Macromolecule Metabolism Research Articles

Gene Expression Profiling Analysis of Castration-Resistant Prostate Cancer

BackgroundProstate cancer is a global health issue. Usually, men with metastatic disease will progress to castration-resistant prostate cancer (CRPC). We aimed to identify the differentially expressed genes (DEGs) in tumor samples from non-castrated and castrated men from LNCaP Orthotopic xenograft models of prostate cancer and to study the mechanisms of CRPC.Material/MethodsIn this work, GSE46218 containing 4 samples from non-castrated men and 4 samples from castrated men was downloaded from Gene Expression Omnibus. We identified DEGs using limma Geoquery in R, the Robust Multi-array Average (RMA) method in Bioconductor, and Bias methods, followed by constructing an integrated regulatory network involving DEGs, miRNAs, and TFs using Cytoscape. Then, we analyzed network motifs of the integrated gene regulatory network using FANMOD. We selected regulatory modules corresponding to network motifs from the integrated regulatory network by Perl script. We preformed gene ontology (GO) and pathway enrichment analysis of DEGs in the regulatory modules using DAVID.ResultsWe identified total 443 DEGs. We built an integrated regulatory network, found three motifs (motif 1, motif 2 and motif 3), and got two function modules (module 1 corresponded to motif 1, and module 2 corresponded to motif 2). Several GO terms (such as regulation of cell proliferation, positive regulation of macromolecule metabolic process, phosphorylation, and phosphorus metabolic process) and two pathways (pathway in cancer and Melanoma) were enriched. Furthermore, some significant DEGs (such as CAV1, LYN, FGFR3 and FGFR3) were related to CPRC development.ConclusionsThese genes might play important roles in the development and progression of CRPC.

Age-Specific Gene Expression Profiles of Rhesus Monkey Ovaries Detected by Microarray Analysis.

The biological function of human ovaries declines with age. To identify the potential molecular changes in ovarian aging, we performed genome-wide gene expression analysis by microarray of ovaries from young, middle-aged, and old rhesus monkeys. Microarray data was validated by quantitative real-time PCR. Results showed that a total of 503 (60 upregulated, 443 downregulated) and 84 (downregulated) genes were differentially expressed in old ovaries compared to young and middle-aged groups, respectively. No difference in gene expression was found between middle-aged and young groups. Differentially expressed genes were mainly enriched in cell and organelle, cellular and physiological process, binding, and catalytic activity. These genes were primarily associated with KEGG pathways of cell cycle, DNA replication and repair, oocyte meiosis and maturation, MAPK, TGF-beta, and p53 signaling pathway. Genes upregulated were involved in aging, defense response, oxidation reduction, and negative regulation of cellular process; genes downregulated have functions in reproduction, cell cycle, DNA and RNA process, macromolecular complex assembly, and positive regulation of macromolecule metabolic process. These findings show that monkey ovary undergoes substantial change in global transcription with age. Gene expression profiles are useful in understanding the mechanisms underlying ovarian aging and age-associated infertility in primates.

In vitro study of genes and molecular pathways differentially regulated by synchrotron microbeam radiotherapy.

The aim of this study was to identify genes and molecular pathways differentially regulated by synchrotron-generated microbeam radiotherapy (MRT) versus conventional broadbeam radiotherapy (CRT) in vitro using cultured EMT6.5 cells. We hypothesized (based on previous findings) that gene expression and molecular pathway changes after MRT are different from those seen after CRT. We found that at 24 h postirradiation, MRT exerts a broader regulatory effect on multiple pathways than CRT. MRT regulated those pathways involved in gene transcription, translation initiation, macromolecule metabolism, oxidoreductase activity and signaling transduction in a different manner compared to CRT. We also found that MRT/CRT alone, or when combined with inflammatory factor lipopolysaccharide, upregulated expression of Ccl2, Ccl5 or Csf2, which are involved in host immune cell recruitment. Our findings demonstrated differences in the molecular pathway for MRT versus CRT in the cultured tumor cells, and were consistent with the idea that radiation plays a role in recruiting tumor-associated immune cells to the tumor. Our results also suggest that a combination of MRT/CRT with a treatment targeting CCL2 or CSF2 could repress the tumor-associated immune cell recruitment, delay tumor growth and/or metastasis and yield better tumor control than radiation alone.

Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to salt stress.

BackgroundSalt stress interferes with plant growth and production. Plants have evolved a series of molecular and morphological adaptations to cope with this abiotic stress, and overexpression of salt response genes reportedly enhances the productivity of various crops. However, little is known about the salt responsive genes in the energy plant physic nut (Jatropha curcas L.). Thus, excavate salt responsive genes in this plant are informative in uncovering the molecular mechanisms for the salt response in physic nut.Methodology/Principal FindingsWe applied next-generation Illumina sequencing technology to analyze global gene expression profiles of physic nut plants (roots and leaves) 2 hours, 2 days and 7 days after the onset of salt stress. A total of 1,504 and 1,115 genes were significantly up and down-regulated in roots and leaves, respectively, under salt stress condition. Gene ontology (GO) analysis of physiological process revealed that, in the physic nut, many “biological processes” were affected by salt stress, particular those categories belong to “metabolic process”, such as “primary metabolism process”, “cellular metabolism process” and “macromolecule metabolism process”. The gene expression profiles indicated that the associated genes were responsible for ABA and ethylene signaling, osmotic regulation, the reactive oxygen species scavenging system and the cell structure in physic nut.Conclusions/SignificanceThe major regulated genes detected in this transcriptomic data were related to trehalose synthesis and cell wall structure modification in roots, while related to raffinose synthesis and reactive oxygen scavenger in leaves. The current study shows a comprehensive gene expression profile of physic nut under salt stress. The differential expression genes detected in this study allows the underling the salt responsive mechanism in physic nut with the aim of improving its salt resistance in the future.

Ionizing radiation-induced gene expression changes in TP53 proficient and deficient glioblastoma cell lines

The genetic heterogeneity presented by different cell lines derived from glioblastoma (GBM) seems to influence their responses to antitumoral agents. Although GBM tumors present several genomic alterations, it has been assumed that TP53, frequently mutated in GBM, may to some extent be responsible for differences in cellular responses to antitumor agents, but this is not clear yet. To directly determine the impact of TP53 on GBM response to ionizing radiation, we compared the transcription profiles of four GBM cell lines (two with wild-type (WT) TP53 and two with mutant (MT) TP53) after 8Gy of gamma-rays. Transcript profiles of cells analyzed 30min and 6h after irradiation showed that WT TP53 cells presented a higher number of modulated genes than MT TP53 cells. Our findings also indicate that there are several pathways (apoptosis, DNA repair/stress response, cytoskeleton organization and macromolecule metabolic process) in radiation responses of GBM cell lines that were modulated only in WT TP53 cells (30min and 6h). Interestingly, the majority of differentially expressed genes did not present the TP53 binding site, suggesting secondary effects of TP53 on transcription. We conclude that radiation-induced changes in transcription profiles of irradiated GBM cell lines mainly depend on the functional status of TP53.

Proteomic Analysis Reveals Novel Extracellular Virulence-Associated Proteins and Functions Regulated by the Diffusible Signal Factor (DSF) in Xanthomonas oryzae pv. oryzicola

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.

Transcriptome Analysis of Spermatogenically Regressed, Recrudescent and Active Phase Testis of Seasonally Breeding Wall Lizards Hemidactylus flaviviridis

