Anasuya Hazra

Estimating vapor pressure curves by thermogravimetry: a rapid and convenient method for characterization of pharmaceuticals.

The purpose of this study was to investigate a rapid method for the evaluation of vaporization characteristics for selected benzoic acid derivatives. The compounds studied in this context were the ortho-, meta- and para-derivatives of hydroxy and amino benzoic acids. Calculations for the order of reaction were first carried out for each of the compounds using methyl paraben as the calibration standard. Those compounds undergoing zero order, non-activated evaporation processes, were analyzed by the Antoine and Langmuir equations, conjointly. The coefficient of vaporization was obtained as 1.2 x 10(5)+/-0.8 Pakg (0.5)mol(0.5)s(-1)m(-2)K(-0.5). The vapor pressure values were used to determine the Antoine constants using the SPSS 10.0 software. This study attempts to outline a comprehensive thermogravimetric technique for vapor pressure characterization of single-component systems.

Pharmacodynamic Modeling of Acute and Chronic Effects of Methylprednisolone on Hepatic Urea Cycle Genes in Rats

In order to investigate the possible mechanisms for eve stripe formation of Drosophila embryo, a spatio-temporal gene/protein interaction network model is proposed to mimic dynamic behaviors of protein synthesis, protein decay, mRNA decay, protein diffusion, transcription regulations and autoregulation to analyze the interplay of genes and proteins at different compartments in early embryogenesis. In this study, we use the maximum likelihood (ML) method to identify the stochastic 3-D Embryo Space-Time (3-DEST) dynamic model for gene/protein interaction network via 3-D mRNA and protein expression data and then use the Akaike Information Criterion (AIC) to prune the gene/protein interaction network. The identified gene/protein interaction network allows us not only to analyze the dynamic interplay of genes and proteins on the border of eve stripes but also to infer that eve stripes are established and maintained by network motifs built by the cooperation between transcription regulations and diffusion mechanisms in early embryogenesis. Literature reference with the wet experiments of gene mutations provides a clue for validating the identified network. The proposed spatio-temporal dynamic model can be extended to gene/protein network construction of different biological phenotypes, which depend on compartments, e.g. postnatal stem/progenitor cell differentiation.Vertebrae and other mammalian repetitive structures are formed from embryonic organs called somites. Somites arise sequentially from the unsegmented presomitic mesoderm (PSM). In mice, a new bilateral pair of somites arise every two hours from the rostral PSM. On the other hand, cells are added to the caudal side of the PSM due to cell proliferation of the tail bud. Somite formation correlates with cycles of cell-autonomous expression in the PSM of genes like Hes7. Because the somitogenesis is a highly dynamic and coordinated process, this event has been subjected to extensive theoretical modeling. Here, we describe the current understanding about the somitogenesis in mouse embryos with an emphasis on insights gained from computer simulations. It is worth noting that the combination of experiments and computer simulations has uncovered dynamical properties of the somitogenesis clock such as the transcription/translation delays, the half-life and the synchronization mechanism across the PSM. Theoretical models have also been useful to provide predictions and rigorous hypothesis about poorly understood processes such as the mechanisms by which the temporal PSM oscillations are arrested and converted into an spatial pattern. We aim at reviewing this theoretical literature in such a way that experimentalists might appreciate the resulting conclusions.Background Previous studies demonstrated that the vascular endothelial growth factor (VEGF) was being implicated in the airways inflammation and remodeling process in patients with asthma. Aims We explored the relationship of three polymorphisms in the VEGF gene with asthma in both case control and family studies. Methods We Genotyped a total of 210 children with asthma, 224 unrelated controls and 160 parents for the +936 C >T (rs3025039), −634 G > C (rs2010963) and −2549 −2567 del 18 of the VEGF promoter region. The Mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis for the +936 C > T, and −634 G > C polymorphisms. Results Of the three polymorphisms studied, a borderline association with asthma was found for the G allele in the −634 G > C polymorphism (p = 0.059). No Statistically significant differences were observed for both +936 C > T, and −2549 −2567 del 18 polymorphisms between asthmatic patients and controls, considering either allelic or genotypic frequencies. The distribution of genotypes according to the severity status revealed a significant differences for the +936 C > T, and −2549 −2567 del 18 polymorphisms. In addition, association was found with the haplotypes inferred by the three polymorphisms and asthma susceptibility. Conclusion We suggest that VEGF Gene polymorphisms can be implicated in asthma.Insulin