Global and threshold-free transcriptional regulatory networks reconstruction through integrating ChIP-Chip and expression data

Inferring transcriptional regulatory networks from high-throughput biological data is a major challenge to bioinformatics today. To address this challenge, we developed TReNGO (Transcriptional Regulatory Networks reconstruction based on Global Optimization), a global and threshold-free algorithm with simulated annealing for inferring regulatory networks by the integration of ChIP-chip and expression data. Superior to existing methods, TReNGO was expected to find the optimal structure of transcriptional regulatory networks without any arbitrary thresholds or predetermined number of transcriptional modules (TMs). TReNGO was applied to both synthetic data and real yeast data in the rapamycin response. In these applications, we demonstrated an improved functional coherence of TMs and TF (transcription factor)- target predictions by TReNGO when compared to GRAM, COGRIM or to analyzing ChIP-chip data alone. We also demonstrated the ability of TReNGO to discover unexpected biological processes that TFs may be involved in and to also identify interesting novel combinations of TFs.

Mining frequent itemsets for protein kinase regulation

Protein kinases, a family of enzymes, have been viewed as an important signaling intermediary by living organisms for regulating critical biological processes such as memory, hormone response and cell growth. The
unbalanced kinases are known to cause cancer and other diseases. With the increasing efforts to collect, store and disseminate information about the entire kinase family, it not only leads to valuable data set to understand cell regulation but also poses a big challenge to extract valuable knowledge about metabolic pathway from the data. Data mining techniques that have been widely used to find frequent patterns in large datasets can be extended and adapted to kinase data as well. This paper proposes a framework for mining frequent itemsets from the collected kinase dataset. An experiment using AMPK regulation data demonstrates that our approaches are useful and efficient in analyzing kinase regulation data.

Keyword extraction for text categorization

Text categorization (TC) is one of the main applications of machine learning. Many methods have been proposed, such as Rocchio method, Naive bayes based method, and SVM based text classification method. These methods learn labeled text documents and then construct a classifier. A new coming text document's category can be predicted. However, these methods do not give the description of each category. In the machine learning field, there are many concept learning algorithms, such as, ID3 and CN2. This paper proposes a more robust algorithm to induce concepts from training examples, which is based on enumeration of all possible keywords combinations. Experimental results show that the rules produced by our approach have more precision and simplicity than that of other methods.

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

A pHMM-ANN based discriminative approach to promoter identification in prokaryote genomic contexts

The computational approach for identifying promoters on increasingly large genomic sequences has led to many false positives. The biological significance of promoter identification lies in the ability to locate true promoters with and without prior sequence contextual knowledge. Prior approaches to promoter modelling have involved artificial neural networks (ANNs) or hidden Markov models (HMMs), each producing adequate results on small scale identification tasks, i.e. narrow upstream regions. In this work, we present an architecture to support prokaryote promoter identification on large scale genomic sequences, i.e. not limited to narrow upstream regions. The significant contribution involved the hybrid formed via aggregation of the profile HMM with the ANN, via Viterbi scoring optimizations. The benefit obtained using this architecture includes the modelling ability of the profile HMM with the ability of the ANN to associate elements composing the promoter. We present the high effectiveness of the hybrid approach in comparison to profile HMMs and ANNs when used separately. The contribution of Viterbi optimizations is also highlighted for supporting the hybrid architecture in which gains in sensitivity (+0.3), specificity (+0.65) and precision (+0.54) are achieved over existing approaches.

Acoustic features extraction for emotion recognition

In the last decade, the efforts of spoken language processing have achieved significant advances, however, the work with emotional recognition has not progressed so far, and can only achieve 50% to 60% in accuracy. This is because a majority of researchers in this field have focused on the synthesis of emotional speech rather than focusing on automating human emotion recognition. Many research groups have focused on how to improve the performance of the classifier they used for emotion recognition, and few work has been done on data pre-processing, such as the extraction and selection of a set of specifying acoustic features instead of using all the possible ones they had in hand. To work with well-selected acoustic features does not mean to delay the whole job, but this will save much time and resources by removing the irrelative information and reducing the high-dimension data calculation. In this paper, we developed an automatic feature selector based on a RF2TREE algorithm and the traditional C4.5 algorithm. RF2TREE applied here helped us to solve the problems that did not have enough data examples. The ensemble learning technique was applied to enlarge the original data set by building a bagged random forest to generate many virtual examples, and then the new data set was used to train a single decision tree, which selects the most efficient features to represent the speech signals for the emotion recognition. Finally, the output of the selector was a set of specifying acoustic features, produced by RF2TREE and a single decision tree.

