidoidea: Eulophidae) and phylogenetic analysis.

Tamarixia radiata plays an important role in biological control of the psyllid Diaphorina citri Kuwayama, vector of the huanglongbing (HLB). In this study, we sequenced and analyzed the mitochondrial genome (mitogenome) of T. radiata, the first mitogenome of species in the family Eulophidae. This mitogenome was 14,752 bp long and encoded 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA unit genes (rRNAs). All 13 PCGs were initiated by the ATN (ATG, ATT, and ATA) codon. Twelve PCGs terminate with the stop codon TAA or TAG except for nad1 which end with the incomplete codon T-. Phylogenetic analysis showed that T. radiata got together with three Pteromalidae species, indicating the close relationship of Eulophidae and Pteromalidae.

Down-regulation of the nuclear factor-kappaB by lidamycin in association with inducing apoptosis in human pancreatic cancer cells and inhibiting xenograft growth.

Pancreatic cancer is now one of the most common causes of cancer death worldwide. K-ras mutations are present in up to 90% of pancreatic cancer cases. The expression of mutant K-ras activates the Akt/protein kinase B pathway, resulting in the activation of the nuclear factor-kappaB (NF-kappaB) transcriptional factor. Constitutive NF-kappaB activity plays a key role in pancreatic carcinoma. NF-kappaB has been shown to inhibit apoptosis in response to chemotherapeutic agents. In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, were investigated on two established pancreatic cell lines, PANC-1 and SW1990. A dose-dependent inhibition of phospho-Akt and NF-kappaB activation was found in the cells treated with LDM as determined by Western blot analysis. Moreover, a down-regulation of K-ras mRNA and a protein expression by LDM were observed in both cell lines as determined by reverse transcription-PCR and Western blot analysis. By MTT assay, a remarkable difference in chemosensitivity to LDM, mitomycin, adriamycin, taxol, and gemcitabine was found in both cell lines. The IC50 values of LDM for the PANC-1 or SW1990 cells were 0.955+/-0.414 or 0.426+/-0.212 nM, respectively, lower than those of the other drugs. Growth inhibition, apoptosis induction and cell cycle arrest were observed in the LDM-treated cells. LDM decreased the invasive potential of pancreatic cancer cells by reducing matrix metalloproteinase-9 activity. Furthermore, LDM was found to suppress the growth of SW1990 xenografts in nude mice. Treatment with an i.v. injection of LDM at the dose of 0.02 and 0.04 mg/kg (once a week for two weeks) inhibited the growth of xenografts by 66 and 72%, respectively. By contrast, an i.p. injection of gemcitabine at the dose of 80 mg/kg inhibited the growth of xenografts by 38%. Our findings suggest that LDM is active in the down-regulation of NF-kappaB and could play a positive role in relevant targeted chemotherapy for pancreatic carcinoma.