sensitizing thiazolidinediones (TZDs) are generally considered to work as agonists for the nuclear receptor peroxisome proliferative activated receptor-gamma (PPARγ). However, TZDs also have acute, non-genomic metabolic effects and it is unclear which actions are responsible for the beneficial pharmacology of these compounds. We have taken advantage of an analog, based on the metabolism of pioglitazone, which has much reduced ability to activate PPARγ. This analog (PNU-91325) was compared to rosiglitazone, the most potent PPARγ activator approved for human use, in a variety of studies both in vitro and in vivo. The data demonstrate that PNU-91325 is indeed much less effective than rosiglitazone at activating PPARγ both in vitro and in vivo. In contrast, both compounds bound similarly to a mitochondrial binding site and acutely activated PI-3 kinase-directed phosphorylation of AKT, an action that was not affected by elimination of PPARγ activation. The two compounds were then compared in vivo in both normal C57 mice and diabetic KKAy mice to determine whether their pharmacology correlated with biomarkers of PPARγ activation or with the expression of other gene transcripts. As expected from previous studies, both compounds improved insulin sensitivity in the diabetic mice, and this occurred in spite of the fact that there was little increase in expression of the classic PPARγ target biomarker adipocyte binding protein-2 (aP2) with PNU-91325 under these conditions. An examination of transcriptional profiling of key target tissues from mice treated for one week with both compounds demonstrated that the relative pharmacology of the two thiazolidinediones correlated best with an increased expression of an array of mitochondrial proteins and with expression of PPARγ coactivator 1-alpha (PGC1α), the master regulator of mitochondrial biogenesis. Thus, important pharmacology of the insulin sensitizing TZDs may involve acute actions, perhaps on the mitochondria, that are independent of direct activation of the nuclear receptor PPARγ. These findings suggest a potential alternative route to the discovery of novel insulin sensitizing drugs.The trans-regulatory circuit is considered as the regulatory interactions between upstream regulatory genes and transcription factor binding site motifs or cis elements. And the cis-regulatory circuit is viewed as a dynamic interactive circuit among binding site motifs with their effective action on the expression scheme of target gene. In brief, gene transcription depends on the trans/cis regulatory circuits. In this study, nonlinear trans/cis regulatory circuits for gene transcription in yeast are constructed using microarray data, translation time delay, and information of transcription factors (TFs) binding sites. We provide a useful nonlinear dynamic modeling and develop a parameter estimating method for the construction of trans/cis regulatory circuits, which is powerful for understanding gene transcription. We apply our method to construct trans/cis regulatory circuits of yeast cell cycle-related genes and successfully quantify their regulatory abilities and find possible cis-element interactions. Not only could the data of yeast be applied by our method, but those of other species also could. The proposed method can provide a quantitative basis for system analysis of gene circuits, which is potential for gene regulatory circuit design with a desired gene expression.The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were “seamlessly” fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2–7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall findings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the post-transcriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.Integrins have been proposed to play a major role in lens morphogenesis. To determine the role of β1-integrin and its down-stream signaling partner, integrin linked kinase (ILK), in lens morphogenesis, eyes of WT mice and mice with a nestin-linked conditional knockout of β1-integrin or ILK were analyzed for defects in lens development. Mice, lacking the genes encoding the β1-integrin subunit (Itgb1) or ILK (Ilk), showed a perinatal degeneration of the lens. Early signs of lens degeneration included vacuolization, random distribution of lens cell nuclei, disrupted fiber morphology and attenuation and separation of the lens capsule. The phenotype became progressively more severe during the first postnatal week eventually leading to the complete loss of the lens. A more severe phenotype was observed in ILK mutants at similar stages. Eyes from embryonic day 13 β1-integrin-mutant embryos showed no obvious signs of lens degeneration, indicating that mutant lens develops normally until peri-recombination. Our findings suggest that β1-integrins and ILK cooperate to control lens cell survival and link lens fibers to the surrounding extracellular matrix. The assembly and integrity of the lens capsule also appears to be reliant on integrin signaling within lens fibers. Extrapolation of these results indicates a novel role of integrins in lens cell-cell adhesions as well as a potential role in the pathogenesis of congenital cataracts.Resistance to radio