Non-quantized minimum free energy in untranslated region exons

In an attempt to improve automated gene prediction in the untranslated region of a gene, we completed an in-depth analysis of the minimum free energy for 8,689 sub-genetic DNA sequences. We expanded Zhang's classification model and classified each sub-genetic sequence into one of 27 possible motifs. We calculated the minimum free energy for each motif to explore statistical features that correlate to biologically relevant sub-genetic sequences. If biologically relevant sub-genetic sequences fall into distinct free energy quanta it may be possible to characterize a motif based on its minimum free energy. Proper characterization of motifs can lead to greater understanding in automated genefinding, gene variability and the role DNA structure plays in gene network regulation.

Our analysis determined: (1) the average free energy value for exons, introns and other biologically relevant sub-genetic sequences, (2) that these subsequences do not exist in distinct energy quanta, (3) that introns exist however in a tightly coupled average minimum free energy quantum compared to all other biologically relevant sub-genetic sequence types, (4) that single exon genes demonstrate a higher stability than exons which span the entire coding sequence as part of a multi-exon gene and (5) that all motif types contain a free energy global minimum at approximately nucleotide position 1,000 before reaching a plateau. These results should be relevant to the biochemist and bioinformatician seeking to understand the relationship between sub-genetic sequences and the information behind them.

Probabilistic reasoning of inconsistent belief in protocol analysis

Security protocols have been recently found with subtle flaws due to incomplete or ambiguous specification. Although formal methods have remarkably assisted in protocol analysis, they ignores the effect of hostile/uncertain environment, which might lead to inconsistent belief that can be held by principals in delivered messages. This discrepant belief may prevent us from representing the insecurity and uncertainty in a real trading situation. Unfortunately, the current approaches lack the ability to handle the inconsistent belief. This article presents a probabilistic method, which intuitively measures the belief from different principals that can be put on the goal of the protocol. The experiments demonstrate our method is useful to enhance the protocol analysis.

Analysis of cardioprotective effects using purified Saliva miltiorrhiza extract on isolated rat hearts

The purpose of the current study is to evaluate the cardioprotective effects of purified Salvia miltiorrhiza extract (PSME) on myocardial ischemia/reperfusion injury in isolated rat hearts. Hearts were excised and perfused at constant flow (7 – 9 ml · min⁻¹) via the aorta. Non-recirculating perfusion with Krebs-Henseleit (KH) solution was maintained at 37°C and continuously gassed with 95% O₂ and 5% CO₂. KH solution with or without PSME (100 mg per liter solution) was used after 30-min zero-flow ischemia for the PSME and control group, respectively. Left ventricular (LV) developed pressure; its derivatives, diastolic pressure, and so on were continuously recorded via a pressure transducer attached to a polyvinylchloride balloon that was placed in the left ventricle through an incision in the left atrium. PSME treated hearts showed significant postischemic contractile function recovery (developed pressure recovered to 44.2 ± 4.9% versus 17.1 ± 5.7%, P<0.05; maximum contraction recovered to 57.2 ± 5.9% versus 15.1 ± 6.3%, P<0.001; maximum relaxation restored to 69.3 ± 7.3% versus 15.4 ± 6.3%, P<0.001 in the PSME and control group, respectively). Significant elevation in end-diastolic pressure, which indicated LV stiffening in PSME hearts might have resulted from the excess high dose of PSME used. Further study will be conducted on the potential therapeutic value with lower dose of PSME on prevention of ischemic heart disease.

Metabolism and transport of oxazphosphorines and the clinical implications

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity. CPA is widely used as an anticancer drug, an immunosuppressant, and for the mobilization of hematopoetic progenitor cells from the bone marrow into peripheral blood prior to bone marrow transplantation for aplastic anemia, leukemia, and other malignancies. New oxazaphosphorines derivatives have been developed in an attempt to improve selectivity and response with reduced toxicity. These derivatives include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). This review highlights the metabolism and transport of these oxazaphosphorines (mainly CPA and IFO, as these two oxazaphosphorine drugs are the most widely used alkylating agents) and the clinical implications. Both CPA and IFO are prodrugs that require activation by hepatic cytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxic nitrogen mustards capable of reacting with DNA molecules to form crosslinks and lead to cell apoptosis and/or necrosis. Such prodrug activation can be enhanced within tumor cells by the CYP-based gene directed-enzyme prodrug therapy (GDEPT) approach. However, those newly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC 612567 and NSC 613060, do not need hepatic activation. They are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by plain phosphodiesterase (PDEs) in plasma and other tissues or by the high-affinity nuclear 3'-5' exonucleases associated with DNA polymerases, such as DNA polymerases and ε. The alternative CYP-catalyzed inactivation pathway by N-dechloroethylation generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Various aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs) are involved in the detoxification of oxazaphosphorine metabolites. The metabolism of oxazaphosphorines is auto-inducible, with the activation of the orphan nuclear receptor pregnane X receptor (PXR) being the major mechanism. Oxazaphosphorine metabolism is affected by a number of factors associated with the drugs (e.g., dosage, route of administration, chirality, and drug combination) and patients (e.g., age, gender, renal and hepatic function). Several drug transporters, such as breast cancer resistance protein (BCRP), multidrug resistance associated proteins (MRP1, MRP2, and MRP4) are involved in the active uptake and efflux of parental oxazaphosphorines, their cytotoxic mustards and conjugates in hepatocytes and tumor cells. Oxazaphosphorine metabolism and transport have a major impact on pharmacokinetic variability, pharmacokinetic-pharmacodynamic relationship, toxicity, resistance, and drug interactions since the drug-metabolizing enzymes and drug transporters involved are key determinants of the pharmacokinetics and pharmacodynamics of oxazaphosphorines. A better understanding of the factors that affect the metabolism and transport of oxazaphosphorines is important for their optional use in cancer chemotherapy.

