A Bioinformatic Algorithm for Analyzing Cell Signaling Using Temporal Proteomic Data.

Significance analysis of proteomic data generated by LC-MS/MS is challenging owing to great data variability originated from biological, operational, and instrumental variations. Protein quantification by LC-MS/MS either in absolute or relative scale is often highly skewed, which put limitations on model-based statistical inference. For this purpose, we have developed an alternative nonparametric statistical algorithm (named IQR algorithm) for significance analysis of temporal proteomic data and have successfully applied our strategy in finding gefitinib-targeted transcription factors and coregulators in Epidermal Growth Factor (EGF)-stimulated HeLa cells. Our strategy relies on a reference group composed of more than a dozen of datasets collected at different experimental times, thus, accurately captures biological variations measured in quartile scale. The algorithm considers six categories and calculates signal strength when performing significance analysis of proteins of different abundances. This stratified strategy allows confident identification of well-characterized EGF responders (e.g. EGR1, JUN, FOSB, BHLHE40, NR4A1, and NR4A2) and unexplored gefitinib induced transcription factors and coregulators in HeLa cells. Gene set enrichment analysis has validated ErbB signaling pathway as the major inhibitory target of gefitinib. The identification of several gefitinib-inducible transcription factors implicates alternative signaling pathways as potential druggable pathways in gefitinib-resistant or insensitive patients.

Development of a nondestructive method for sexing live adult Sternoplax souvorowiana (Coleoptera: Tenebrionidae).

The darkling beetle, Sternoplax souvorowiana (Reitter) (Coleoptera: Tenebrionidae), is flightless and lives in the Guerbantonggut desert in northwestern China. Its special eggshell structure, day-active habit, large body size, short life cycle, and ease of rearing under laboratory conditions make it an excellent model for advanced studies on desert adaptation. Determining the sex of this beetle is usually complicated by the lack of a discreet, externally visible gender-specific character. To date, dissection has been used for sex identification in this species, whereas a nondestructive means is needed for further studies of sexual dimorphism. Here, a new method based on the difference of the pigmentation pattern on the eighth tergite of each sex is described and illustrated. This method can be quickly learned, is nondestructive, is 100% accurate, and is fast enough for most applications in both the field and the laboratory. Experienced users in our laboratory routinely sex 8-10 beetles per minute.

A novel function - thermal protective properties of an antifreeze protein from the summer desert beetle Microdera punctipennis.

It is well known that antifreeze proteins play an important role in protecting poikilothermic organisms from freezing. However, the transcripts of antifreeze protein genes, Mpafps, were observed in the desert beetle Microdera punctipennis in summer. The mRNA levels of Mpafps increased significantly after heat shock at 50°C, which implies that a novel function may exist in the antifreeze proteins from M. punctipennis. Southern blot analysis suggested the presence of multiple copies of the Mpafps family in the genome. Transcripts of two cDNAs encoding antifreeze proteins (Mpafps77 and Mpafps52) were isolated from beetles collected in the summer. The deduced amino acid sequences of the MpAFPs expressed in the summer are shorter by one 12-residue repeat and have significantly different C-terminal end sequences relative to the AFPs expressed in winter. Mpafps77 was constructed and expressed in Escherichia coli as a fusion protein, maltose-binding protein (MBP)-MpAFPS77. An in vitro heat protection assay was done by measuring the survival of bacteria and yeast that were exposed to 50 and 42°C, respectively and showed that the fusion protein significantly increased the thermal tolerance of these cells. It also increased the thermotolerance of the lactate dehydrogenase (LDH) enzyme at 65°C. These studies are the first biochemical demonstration of a thermal protective function for MpAFP and suggest some novel protective mechanisms may be present in M. punctipennis.

Chemodivergence in Pd-catalyzed desymmetrization of allenes: enantioselective [4+3] cycloaddition, desymmetric allenylic substitution and enynylation.