BackgroundReptiles are phylogenically important group of organisms as mammals have evolved from them. Wall lizard testis exhibits clearly distinct morphology during various phases of a reproductive cycle making them an interesting model to study regulation of spermatogenesis. Studies on reptile spermatogenesis are negligible hence this study will prove to be an important resource.Methodology/Principal FindingsHistological analyses show complete regression of seminiferous tubules during regressed phase with retracted Sertoli cells and spermatognia. In the recrudescent phase, regressed testis regain cellular activity showing presence of normal Sertoli cells and developing germ cells. In the active phase, testis reaches up to its maximum size with enlarged seminiferous tubules and presence of sperm in seminiferous lumen. Total RNA extracted from whole testis of regressed, recrudescent and active phase of wall lizard was hybridized on Mouse Whole Genome 8×60 K format gene chip. Microarray data from regressed phase was deemed as control group. Microarray data were validated by assessing the expression of some selected genes using Quantitative Real-Time PCR. The genes prominently expressed in recrudescent and active phase testis are cytoskeleton organization GO 0005856, cell growth GO 0045927, GTpase regulator activity GO: 0030695, transcription GO: 0006352, apoptosis GO: 0006915 and many other biological processes. The genes showing higher expression in regressed phase belonged to functional categories such as negative regulation of macromolecule metabolic process GO: 0010605, negative regulation of gene expression GO: 0010629 and maintenance of stem cell niche GO: 0045165.Conclusion/SignificanceThis is the first exploratory study profiling transcriptome of three drastically different conditions of any reptilian testis. The genes expressed in the testis during regressed, recrudescent and active phase of reproductive cycle are in concordance with the testis morphology during these phases. This study will pave the way for deeper insight into regulation and evolution of gene regulatory mechanisms in spermatogenesis.

Microarray Analysis of Transcriptome of Medulla Identifies Potential Biomarkers for Parkinson’s Disease

To complement the molecular pathways contributing to Parkinson's disease (PD) and identify potential biomarkers, gene expression profiles of two regions of the medulla were compared between PD patients and control. GSE19587 containing two groups of gene expression profiles [6 dorsal motor nucleus of the vagus (DMNV) samples from PD patients and 5 from controls, 6 inferior olivary nucleus (ION) samples from PD patients and 5 from controls] was downloaded from Gene Expression Omnibus. As a result, a total of 1569 and 1647 differentially expressed genes (DEGs) were, respectively, screened in DMNV and ION with limma package of R. The functional enrichment analysis by DAVID server (the Database for Annotation, Visualization and Integrated Discovery) indicated that the above DEGs may be involved in the following processes, such as regulation of cell proliferation, positive regulation of macromolecule metabolic process, and regulation of apoptosis. Further analysis showed that there were 365 common DEGs presented in both regions (DMNV and ION), which may be further regulated by eight clusters of microRNAs retrieved with WebGestalt. The genes in the common DEGs-miRNAs regulatory network were enriched in regulation of apoptosis process via DAVID analysis. These findings could not only advance the understandings about the pathogenesis of PD, but also suggest potential biomarkers for this disease.

Comparative antibacterial mode of action of purified alcalase- and tryptic-hydrolyzed palm kernel cake proteins on the food-borne pathogen Bacillus cereus

Purified alcalase- and tryptic-hydrolyzed palm kernel cake proteins, PAH and PTH, the value-added products derived from palm kernel cake, a co-product of palm kernel oil industry were proven to possess potent bacteriostatic effect against spore-forming bacteria particularly the Bacillus cereus. Comparatively, PAH exhibited slightly better inhibition effect against B. cereus than PTH. Both compounds inhibited the bacterial growth by altering the membrane permeability of the bacterial cells. Incubation for 30 min in the presence of PAH or PTH at minimum inhibitory concentrations (MIC, 250 μg/ml or 350 μg/ml respectively) rapidly induced K+ leakage of the cells. Intracellular ATP was also depleted after 30 min treatment with PTH at MIC level. The total ATP level of the cells was reduced by 5-fold after 30 min of treatment with PAH compared to control, thus suggesting that PAH tends to trigger ATP hydrolysis rather than causing ATP efflux as occurred in cells treated with PTH. Analysis of the biosynthesis of macromolecules demonstrated that RNA is the sole inhibition target of PAH and PTH. From this study, it could be concluded that both PAH and PTH appear to disrupt the membrane integrity, allowing efflux of K+, deplete the ATP molecules and inhibit the intracellular macromolecule metabolism especially the RNA of B. cereus.