and chemotherapy is one of the major drawbacks in the progression of head and neck squamous cell cancer (HNSCC) patients, evidencing the importance of finding optimum molecular prognosis markers to develop personalized treatment schedules. TGF-β effector TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al. 1998; Edlund et al. 2003; Kaur et al. 2005) and, although there has been described no significant implication of TAK1 in HNSCC development, we have further examined the role of its mRNA expression as a marker of prognosis in HNSCC. Fifty-nine advanced HNSCC patients were recruited for the study. The tumor expression of TAK1 mRNA was analyzed with RT-PCR using Taqman technology and its relationship with the clinical outcome of the patients studied. TAK1 mRNA expression was lower in patients that relapsed than in those that did not, but the difference was only significant between the patients that showed response to treatment (p < 0.001). ROC curve analyses pointed a 0.5 expression ratio TAK1/B2M value as an optimum cut-off point for relapse and response. Our data suggest the TAK1 mRNA analysis by Taqman RT-PCR can predict the risk of relapse in HNSCC patients.In microarray studies several statistical methods have been proposed with the purpose of identifying differentially expressed genes in two varieties. A commonly used method is an analysis of variance model where only the effect of interaction between variety and gene is tested. In this paper we argue that in addition to the interaction effects, the main effect of variety should simultaneously also be taken into account when posting the hypothesis.Prostanoids have a broad spectrum of biological activities in a variety of organs including the brain. However, their effects on synaptic plasticity in the brain, which have been recently revealed, are ambiguous in comparison to those in the other organs. Prostaglandin E2 (PGE2) is a prostanoid produced from arachidonic acid in the cellular membrane, and knowledge about its functions is increasing. Recently, a novel function of PGE2 in the brain has shed light on aspects of synaptic plasticity such as long-term potentiation (LTP). More recently, we have proposed a hypothesis for the mechanisms of this PGE2-related form of synaptic plasticity in the visual cortex. This involves the dynamics of two subtypes of PGE2 receptors that have opposing functions in intracellular signal transduction. Consequently, mechanisms that increase the level of cyclic AMP in the cytosol may explain for the mechanisms of LTP in the visual cortex. The current notion of bidirectional trafficking of PGE2 receptors under this hypothesis is reminiscent of the “silent synapse” mechanism of LTP on the trafficking of the AMPA receptors between the membrane and cytosol. Moreover, we propose the hypothesis that PGE2 acts as a “post-to-postsynaptic messenger” for the induction of LTP in the visual cortex. This review describes a complex mode of action of PGE2 receptors in synaptic plasticity in the brain.Purpose The present study predicts and tests genetic networks that modulate gene expression during the retinal wound-healing response. Methods Upstream modulators and target genes were defined using meta-analysis and bioinformatic approaches. Quantitative trait loci (QTLs) for retinal acute phase genes (Vazquez-Chona et al. 2005) were defined using QTL analysis of CNS gene expression (Chesler et al. 2005). Candidate modulators were defined using computational analysis of gene and motif sequences. The effect of candidate genes on wound healing was tested using animal models of gene expression. Results A network of early wound-healing genes is modulated by a locus on chromosome 12. The genetic background of the locus altered the wound-healing response of the retina. The C57BL/6 allele conferred enhanced expression of neuronal marker Thy1 and heat-shock-like crystallins, whereas the DBA/2J allele correlated with greater levels of the classic marker of retinal stress, glial fibrillary acidic protein (GFAP). Id2 and Lpin1 are candidate upstream modulators as they strongly correlated with the segregation of DBA/2J and C57BL/6 alleles, and their dosage levels correlated with the enhanced expression of survival genes (Thy1 and crystallin genes). Conclusion We defined a genetic network associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and can didate modulators that control transcript levels of acute phase genes. Our results support the convergence of gene expression profiling, QTL analysis, and bioinformatics as a rational approach to discover molecular pathways controlling retinal wound healing.Introduction advent of molecular biology caused a reductionist “fever” to spread throughout the biological research community that continues to this day. The new molecular insights and techniques enabled researchers to probe the constituent parts of complex biological systems at unprecedented scale and detail. The reductionist approach naturally emerged: if we could now isolate and study the component parts of a system, we should be able to synthesize the information about the individual components into a unifi ed understanding of the whole system. However, this “naive reductionism” (Bloom, 