A class of prochiral allenylic di-electrophiles have been introduced for the first time as three-atom synthons in cycloadditions, and a new type of [4+3] cycloaddition involving transition metal-catalyzed enantioselective sequential allenylic substitution has been successfully developed, enabling challenging seven-membered exocyclic axially chiral allenes to be accessed in good yields with good enantioselectivity. Through the addition of a catalytic amount of ortho-aminoanilines or ortho-aminophenols, the racemization of the [4+3] cycloaddition products is effectively suppressed. Mechanistic studies reveal that elusive Pd-catalyzed enantioselective intramolecular allenylic substitution rather than intermolecular allenylic substitution is the enantio-determining step in this cycloaddition. By tuning the ligands, a Pd-catalyzed enantioselective desymmetric allenylic substitution leading to linear axially chiral tri-substituted allenes or a Pd-catalyzed tandem desymmetric allenylic substitution/ß-vinylic hydrogen elimination (formal enynylation) leading to multi-functionalized 1,3-enynes is achieved chemodivergently.

RNAi Mediated Gene Silencing of Detoxification Related Genes in the Ectropis oblique.

Ectropis oblique is one of the main pests that feed on tea leaves. At present, the main control method is chemical control, but the long-term use of insecticides has been related to the development of insect resistance. One of the resistance mechanisms is the upregulation of relevant detoxification enzymes for defense. In this study, four genes with increased expression were screened from the gene sequences annotated from the transcriptome data of deltamethrin-treated larvae of E. oblique, which are acid phosphatase EoACP138, and cytochrome P450 EoCYP316, carboxylesterase EoCarE592 and acetylcholine esterase EoAchE989, respectively. The fourth instar larvae of E. oblique were stimulated by deltamethrin, chlorpyrifos and fenpropathrin respectively, and the expression levels of the genes were detected by qRT-PCR. The result showed that all four genes' expression had significantly increased under the stimulation of three insecticides. RNAi technology was used to silence the expression of genes of EoACP138, EoCYP316, EoCarE592 and EoAchE989 in the fourth instar larvae of E. oblique. The change in the expression levels of the above genes in the larvae treated with dsRNA and stimulated with pesticides was determined by qRT-PCR. The target genes have been effectively silenced after feeding on dsRNA and higher sensitivity with higher mortality to pesticides was observed in the larvae interfered with dsRNA. The above genes are related to the detoxification and metabolism of resistance of E. oblique, which lays a foundation for further study on the mechanism of insecticide resistance in E. oblique.

Mechanisms of Action of the Antimicrobial Peptide Cecropin in the Killing of Candida albicans.

The development of drug resistance has caused fungal infections to become a global health concern. Antimicrobial peptides (AMPs) offer a viable solution to these pathogens due to their resistance to drug resistance and their diverse mechanisms of actions, which include direct killing and immunomodulatory properties. The peptide Cecropin, which is expressed by genetically engineered bacteria, has antifungal effects on Candida albicans. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of Candida albicans were 0.9 µg/mL and 1.8 µg/mL, respectively, detected by the micro-broth dilution method. According to the killing kinetics, the MFC of Cecropin could kill Candida albicans in 40 min. The electron microscope indicated that Cecropin could cause the cell wall to become rough and nicked, eventually killing Candida albicans. The effects of Cecropin on the cell membrane of treated C. albicans, using the 1,6-diphenyl-1,3,5-hexatriene and propidium iodide protocol, showed that they could change the permeability and fluidity, destroy it, and lead to cell necrosis. In addition, Cecropin can also induce cells to produce excessive reactive oxygen species, causing changes in the mitochondrial membrane potential. Therefore, this study provides a certain theoretical basis for the antifungal infection of new antifungal agents.

Characterization of a novel ß-helix antifreeze protein from the desert beetle Anatolica polita.

Many ectotherms organisms produce antifreeze proteins (AFPs) which inhibit the growth of ice by binding to the surface of ice crystals. In this study, a novel antifreeze protein gene from the desert beetle Anatolica polita (named as Apafp752) was expressed in a high level in Escherichia coli strain BL21 (DE3). An approximately 30kDa fusion protein thioredoxin (Trx)-ApAFP752 was purified through Ni-NTA affinity chromatography and gel filtration chromatography. The activity of the purified fusion protein Trx-ApAFP752 was analyzed by thermal hysteresis activity (THA) and cryoprotection assay. The results suggested that Trx-ApAFP752 conferred freeze resistance on bacterium in a concentration- and time-dependent manner and the cryoprotective effect increased under alkaline conditions. Circular Dichroism (CD) spectrum analysis showed that the recombinant protein of ApAFP752 possessing ß-sheet as the main structure was stable under a wide range of pH from 2.0 to 11.0 and thermal stability below 50°C. The predicted 3D structure showed that Trx-ApAFP752 could form a ß-helix structure on the antifreeze protein part, which placed most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.