Distinct and Competitive Regulatory Patterns of Tumor Suppressor Genes and Oncogenes in Ovarian Cancer

BackgroundSo far, investigators have found numerous tumor suppressor genes (TSGs) and oncogenes (OCGs) that control cell proliferation and apoptosis during cancer development. Furthermore, TSGs and OCGs may act as modulators of transcription factors (TFs) to influence gene regulation. A comprehensive investigation of TSGs, OCGs, TFs, and their joint target genes at the network level may provide a deeper understanding of the post-translational modulation of TSGs and OCGs to TF gene regulation.Methodology/Principal FindingsIn this study, we developed a novel computational framework for identifying target genes of TSGs and OCGs using TFs as bridges through the integration of protein-protein interactions and gene expression data. We applied this pipeline to ovarian cancer and constructed a three-layer regulatory network. In the network, the top layer was comprised of modulators (TSGs and OCGs), the middle layer included TFs, and the bottom layer contained target genes. Based on regulatory relationships in the network, we compiled TSG and OCG profiles and performed clustering analyses. Interestingly, we found TSGs and OCGs formed two distinct branches. The genes in the TSG branch were significantly enriched in DNA damage and repair, regulating macromolecule metabolism, cell cycle and apoptosis, while the genes in the OCG branch were significantly enriched in the ErbB signaling pathway. Remarkably, their specific targets showed a reversed functional enrichment in terms of apoptosis and the ErbB signaling pathway: the target genes regulated by OCGs only were enriched in anti-apoptosis and the target genes regulated by TSGs only were enriched in the ErbB signaling pathway.Conclusions/SignificanceThis study provides the first comprehensive investigation of the interplay of TSGs and OCGs in a regulatory network modulated by TFs. Our application in ovarian cancer revealed distinct regulatory patterns of TSGs and OCGs, suggesting a competitive regulatory mechanism acting upon apoptosis and the ErbB signaling pathway through their specific target genes.

Bacteriostatic Mode of Action of Trypsin-Hydrolyzed Palm Kernel Expeller Peptide Against Bacillus cereus.

Palm kernel expeller (PKE), the by-product derived from the palm kernel oil milling industry, is commonly added to ruminant feed as a source of protein. Recent research has demonstrated that the enzymatically hydrolyzed protein is inhibitory to spore-forming bacteria including Bacillus cereus. The trypsin-hydrolyzed PKE peptide appears to disrupt the membrane integrity and inhibit the intracellular macromolecule metabolism of B. cereus. The addition of the PKE peptide (350 and 700μg/ml) to B. cereus cultures triggered the efflux of K(+) and caused the depletion of the intracellular ATP. However, no proportional increase in cell's extracellular ATP was observed. Analysis of the biosynthesis of macromolecules demonstrated that RNA was affected by the PKE peptide. Results of this study suggest that the PKE peptide is bacteriostatic interfering with membrane integrity and forming membrane pores permitting the efflux of K(+) and interferes with intracellular biopolymer synthesis.

A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides

Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode

Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5-7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: "macromolecule metabolic process", "developmental process", "response to stress", and "biological regulation". Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection.

Proteomic modification in gills and brains of medaka fish ( Oryzias melastigma) after exposure to a sodium channel activator neurotoxin, brevetoxin-1

Although brevetoxins (PbTxs) produced by the marine dinoflagellate Karenia brevis are known to be absorbed across gill membranes and exert their acute toxic effects through an ion-channel mediated pathway in neural tissue, the exact biochemical mechanism concerning PbTxs neurotoxicity in neural tissue and gas-exchange organs has not been well elucidated. In this study, we calculated the LC 50 value of PbTx-1 using the medaka fish model, and presented the molecular responses of sub-acute exposure to PbTx-1 with proteomic method. By adopting two-dimensional electrophoresis, the abundances of 14 and 24 proteins were found to be remarkably altered in the gills and brains, respectively, in response to toxin exposure. Thirteen gill and twenty brain proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis. These proteins could be categorized into diverse functional classes such as cell structure, macromolecule metabolism, signal transduction and neurotransmitter release. These findings can help to elucidate the possible pathways by which aquatic toxins affect marine organisms within target organs.