2001) needs to be balanced with a systems approach for a simple reason: the complex dynamics of a biological system often produce behaviors and properties that cannot be explained by the presence of a single component, but rather emerge from the interactions of the components of the system (so-called emergentThe aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA micorarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells.With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License.Bacterial RNA polymerase is composed of a core of subunits (β, β′, α1, α2, ω), which have RNA synthesizing activity, and a specificity factor (σ), which identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. Four core promoter consensus sequences, the −10 element, the extended −10 (TGn) element, the −35 element, and the UP elements, have been known for many years; the importance of a nontemplate G at position −5 has been recognized more recently. However, the functions of these elements are not the same. The AT-rich UP elements, the −35 elements (−35TTGACA−30), and the extended −10 (−15TGn−13) are recognized as double-stranded binding elements, whereas the −5 nontemplate G is recognized in the context of single-stranded DNA at the transcription bubble. Furthermore, the −10 element (−12TATAAT−7) is recognized as both double-stranded DNA for the T:A bp at position −12 and as nontemplate, single-stranded DNA from positions −11 to −7. The single-stranded sequences at positions −11 to −7 as well as the −5 contribute to later steps in transcription initiation that involve isomerization of polymerase and separation of the promoter DNA around the transcription start site. Recent work has demonstrated that the double-stranded elements may be used in various combinations to yield an effective promoter. Thus, while some minimal number of contacts is required for promoter function, polymerase allows the elements to be mixed and matched. Interestingly, which particular elements are used does not appear to fundamentally alter the transcription bubble generated in the stable complex. In this review, we discuss the multiple steps involved in forming a transcriptionally competent polymerase/promoter complex, and we examine what is known about polymerase recognition of core promoter elements. We suggest that considering promoter elements according to their involvement in early (polymerase binding) or later (polymerase isomerization) steps in transcription initiation rather than simply from their match to conventional promoter consensus sequences is a more instructive form of promoter classification.To perform a quantitative analysis with gene-arrays, one must take into account inaccuracies (experimental variations, biological variations and other measurement errors) which are seldom known. In this paper we investigated amplification and noise propagation related errors by measuring intensity dependent variations. Based on a set of control samples, we create confidence intervals for up and down regulations. We validated our method through a qPCR experiment and compared it to standard analysis methods (including loess normalization and filtering methods based on genetic variability). The results reveal that amplification related errors are a major concern.Fanconi anemia (FA) is an autosomal recessive disorder characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. At least eleven members of the FA gene family have been identified using complementation experiments. Ubiquitin-proteasome has been shown to be a key regulator of FA proteins and their involvement in the repair of DNA damage. Here, we identified a novel functional link between the FA/BRCA pathway and E2F-mediated cell cycle regulome. In silico mining of a transcriptome database and promoter analyses revealed that a significant number of FA gene members were regulated by E2F transcription factors, known to be pivotal regulators of cell cycle progression – as previously described for BRCA1. Our findings suggest that E2Fs partly determine cell fate through the FA/BRCA pathway.Microchimerism refers to the status of harboring cells from another individual at low levels. It is well known that cells traffic bidirectionally between fetus and mother during pregnancy. This situation resembles a naturally occurring long lasting fetal stem cell transplantation. The fetus acts as the donor and the mother acts as the recipient. To study the role of microchimerism in tissue regeneration, we constructed a murine microchimerism model with wild type C57BL/6J female mice carrying progenies which expressed green fluorescent proteins (GFP). Our data indicated that skin injuries in the female mice during pregnancy increased microchimerism of GFP expressing cells from the GFP transgenic progenies. The GFP positive cells also appeared at the site of spinal cord where injury occurred during pregnancy. Our study suggests that the amount of fetal cells in maternal mice significantly increased if injuries occurred during pregnancy. Fetal stem cells appear to respond to maternal injury signals and may play a role in maternal tissue regeneration during pregnancy.Receptor-like kinases (RLKs) in plants are a large superfamily of