Crystallization and preliminary X-ray analysis of cecropin B from Bombyx mori.

Cecropin B is a 37-residue cationic antimicrobial peptide derived from the haemolymph of Bombyx mori. The precise mechanism by which cecropins exert their antimicrobial and cytolytic activities is not well understood. Crystals of cecropin B were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant at 289 K. The crystal diffracted to 1.43 A resolution using X-ray radiation and belonged to the orthorhombic space group P1, with unit-cell parameters a = 15.08, b = 22.75, c = 30.20 A, alpha = 96.9, beta = 103.1, gamma = 96.5 degrees. The asymmetric unit contained only one molecule of cecropin B, with a calculated Matthews coefficient of 2.48 A(3) Da(-1) and a solvent content of 50.4%.

Molecular and functional characterization of ApPGRP from Anatolica polita in the immune response to Escherichia coli.

Peptidoglycan recognition proteins (PGRPs) widely distributed in invertebrates and vertebrates are pattern-recognition molecules in innate immunity. In the present study, a novel short PGRP gene, designated as ApPGRP was identified from Anatolica polita. The deduced amino acid sequence of ApPGRP is composed of 196 residues and contains a conserved PGRP domain at the C-terminus. The phylogenetic tree showed that ApPGRP shared high homology of amino acid sequence with TcPGRP2 from Tribolium castaneum. The recombinant protein of ApPGRP exhibited binding activity toward Escherichia coli and DAP-type PGN from E. coli. Quantitative real time PCR (RT-qPCR) analysis indicated that the relative expression level of ApPGRP was up-regulated significantly after E. coli and DAP-type PGN challenge in the fifth instar larvae of A. polita. Moreover, the expressions of three antimicrobial peptides (AMPs) (ApAttacin 1, ApAttacin 2 and ApColeoptericin) were significantly increased after E. coli and DAP-type PGN challenge. RNA interference (RNAi) experiments showed that the expressions of three AMPs in the larvae of A. polita were significantly decreased after injection of ApPGRP dsRNA. Furthermore, the expressions of three AMPs in the larvae injected were still significantly decreased after E. coli challenge compared to the control samples without dsRNA injection. The predicted 3D structure showed that the ApPGRP could form the protein core of five ß-sheet and three ɑ-helices, which would be involved in specific PGN recognition. These results suggested that ApPGRP may play an important role in the immune response to E. coli infection and function as a receptor for antimicrobial peptide gene induction in Anatolica polita.

Molecular cloning and characterization of the ß-1,3-glucan recognition protein in Anatolica polita.

ß-1,3-Glucan recognition protein (ßGRP) is an important pattern recognition protein, which could trigger immune response to eliminate pathogens by identifying and combining the pathogenic bacteria. In the present study, a ß-1,3-glucan recognition protein gene (ApßGRP) was cloned from a desert beetle Anatolica polita based on the EST sequence of ApßGRP in the suppression subtractive cDNA library. Quantitative real-time PCR (qRT-PCR) results showed that ApßGRP transcript in A. polita was significantly upregulated under the challenge of Escherichia coli and Staphylococcus aureus. Western blot analysis indicated that recombinant ApßGRP expressed in E. coli BL21, has the ability of binding to E. coli and S. aureus. Moreover, agglutination assay suggested that recombinant ApßGRP could agglutinate E. coli, S. aureus and Saccharomyces cerevisiae. The predicted 3D structure showed that ApßGRP possesses a typical ß-glucan recognition domain with seven ß-strands structures and conserved amino acid sequence. These data indicate that ApßGRP may be involved in immune defense in A. polita and could recognize and bind the bacteria against the invasion of external pathogens.

Pretreatment Donors after Circulatory Death with Simvastatin Alleviates Liver Ischemia Reperfusion Injury through a KLF2-Dependent Mechanism in Rat.