A Systemic Network Triggered by Human Cytomegalovirus Entry

Virus entry is a multistep process that triggers various cellular pathways that interconnect into a complex network; yet the molecular complexity of this network remains largely elusive. Here, by employing systems biology approaches, we reveal a systemic virus-entry network initiated by human cytomegalovirus (HCMV), a widespread opportunistic pathogen. This network contains ten functional modules (i.e., groups of proteins) that coordinately respond to HCMV entry. Functional modules activated (up- and downregulated) in this network dramatically decline shortly within 25 minutes post infection. While modules annotated as receptor system, ion transport, and immune response are continuously activated during the entire process of HCMV entry, those annotated for cell adhesion and skeletal movement are specifically activated during viral early attachment. The up-regulated network contains various functional modules, such as cell surface receptors, skeletal development, endocytosis, ion transport, and chromatin remodeling. Interestingly, macromolecule metabolism and chromatin remodeling module predominates this over-expressed system, suggesting that the fundamental nuclear process modulation is one of the most important events in HCMV entry. The entire up-regulated network is primarily controlled by multiple elements like SLC10A1. Thus, virus entry triggers multiple cellular processes especially nuclear processes to facilitate its entry.

185 HIGH-THROUGHPUT PROTEOMIC ASSESSMENT OF FROZEN - THAWED BOAR SPERMATOZOA

Cryopreservation is a tool of choice for seedstock constitution of genetically superior males. Its successful application in swine AI industries is limited because of the poor freezability of boar semen. Indeed, a subset of boars exists that can be successfully frozen–thawed for AI, whereas another group appears highly cryosusceptible, and therefore unusable for long-term semen storage. The reasons for such differences are unknown, and the full characterisation of the protein composition of boar spermatozoa will help determine potential cryosensitive proteins. Here, we performed high-throughput proteomic analyses of boar spermatozoa and compared the proteome profiles of ‘good’ and ‘poor’ freezer boars. Eight commercially proven fertile boars were selected based on conception rates after AI using fresh semen. Semen from 3 independent ejaculations was collected from 4 good and 4 poor freezer boars and frozen in 5-mL straws for the study. Frozen–thawed semen was diluted in the thawing solution and centrifuged through a discontinuous Percoll gradient (90/45) to remove seminal plasma, freezing extender, somatic cells, and dead sperm cells. Purified motile spermatozoa were washed 3 times with cold PBS and pooled in pellets of 3 × 108 spermatozoa per boar. Protein samples were digested with trypsin and prepared for LC-MS/MS analysis. Peptides yielding probability scores lower than 0.05 were subjected to protein identification, and the significance of differentially expressed protein was fixed at P < 0.05. More than 3000 proteins were identified in each group of spermatozoa. Proportions of 63 and 61% total proteins were exclusively detected in good and poor freezer boars, respectively. Many of the identified proteins were related to different cellular compartments and important molecular mechanisms related to sperm function, such as cell death regulation, macromolecule metabolism, and energy-related pathways. Approximately 5% of total proteins, representing 163 to 182 individual proteins, were detected at higher levels in both semen groups. Half of these highly abundant proteins were differentially expressed between good and poor freezer boars. Only 8 appeared partially annotated and 11 were predicted. The remaining list of fully annotated proteins included candidates such as transferrin, albumin, and fascin3, which were significantly (P < 0.05) abundant in good freezer boars, and outer dense fibre (ODF) 2, protamine (PRM) 2, and calmodulin (CALM) 1, which were significantly (P < 0.05) abundant in poor freezer boars. Overall, the results indicate that boar spermatozoa contain large amount of proteins whose susceptibility to cryopreservation and implications for sperm function are still to be characterised. Our findings are particularly important for 1) the search for potential biomarkers of semen freezability, and 2) improvement of semen freezing-thawing extenders for boars and other species with similar issues. Funded by USDA-ARS Special Initiative No. 58-6402-3-0120 and Mississippi Agriculture and Forestry Experiment Station.

Comparative Gene Expression Profiling of In Vivo-Derived Expanded and Hatched Porcine Blastocyst-Stage Embryos.

