Genetic study of alcoholism and novel gene expression in the alcoholic brain

Alcohol dependence may result from neuroadaptation involving alteration of gene expression after long-term alcohol exposure. The systematic study of gene expression profiles of the human alcoholic brain was initiated using the method of polymerase chain reaction (PCR)-differential display and was followed by DNA microarray. To date, more than 100 alcohol-responsive genes have been identified from the frontal cortex, motor cortex and nucleus accumbens of the human brain. These genes have a wide range of functions in the brain and indicate diverse actions of alcohol on neuronal function. This review discusses the current information on the genetic basis of alcoholism and the induction and characterization of these alcohol-responsive genes.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

Cell cycle alterations in biopsied olfactory neuroepithelium in schizophrenia and bipolar I disorder using cell culture and gene expression analyses

We previously demonstrated that olfactory cultures front individuals with schizophrenia had increased cell proliferation compared to Cultures from healthy controls. The aims of this study were to (a) replicate this observation in a new group Of individuals with schizophrenia, (b) examine the specificity of these findings by including individuals with bipolar I disorder and (c) explore gene expression differences that may underlie cell cycle differences in these diseases. Compared to controls (n = 10), there was significantly more mitosis in schizophrenia patient cultures (it = 8) and significantly more cell death in the bipolar I disorder patient cultures (n=8). Microarray data showed alterations to the cell cycle and phosphatidylinositol signalling pathways in schizophrenia and bipolar I disorder, respectively. Whilst caution is required in the interpretation of the array results, the study provides evidence indicating that cell proliferation and cell death in olfactory neuroepithelial cultures is differentially altered in schizophrenia and bipolar disorder. (c) 2005 Elsevier B.V. All rights reserved.

Development of the Minimum Information Specification for in situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community. This paper is part of the special issue of OMICS on data standards.

Knowledge integration for analyzing ChIP-seq

To capture the genomic profiles for histone modification, chromatin immunoprecipitation (ChIP) is combined with next generation sequencing, which is called ChIP-seq. However, enriched regions generated from the ChIP-seq data are only evaluated on the limited knowledge acquired from manually examining the relevant biological literature. This paper proposes a novel framework, which integrates multiple knowledge sources such as biological literature, Gene Ontology, and microarray data. In order to precisely analyze ChIP-seq data for histone modification, knowledge integration is based on a unified probabilistic model. The model is employed to re-rank the enriched regions generated from peak finding algorithms. Through filtering the reranked enriched regions using some predefined threshold, more reliable and precise results could be generated. The combination of the multiple knowledge sources with the peaking finding algorithm produces a new paradigm for ChIP-seq data analysis. © (2012) Trans Tech Publications, Switzerland.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Evaluation of Some Statistical Methods for the Identification of Differentially Expressed Genes

Microarray platforms have been around for many years and while there is a rise of new technologies in laboratories, microarrays are still prevalent. When it comes to the analysis of microarray data to identify differentially expressed (DE) genes, many methods have been proposed and modified for improvement. However, the most popular methods such as Significance Analysis of Microarrays (SAM), samroc, fold change, and rank product are far from perfect. When it comes down to choosing which method is most powerful, it comes down to the characteristics of the sample and distribution of the gene expressions. The most practiced method is usually SAM or samroc but when the data tends to be skewed, the power of these methods decrease. With the concept that the median becomes a better measure of central tendency than the mean when the data is skewed, the tests statistics of the SAM and fold change methods are modified in this thesis. This study shows that the median modified fold change method improves the power for many cases when identifying DE genes if the data follows a lognormal distribution.

Classificação de atividade eletromiográfia facial de indivíduos saudáveis e com hanseníese por meio de máquina de vetores de suporte

A number of studies in the areas of Biomedical Engineering and Health Sciences have employed machine learning tools to develop methods capable of identifying patterns in different sets of data. Despite its extinction in many countries of the developed world, Hansen’s disease is still a disease that affects a huge part of the population in countries such as India and Brazil. In this context, this research proposes to develop a method that makes it possible to understand in the future how Hansen’s disease affects facial muscles. By using surface electromyography, a system was adapted so as to capture the signals from the largest possible number of facial muscles. We have first looked upon the literature to learn about the way researchers around the globe have been working with diseases that affect the peripheral neural system and how electromyography has acted to contribute to the understanding of these diseases. From these data, a protocol was proposed to collect facial surface electromyographic (sEMG) signals so that these signals presented a high signal to noise ratio. After collecting the signals, we looked for a method that would enable the visualization of this information in a way to make it possible to guarantee that the method used presented satisfactory results. After identifying the method's efficiency, we tried to understand which information could be extracted from the electromyographic signal representing the collected data. Once studies demonstrating which information could contribute to a better understanding of this pathology were not to be found in literature, parameters of amplitude, frequency and entropy were extracted from the signal and a feature selection was made in order to look for the features that better distinguish a healthy individual from a pathological one. After, we tried to identify the classifier that best discriminates distinct individuals from different groups, and also the set of parameters of this classifier that would bring the best outcome. It was identified that the protocol proposed in this study and the adaptation with disposable electrodes available in market proved their effectiveness and capability of being used in different studies whose intention is to collect data from facial electromyography. The feature selection algorithm also showed that not all of the features extracted from the signal are significant for data classification, with some more relevant than others. The classifier Support Vector Machine (SVM) proved itself efficient when the adequate Kernel function was used with the muscle from which information was to be extracted. Each investigated muscle presented different results when the classifier used linear, radial and polynomial kernel functions. Even though we have focused on Hansen’s disease, the method applied here can be used to study facial electromyography in other pathologies.