OBJECTIVE: Severe hepatic ischemia reperfusion injury (IRI) can result in poor short- and long-term graft outcome after transplantation. The way to improve the viability of livers from donors after circulatory death (DCD) is currently limited. The aim of the present study was to explore the protective effect of simvastatin on DCD livers and investigate the underlying mechanism. METHODS: 24 male rats randomly received simvastatin or its vehicle. 30 min later, rat livers were exposed to warm ischemia in situ for 30 min. Livers were removed and cold-stored in UW solution for 24 h, subsequently reperfused for 60 min with an isolated perfused rat liver system. Liver injury was evaluated during and after warm reperfusion. RESULTS: Pretreatment of DCD donors with simvastatin significantly decreased IRI liver enzyme release, increased bile output and ATP, and ameliorated hepatic pathological changes. Simvastatin maintained the expression of KLF2 and its protective target genes (eNOS, TM, and HO-1), reduced oxidative stress, inhibited innate immune responses and inflammation, and increased the expression of Bcl-2/Bax to suppress hepatocyte apoptosis compared to DCD control group. CONCLUSION: Pretreatment of DCD donors with simvastatin improves DCD livers' functional recovery probably through a KLF2-dependent mechanism. These data suggest that simvastatin may provide a potential benefit for clinical DCD liver transplantation.

Novel multi-targeted ErbB family inhibitor afatinib blocks EGF-induced signaling and induces apoptosis in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.

Cloning and characterization of metallothionein gene (HcMT) from Halostachys caspica and its expression in E. coli.

Halostachys caspica is a short shrub distributed in the semi-arid and saline-alkali area, which evolved various mechanisms for modulating salt and metal level. In the present study, a Type 2 metallothionein (HcMT) gene was cloned from the salt induced suppression subtractive hybridization (SSH) cDNA library of H.caspica. Quantitative real time PCR (qRT-PCR) analysis indicated that HcMT gene was up-regulated under the stress of Cu(2+), Zn(2+) and Cd(2+), and the tolerance of E. coli strain harboring with the recombinant HcMT (pET-32a-HcMT) to Cu(2+), Zn(2+) and Cd(2+) was enhanced compared to strain with control vector (pET-32a). Moreover, the purified TrxA-HcMT fusion protein from E. coli cells grown in the presence of 0.3mM CuSO4, 0.3mM ZnSO4, or 0.1mM CdCl2 could bind more metal ions than TrxA alone. The predicted 3D structure showed that HcMT could form a single metal-thiolate cluster, which confers the ability to bind five divalent metal ions through fourteen cysteine residues. These data indicate that HcMT may be involved in processes of metal tolerance in H. caspica and could be employed as a potential candidate for heavy metal phytoremediation.

Second-generation proteasome inhibitor carfilzomib sensitizes neuroblastoma cells to doxorubicin-induced apoptosis.

Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common extracranial malignant neoplasm in children. Elevated level of proteasome activity promotes cancer development and the inhibition of proteasome activity is a promising strategy for cancer treatment. Therefore, targeting proteasome by small molecule inhibitors may be a viable option for NB therapy. Here in this study, we show that a novel proteasome inhibitor Carfilzomib (CFZ) exerts anti-tumor effect on NB. CFZ caused decreased cell viability and attenuated colony formation ability of a subset of NB cell lines. CFZ induced cell apoptosis in NB cells. Moreover, CFZ enhanced the cytotoxic effect of doxorubicin (Dox) on NB cells and Dox-induced p38 and JNK phosphorylation. In addition, CFZ inhibited Dox-induced NF-κB activation by stabilizing the protein level of IκBα. Furthermore, CFZ induced apoptosis and augmented Dox-induced apoptosis in NB tumor cells in orthotopic xenograft mouse models. In summary, our study suggests that proteasome is a therapeutic target in NB and proteasome inhibition by CFZ is a potential therapeutic strategy for treating NB patients.

Novel MDM2 inhibitor SAR405838 (MI-773) induces p53-mediated apoptosis in neuroblastoma.

Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common childhood extracranial malignant tumor. In NB, somatic mutations of the tumor suppressor, p53, are exceedingly rare. Unlike in adult tumors, the majority of p53 downstream functions are still intact in NB cells with wild-type p53. Thus, restoring p53 function by blocking its interaction with p53 suppressors such as MDM2 is a viable therapeutic strategy for NB treatment. Herein, we show that MDM2 inhibitor SAR405838 is a potent therapeutic drug for NB. SAR405838 caused significantly decreased cell viability of p53 wild-type NB cells and induced p53-mediated apoptosis, as well as augmenting the cytotoxic effects of doxorubicin (Dox). In an in vivo orthotopic NB mouse model, SAR405838 induced apoptosis in NB tumor cells. In summary, our data strongly suggest that MDM2-specific inhibitors like SAR405838 may serve not only as a stand-alone therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact MDM2-p53 axis.

[Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli].

To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.