proteins that are structurally similar. RLKs are involved in a diverse array of plant responses including development, growth, hormone perception and the response to pathogens. Current studies have focused attention on plant receptor-like kinases as an important class of sentinels acting in plant defense responses. RLKs have been identified that act in both broad-spectrum, elicitor-initiated defense responses and as dominant resistance (R) genes in race-specific pathogen defense. Most defense-related RLKs are of the leucine-rich repeat (LRR) subclass although new data are highlighting other classes of RLKs as important players in defense responses. As our understanding of RLK structure, activation and signaling has expanded, the role of the ubiquitin/proteasome system in the regulation of these receptors has emerged as a central theme.Purpose Epileptic mutant EL mice show secondary generalized seizures. Seizure discharges initiate in the parietal cortex and generalize through the hippocampus. We have previously demonstrated an increase in the activity of inducible nitric oxide synthetase (iNOS) as well as a decrease in the activity of superoxide dismutase (SOD) in the hippocampus of EL mice, suggesting that cell toxic free radicals are increased in the brain of EL mice. In parallel with this, neurotrophic factors were significantly increased in the hippocampus of EL mice in earlier developmental stages before exhibiting frequent seizures. These findings were no longer present after frequent seizures, suggesting that these events may trigger ictogenesis. On the other hand, it is reported that limbic seizures rapidly induce cytokines and related inflammatory mediators. It remains to be seen, however, whether cytokines contribute to the transition from interictal to ictal state. The present study was designed to address this issue using EL mice. Methods EL mice at the age from 4 to 23 weeks and their control animal, DDY mice at the age of 10 and 20 weeks were used. Seizures were induced in EL mice once every week since 5 weeks. Cytokines, such as interleukin-1 alpha (IL-1a), interleukin 1-beta (IL-1b), IL-6, IL-1 receptor (IL-1r), IL-1 receptor antagonist (IL-ra) and tumor necrosis factor alpha (TNF-a) were examined by Western blotting in the ‘focus complex’ of brain (namely, in the parietal cortex and hippocampus) of EL mice in the interictal period at different developmental stages. In 15 week old EL mice, which show seizures once a week, these cytokines were similarly determined 5 min, 2 hr, 4 hr, 11 hr, 24 hr, 3 days and 6 days after the last seizure induced. Results A significant increase in the level of cytokines was observed in the brain of EL mice at any stages during development, compared with the level of cytokines in the brain of control DDY. Cytokines were increased predominantly before experiencing frequent seizures. In EL mice at the age of 15 weeks, the level of cytokines in the hippocampus was highest 6 days after seizures. In the parietal cortex, cytokines were most highly expressed 2 hr after seizures. The results indicate that cytokines were kept up-regulated until next seizures in the hippocampus, whereas they were transiently up-regulated immediately after seizures in the parietal cortex. Conclusion It is concluded that in the brain of EL mice, pro-inflammatory cytokines are increased progressively and periodically in association with the development and the seizure activity, respectively. A periodic increase of cytokines prior to the next seizure episode may play a role in triggering the ictal activity. Namely, alteration of region-specific cytokines may induce ictal activities from the interictal state. It is conceivable that inflammatory cytokines may work together with neuronal factors during epileptogenesis and in the transition from interictal to ictal state.Large-scale expression profiling using DNA microarrays enables identification of tissue-selective genes for which expression is considerably higher and/or lower in some tissues than in others. Among numerous possible methods, only two outlier-detection-based methods (an AIC-based method and Sprent’s non-parametric method) can treat equally various types of selective patterns, but they produce substantially different results. We investigated the performance of these two methods for different parameter settings and for a reduced number of samples. We focused on their ability to detect selective expression patterns robustly. We applied them to public microarray data collected from 36 normal human tissue samples and analyzed the effects of both changing the parameter settings and reducing the number of samples. The AIC-based method was more robust in both cases. The findings confirm that the use of the AIC-based method in the recently proposed ROKU method for detecting tissue-selective expression patterns is correct and that Sprent’s method is not suitable for ROKU.DNA microarray is a powerful tool in biomedical research. However, transcriptomic profiling using DNA microarray is subject to many variations including biological variability. To evaluate the different sources of variation in mRNA gene expression profiles, gene expression profiles were monitored using the Affymetrix