During the pre-implantation period of embryonic development, the mammalian embryo exhibits dramatic changes and many key events take place. The developmental stage at which porcine embryos are most commonly manipulated is the blastocyst, and the quality of the embryos used is critical to the success of reproductive technologies such as embryo transfer. Despite the importance of this critical period, our ability to determine early embryonic quality based on morphological criteria is limited. As an alternative, characterization of the gene expression profile of the early porcine embryo could identify gene markers of embryonic quality. The objective of this study was to perform comparative gene expression profiling analysis of in vivo-derived expanded and hatched porcine blastocysts using two-colored microarray (Pig-Oligo Array, USDA Pig Genome Consortium). Microarray data were interpreted using WebArray and DAVID functional annotation tools. The microarray results identified 1783 genes that were commonly expressed in both expanded and hatched porcine blastocysts. A further 148 genes were differentially expressed (p<0.05) and in comparison to expanded blastocysts, 35 genes were down-regulated and 113 genes were up-regulated after hatching. Many of the differentially expressed genes are related to important molecular mechanisms, such as macromolecule metabolism and energy related pathways. A total of 14 genes of interest (LDHA, LDHB, SLC16A7 (previously known as MCT2), BSG, SLC2A1, SLC2A3, SLC2A5, POU5F1 (previously known as OCT4), ACTB, GAPDH, GRB2, SETX, SPG7, and YWHAZ) were selected from the gene list generated by the microarray analysis, and their expressions were verified using SYBR Green-based real-time PCR. For the genes tested, the real-time PCR results were consistent with the microarray results. The differences detected in the gene expression profiles between in vivo-derived expanded and hatched porcine blastocysts increases our understanding of the molecular mechanisms involved in the hatching process and identifies potential candidate gene markers of porcine embryo quality. This research was supported by the Natural Sciences and Engineering Research Council (NSERC) and is a part of the activities of the EmbryoGENE NSERC Strategic Research Network. (poster)

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium’s survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism ( leuD, guaB1, plcC, hupB and glyS), polyketide synthases ( pks6 and pks9), cell processes ( ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.

Protein profiles in zebrafish ( Danio rerio) brains exposed to chronic microcystin-LR

Microcystin-LR (MCLR) is a commonly encountered blue-green algal hepatotoxin and a known inhibitor of cellular protein phosphatase (PP), however, little is known about its neurotoxicity. This study investigated the protein profiles of zebrafish ( Danio rerio) brains chronically exposed to MCLR concentrations (2 or 20 μg L −1) using the proteomic approach. The results showed that MCLR strikingly enhanced toxin accumulation and the PP activity in zebrafish brains after 30 d exposure. Comparison of two-dimensional electrophoresis protein profiles of MCLR exposed and non-exposed zebrafish brains revealed that the abundance of 30 protein spots was remarkably altered in response to MCLR exposure. These proteins are involved in cytoskeleton assembly, macromolecule metabolism, oxidative stress, signal transduction, and other functions (e.g. transporting, protein degradation, apoptosis and translation), indicating that MCLR toxicity in the fish brain is complex and diverse. The chronic neurotoxicity of MCLR might initiate the PP pathway via an upregulation of PP2C in the zebrafish brain, in addition to the reactive oxygen species pathway. Additionally, the increase of vitellogenin abundance in MCLR exposed zebrafish brains suggested that MCLR might mimic the effects of endocrine disrupting chemicals. This study demonstrated that MCLR causes neurotoxicity in zebrafish at the proteomic level, which provides a new insight into MCLR toxicity in aquatic organisms and human beings.

The Effects of Two Different Ganoderma Species (Lingzhi) on Gene Expression in Human Monocytic THP-1 Cells

Lingzhi (ganoderma) is an important woody mushroom that is known for its medicinal benefits in China since ancient times. The mode of action in humans is still not clear. Using microarray technology, we have compared the ethanol extracts of two different lingzhi (red lingzhi, G. lucidum; and purple lingzhi, G. sinense) for their effects on gene expression profile in human monocytic cells. Our results suggest that at best ≈25% of target genes are common to the two lingzhi: functionally ranging from cell development, negative regulation of cellular process, and cellular protein metabolic process to signal transduction and transcription. The pathways mediated by purple lingzhi focus on inflammation and immune response, whereas red lingzhi modestly increases levels of expression for genes involved in macromolecule metabolism. Furthermore, our ethanolic extracts of both red and purple lingzhi do not inhibit monocytic cell growth. The extract of red lingzhi does not have significant effect on the genes in the nuclear factor kappa B (NFκB) pathway (an important inflammation pathway), whereas the extract of purple lingzhi can increase multiple key genes in the NFκB pathway. Altogether, our results suggest that the common mode of action for lingzhi is complex; and different species of Ganoderma can modulate different pathways in human cells.