Classificação de atividade eletromiográfia facial de indivíduos saudáveis e com hanseníase por meio de máquina de vetores de suporte

A number of studies in the areas of Biomedical Engineering and Health Sciences have employed machine learning tools to develop methods capable of identifying patterns in different sets of data. Despite its extinction in many countries of the developed world, Hansen’s disease is still a disease that affects a huge part of the population in countries such as India and Brazil. In this context, this research proposes to develop a method that makes it possible to understand in the future how Hansen’s disease affects facial muscles. By using surface electromyography, a system was adapted so as to capture the signals from the largest possible number of facial muscles. We have first looked upon the literature to learn about the way researchers around the globe have been working with diseases that affect the peripheral neural system and how electromyography has acted to contribute to the understanding of these diseases. From these data, a protocol was proposed to collect facial surface electromyographic (sEMG) signals so that these signals presented a high signal to noise ratio. After collecting the signals, we looked for a method that would enable the visualization of this information in a way to make it possible to guarantee that the method used presented satisfactory results. After identifying the method's efficiency, we tried to understand which information could be extracted from the electromyographic signal representing the collected data. Once studies demonstrating which information could contribute to a better understanding of this pathology were not to be found in literature, parameters of amplitude, frequency and entropy were extracted from the signal and a feature selection was made in order to look for the features that better distinguish a healthy individual from a pathological one. After, we tried to identify the classifier that best discriminates distinct individuals from different groups, and also the set of parameters of this classifier that would bring the best outcome. It was identified that the protocol proposed in this study and the adaptation with disposable electrodes available in market proved their effectiveness and capability of being used in different studies whose intention is to collect data from facial electromyography. The feature selection algorithm also showed that not all of the features extracted from the signal are significant for data classification, with some more relevant than others. The classifier Support Vector Machine (SVM) proved itself efficient when the adequate Kernel function was used with the muscle from which information was to be extracted. Each investigated muscle presented different results when the classifier used linear, radial and polynomial kernel functions. Even though we have focused on Hansen’s disease, the method applied here can be used to study facial electromyography in other pathologies.

Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.

Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

BACKGROUND: Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles.

METHODS: SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA.

RESULTS: SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum.

CONCLUSIONS: The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible association with disease pathology, while the strong reactivity of sera from experimentally infected rats against rSjB6 suggests that native SjB6 is released into host tissue and induces an immune response. This study presents a comprehensive demonstration of sequence and structural-based analysis of a secretory serpin from a trematode and suggests SjB6 may be associated with important functional roles in S. japonicum, particularly in parasite modulation of the host microenvironment.

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01

FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity1, 2, 3. Since breast cancer stem cells (BCSCs) are CD44 positive, we wanted to explore if AD-01 could specifically target BCSCs. FKBPL stable overexpression or AD-01 treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- subpopulation, validated these results. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation (p<0.001). Clonogenic assays suggested that AD-01 mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog and Oct4 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). When AD-01 was combined with other agents, we observed synergistic activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using ‘gold standard’ in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, AD-01 appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Targeting treatment resistant cancer stem cells with FKBPL and its peptide therapeutics

FKBPL and its peptide derivatives have already demonstrated well-established inhibitory effects on cancer growth and CD44-dependent anti-angiogenic activity. Since cancer stem cells (CSCs) are CD44 positive, we wanted to explore if these therapeutics could specifically target CSCs in breast and ovarian cancer. In a tumoursphere assay, FKBPL stable overexpression or FKBPL-based peptide (AD-01, preclinical peptide or ALM201, clinical peptide candidate) treatment were highly effective at reducing the CSC population measured by inhibiting tumoursphere forming efficiency in breast and ovarian cancer cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- and ALDH+ cell subpopulations representative of CSCs, validated these results. The ability of AD-01 and ALM201 to inhibit the self-renewal capacity of CSCs was confirmed across three generations, eradicating CSC completely by the third generation (p<0.001). Furthermore, clonogenic assay demonstrated that FKBPL-based peptides mediated CSC differentiation, with a significant decrease in the number of CSCs or holoclones and an associated increase in differentiated cancer cells or meroclones/paraclones. In addition, AD-01 treatment in vitro and in vivo led to a significant reduction in the stem cell markers, Nanog, Sox2 and Oct4 protein and mRNA levels; whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in tumoursphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). Additionally, when AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in CSCs. Importantly, using gold standard in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, FKBPL-based peptides appear to have dual anti-angiogenic and anti-CSC activity which will be advantageous as this agent enters clinical trial.