RatTox U34 arrays in cultured primary hepatocytes derived from six rats over a 26 hour period at 6 time points (0h, 2h, 5h, 8h, 14h and 26h) with two replicate arrays at each time point for each animal. In addition, the impact of sample size on the variability of differentially expressed gene lists and the consistency of biological responses were also investigated. Excellent intra-animal reproducibility was obtained at all time points with 0 out of 370 present probe sets across all time points showing significant difference between the 2 replicate arrays (3-way ANOVA, p ≤ 0.0001). However, large inter-animal biological variation in mRNA expression profiles was observed with 337 out of 370 present probe sets showing significant differences among 6 animals (3-way ANOVA, p ≤ 0.05). Principal Component Analysis (PCA) revealed that time effect (PC1) in this data set accounted for 47.4% of total variance indicating the dynamics of transcriptomics. The second and third largest effects came from animal difference, which accounted for 16.9% (PC2 and PC3) of the total variance. The reproducibility of gene lists and their functional classification was declined considerably when the sample size was decreased. Overall, our results strongly support that there is significant inter-animal variability in the time-course gene expression profiles, which is a confounding factor that must be carefully evaluated to correctly interpret microarray gene expression studies. The consistency of the gene lists and their biological functional classification are also sensitive to sample size with the reproducibility decreasing considerably under small sample size.Corticosteroids (CS) regulate many enzymes at both mRNA and protein levels. This study used microarrays to broadly assess regulation of various genes related to the greater urea cycle and employs pharmacokinetic/pharmacodynamic (PK/PD) modeling to quantitatively analyze and compare the temporal profiles of these genes during acute and chronic exposure to methylprednisolone (MPL). One group of adrenalectomized male Wistar rats received an intravenous bolus dose (50 mg/kg) of MPL, whereas a second group received MPL by a subcutaneous infusion (Alzet osmotic pumps) at a rate of 0.3 mg/kg/hr for seven days. The rats were sacrificed at various time points over 72 hours (acute) or 168 hours (chronic) and livers were harvested. Total RNA was extracted and Affymetrix® gene chips (RG_U34A for acute and RAE 230A for chronic) were used to identify genes regulated by CS. Besides five primary urea cycle enzymes, many other genes related to the urea cycle showed substantial changes in mRNA expression. Some genes that were simply up- or down-regulated after acute MPL showed complex biphasic patterns upon chronic infusion indicating involvement of secondary regulation. For the simplest patterns, indirect response models were used to describe the nuclear steroid-bound receptor mediated increase or decrease in gene transcription (e.g. tyrosine aminotransferase, glucocorticoid receptor). For the biphasic profiles, involvement of a secondary biosignal was assumed (e.g. ornithine decarboxylase, CCAAT/enhancer binding protein) and more complex models were derived. Microarrays were used successfully to explore CS effects on various urea cycle enzyme genes. PD models presented in this report describe testable hypotheses regarding molecular mechanisms and quantitatively characterize the direct or indirect regulation of various genes by CS.Genes mostly interact with each other to form transcriptional modules for performing single or multiple functions. It is important to unravel such transcriptional modules and to determine how disturbances in them may lead to disease. Here, we propose a non-negative independent component analysis (nICA) approach for transcriptional module discovery. nICA method utilizes the non-negativity constraint to enforce the independence of biological processes within the participated genes. In such, nICA decomposes the observed gene expression into positive independent components, which fits better to the reality of corresponding putative biological processes. In conjunction with nICA modeling, visual statistical data analyzer (VISDA) is applied to group genes into modules in latent variable space. We demonstrate the usefulness of the approach through the identification of composite modules from yeast data and the discovery of pathway modules in muscle regeneration.Skin irritation is a complex phenomenon, and keratinocytes play an important role in it. We have recently characterized the expression and protective role of adipose differentiation related protein (ADRP) in skin irritation. In particular, ADRP expression is induced to recover functional cell membrane following the cell damage caused by skin irritants. The purpose of this study was to characterize in a human keratinocyte cells line (NCTC 2544) the biochemical events that lead to ADRP expression following SDS treatment, and in particular, to investigate the role of transcription factor SP-1. Analysis of ADRP promoter region revealed the presence of a potential binding site for the transcription factor SP-1 close to the start site. Evaluated by measuring the