A mechanistic study on reduced toxicity of irontecan by coadministered thalidomide, a tumor necrosis factor-a inhibitor

Dose-limiting diarrhea and myelosuppression compromise the success of irinotecan (7-ethyl-10-[4-[1-piperidino]-1-piperidino] carbonyloxycamptothecin) (CPT-11)-based chemotherapy. A recent pilot study indicates that thalidomide attenuates the toxicity of CPT-11 in cancer patients. This study aimed to investigate whether coadministered thalidomide modulated the toxicities of CPT-11 and the underlying mechanisms using several in vivo and in vitro models. Diarrhea, intestinal lesions, cytokine expression, and intestinal epithelial apoptosis were
monitored. Coadministered thalidomide (100 mg/kg i.p. for 8 days) significantly attenuated body weight loss, myelosuppression, diarrhea, and intestinal histological lesions caused by CPT-11 (60 mg/kg i.v. for 4 days). This was accompanied by inhibition of tumor necrosis factor-, interleukins 1 and 6 and interferon-, and intestinal epithelial apoptosis. Coadministered
thalidomide also significantly increased the systemic exposure of CPT-11 but decreased that of SN-38 (7-ethyl-10-hydroxycampothecin). It significantly reduced the biliary excretion and cecal exposure of CPT-11, SN-38, and SN-38 glucuronide. Thalidomide hydrolytic products inhibited hydrolysis of CPT-11 in rat liver microsomes but not in primary rat hepatocytes. In addition, thalidomide and its major hydrolytic products, such as phthaloyl glutamic acid (PGA), increased the intracellular accumulation of CPT-11 and SN-38 in primary rat hepatocytes. They also significantly decreased the transport of CPT-11 and SN-38 in Caco-2 and parental MDCKII cells. Thalidomide and PGA also significantly inhibited P-glycoprotein (PgP/MDR1), multidrug resistance-associated protein (MRP1)- and MRP2-mediated CPT-11 and SN-38 transport in MDCKII cells. These results provide insights into the pharmacodynamic and  pharmacokinetic mechanisms for the protective effects of thalidomide against CPT-11-induced intestinal toxicity.

Topotecan is a substrate for multidrug resistance associated protein 4

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both doselimiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated Km of 1.66 mM and Vmax of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 μM) for 24 hr, celecoxib (50 mM), or MK-571 (100 mM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POMPMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.

Human multidrug resistance associated protein 4 resitance to camtothecins

Purpose The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included.
Methods HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods.
Results Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine.
Conclusions Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.

St Johns wort modulates the toxicities and parmacokinetics of CPT-11 (Irontecan) in rats

CPT-11 is a DNA topoisomerase I inhibitor for the therapy of colorectal cancer, whereas St. Johnrsquos Wort (Hypericum perforatum, SJW) is a widely used herbal anti-depressant. This study aimed to investigate the effects of co-administered SJW on the toxicities and pharmacokinetics of CPT-11 and the underlying mechanisms. The body weight loss, gastrointestinal and hematological toxicities induced by CPT-11, and the pharmacokinetic parameters of CPT-11 were evaluated in rats pretreated with SJW or vehicle. Rats treated with CPT-11 alone experienced rapid decrease in body weight, whereas co-administration of SJW with CPT-11 resulted in lesser body weight loss. The gastrointestinal and hematological toxicities following CPT-11 injection were both alleviated in the presence of SJW. The rat pharmacokinetics of both CPT-11 and its metabolite SN-38 were significantly altered in presence of SJW. In conclusion, co-administered SJW significantly ameliorated the toxicities induced by CPT-11. The protective effect of SJW may be partially due to pharmacokinetic interaction between CPT-11 and SJW.