Avaliação do comportamento ingestivo utilizando eletromiografia de superfície do músculo masseter

Physiologists and animal scientists try to understand the relationship between ruminants and their environment. The knowledge about feeding behavior of these animals is the key to maximize the production of meat and milk and their derivatives and ensure animal welfare. Within the area called precision farming, one of the goals is to find a model that describes animal nutrition. Existing methods for determining the consumption and ingestive patterns are often time-consuming and imprecise. Therefore, an accurate and less laborious method may be relevant for feeding behaviour recognition. Surface electromyography (sEMG) is able to provide information of muscle activity. Through sEMG of the muscles of mastication, coupled with instrumentation techniques, signal processing and data classification, it is possible to extract the variables of interest that describe chewing activity. This work presents a new method for chewing pattern evaluation, feed intake prediction and for the determination of rumination, food and daily rest time through ruminant animals masseter muscle sEMG signals. Short-term evaluation results are shown and discussed, evidencing employed methods viability.

TRIB2 in human AML: a biological and clinical investigation

Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest of cells at a very early stage of myeloid cell differentiation. The biological and clinical heterogeneity of this disease complicates treatment and highlights the significance of understanding the underlying causes of AML, which may constitute potential therapeutic targets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potent murine oncogene capable of inducing transplantable AML with complete penetrance. The pathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of the full length isoform of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα p42). The role of TRIB2 in human AML cells, however, has not been systematically investigated or targeted. Across human cancers, TRIB2 oncogenic activity was found to be associated with its elevated expression. In the context of AML, TRIB2 overexpression was suggested to be associated with the large and heterogeneous subset of cytogenetically normal AML patients. Based upon the observation that overexpression of TRIB2 has a role in cellular transformation, the effect of modulating its expression in human AML was examined in a human AML cell line that expresses high levels of TRIB2, U937 cells. Specific suppression of TRIB2 led to impaired cell growth, as a consequence of both an increase in apoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2 silencing strongly reduced progression of the U937 in vivo xenografts, accompanied by detection of a lower spleen weight when compared with mice transplanted with TRIB2- expressing control cells. Gene expression analysis suggested that TRIB2 modulates apoptosis and cell-cycle sensitivity by influencing the expression of a subset of genes known to have implications on these phenotypes. Furthermore, TRIB2 was found to be expressed in a significant subset of AML patient samples analysed. To investigate whether increased expression of this gene could be afforded prognostic significance, primary AML cells with dichotomized levels of TRIB2 transcripts were evaluated in terms of their xenoengraftment potential, an assay reported to correlate with disease aggressiveness observed in humans. A small cohort of analysed samples with higher TRIB2 expression did not associate with preferential leukaemic cell engraftment in highly immune-deficient mice, hence, not predicting for an adverse prognosis. However, further experiments including a larger cohort of well characterized AML patients would be needed to clarify TRIB2 significance in the diagnostic setting. Collectively, these data support a functional role for TRIB2 in the maintenance of the oncogenic properties of human AML cells and suggest TRIB2 can be considered a rational therapeutic target. Proteasome inhibition has emerged as an attractive target for the development of novel anti-cancer therapies and results from translational research and clinical trials support the idea that proteasome inhibitors should be considered in the treatment of AML. The present study argued that proteasome inhibition would effectively inhibit the function of TRIB2 by abrogating C/EBPα p42 protein degradation and that it would be an effective pharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cell models expressing high levels of TRIB2 were successfully targeted by treatment with proteasome inhibitors, as demonstrated by multiple measurements that included increased cytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo. Mechanistically, it was shown that block of the TRIB2 degradative function led to an increase of C/EBPα p42 and that response was specific to the TRIB2-C/EBPα axis. Specificity was addressed by a panel of experiments showing that U937 cells (express detectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitor bortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression and that ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negative leukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased following TRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPα axis. Together these findings provide pre-clinical evidence that TRIB2- expressing AML cells can be pharmacologically targeted with proteasome inhibition due, in part, to blockage of the TRIB2 proteolytic function on C/EBPα p42. A large body of evidence indicates that AML arises through the stepwise acquisition of genetic and epigenetic changes. Mass spectrometry data has identified an interaction between TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5 (PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effective targeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5 cooperation was investigated in order to gain a deeper understanding of the molecular network in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data set was interrogated for PRMT5 expression levels and the primary enzyme responsible for symmetric dimethylation was found to be transcribed at significantly higher levels in AML patients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cell line was shown to reduce the transformative phenotype in the high expressing TRIB2 AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain the leukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interacting protein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlying mechanism and the functional significance of this interplay. In summary, the present study provides experimental evidence that TRIB2 has an important oncogenic role in human AML maintenance and, importantly in such a molecularly heterogeneous disease, provides the rational basis to consider proteasome inhibition as an effective targeting strategy for AML patients with high TRIB2 expression. Finally, the identification of PRMT5 as a TRIB2-interacting protein opens a new level of regulation to consider in AML. This work may contribute to our further understanding and therapeutic strategies in acute leukaemias.