DNA binding activity, we found that SDS induced a dose and time related SP-1 activation, which was correlated with SDS-induced ADRP mRNA expression. Furthermore, SDS-induced SP-1 activation, ADRP mRNA expression and lipid droplets accumulation could be modulated by mithramycin A, an antibiotic that selectively binds to the GC box preventing SP-1 binding and gene expression. This demonstrated that SDS-induced ADRP expression was mediated in part through the transcription factor SP-1. In addition, SDS-induced SP-1 activation and ADRP expression could be modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP-induction. Thus, every time an irritant perturbs the membrane barrier, it renders the membrane leaky and allows extracellular calcium to enter the cells, an event that provides the upstream mechanisms initiating the signaling cascade that triggers the activation of SP-1 and culminates in the enhancement of ADRP expression, which helps to restore the normal homeostasis and ultimately repairs the to membrane.Intergenic repeat units of 127-bp (RU-1) and 168-bp (RU-2), as well as a newly-found class of 103-bp (RU-3), represent small mobile sequences in enterobacterial genomes present in multiple intergenic regions. These repeat sequences display similarities to eukaryotic miniature inverted-repeat transposable elements (MITE). The RU mobile elements have not been reported to encode amino acid sequences. An in silico approach was used to scan genomes for location of repeat units. RU sequences are found to have open reading frames, which are present in annotated gene loci whereby the RU amino acid sequence is maintained. Gene loci that display repeat units include those that encode large proteins which are part of super families that carry conserved domains and those that carry predicted motifs such as signal peptide sequences and transmembrane domains. A putative exported protein in Y. pestis and a phylogenetically conserved putative inner membrane protein in Salmonella species represent some of the more interesting constructs. We hypothesize that a major outcome of RU open reading frame fusions is the evolutionary emergence of new proteins.In recent years it has become clear that carcinogenesis is a complex process, both at the molecular and cellular levels. Understanding the origins, growth and spread of cancer, therefore requires an integrated or system-wide approach. Computational systems biology is an emerging sub-discipline in systems biology that utilizes the wealth of data from genomic, proteomic and metabolomic studies to build computer simulations of intra and intercellular processes. Several useful descriptive and predictive models of the origin, growth and spread of cancers have been developed in an effort to better understand the disease and potential therapeutic approaches. In this review we describe and assess the practical and theoretical underpinnings of commonly-used modeling approaches, including ordinary and partial differential equations, petri nets, cellular automata, agent based models and hybrid systems. A number of computer-based formalisms have been implemented to improve the accessibility of the various approaches to researchers whose primary interest lies outside of model development. We discuss several of these and describe how they have led to novel insights into tumor genesis, growth, apoptosis, vascularization and therapy.

Mathematical Assessment of Properties of Precursor-Dependent Indirect Pharmacodynamic Response Models

Precursor-dependent indirect response (PDIDR) models may describe the tolerance and rebound phenomenon observed for many pharmacodynamic responses where these characteristics are manifested due to the depletion or accumulation of a physiological precursor or cofactor pool responsible for generating drug effects. The purpose of this report is to extend the concepts and applications of these models and to approximate responses and limiting conditions for very large doses of drugs. Asymptotic analysis was performed for qualitative determination of various parameters, such as maximum response (Rmax) and rebound (RBmax), time to maximum response and rebound (TRmax and TRBmax), and area under the effect and rebound curve (ABEC and ABRC) for large doses. Computer simulations were performed to assess the role of dose for both cases where drugs act either by depleting (Model V) or by blocking (Model VI) the endogenous precursor. Simulations showed that Rmax, RBmax, TRBmax, ABEC and ABRC increase with dose, eventually reaching a plateau when Dose/V is very large compared to the efficacy parameters (SC50 or IC50) of the drug. However, TRmax either increased or decreased with dose depending on various system and drug parameters. The limits for these parameters at large doses qualitatively determined by asymptotic analysis closely approximated the plateaus observed from the simulated curves. At large doses, the drug response could be approximated by a Bateman-like function for both Models V and VI. Qualitative analyses along with simulation studies provide a fundamental basis for understanding the temporal aspects of the PDIDR models especially at large doses to describe the tolerance and rebound phenomenon.

THU0178 Relationship Between NK Cell Count and Important Safety Events in Rheumatoid Arthritis Patients Treated with Tofacitinib

Background Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). Cytokines (eg interleukin [IL]-2, -4, -7, -15, -21) involved in lymphocyte development, function and homeostasis are known to signal through JAK. Objectives To characterise changes in natural killer (NK) cell counts following tofacitinib treatment and evaluate the relationship between NK counts and rates of infection and malignancy. Methods Lymphocyte subset data, enumerated by flow cytometric analyses, were pooled from 3 double-blind, placebo-controlled, Phase 2 (P2) studies (two monotherapy and one background methotrexate [MTX]) in MTX inadequate responders, and from a sub-study of an ongoing long-term extension (LTE) study (study is ongoing; database not locked). RA patients (pts) received tofacitinib (1 to 30 mg twice daily [BID]) or placebo for 6 to 24 weeks in P2 and tofacitinib 5 or 10 mg BID for 22 months (median) in LTE. 928 and 161 pts contributed NK cell data in P2 and LTE, respectively. Correlations between baseline or nadir NK cell count and serious infection (SIE, any infection that requires hospitalisation for treatment or parenteral antimicrobial therapy), herpes zoster (HZ) and malignancy (excluding non-melanoma skin cancer) were assessed by sub-dividing NK cell data into deciles and calculating incidence rate (events/100 pt-years) of adverse events for each decile. Results Following tofacitinib administration, NK cell counts decreased in a dose-dependent manner by Week 2 (Figure 1A). Median NK cells counts returned to baseline 2 to 6 weeks after treatment discontinuation (dc). At the 5 mg BID dose, pts with the largest (>90th percentile) decrease in NK cell counts recovered from ∼70% below baseline to ∼18% by 6 weeks after treatment dc (n=4). The estimated median decrease for 5 mg BID at Week 24 was ∼35%. Cross-sectional analyses in different groups of pts showed similar median NK cell count in the LTE (141 cells/μL) compared to pre-treatment baseline (135 cells/μL). No clear association between baseline or nadir NK cell counts (predominantly collected within first 6 months of treatment) and the incidence of SIE, HZ or malignancy (Figure 1B) was seen over the period of observation (median duration 1.9 years). Conclusions Tofacitinib treatment is associated with a dose-dependent decrease in NK cell counts. No association between baseline or nadir NK cell counts and the incidence of SIE, HZ or malignancy was observed. Long-term lymphocyte subset data are being collected in the ongoing LTE study to confirm these relationships. Acknowledgements Previously presented (van Vollenhoven R et al. Arthritis Rheum 2014; 66(11): S220 abs 508) and reproduced with permission from Arthritis and Rheumatism. All aspects of this work were funded by Pfizer Inc. Editorial support under the direction of the authors was provided by Amanda Pedder, of Complete Medical Communications, and funded by Pfizer Inc. Disclosure of Interest R. F. van Vollenhoven Grant/research support from: Pfizer, Consultant for: Pfizer, Y. Tanaka Consultant for: Abbvie, Chugai, Astellas, Takeda, Santen, Mitsubishi Tanabe, Pfizer, Janssen, Eisai, Daiichi-Sankyo, UCB Japan, GlaxoSmithKline, Bristol-Myers Squibb, Speakers bureau: Abbvie, Chugai, Astellas, Takeda, Santen, Mitsubishi Tanabe, Pfizer, Janssen, Eisai, Daiichi-Sankyo, UCB Japan, GlaxoSmithKline, Bristol-Myers Squibb, M. Lamba Shareholder of: Pfizer, Employee of: Pfizer, M. Collinge Shareholder of: Pfizer, Employee of: Pfizer, T. Hendrikx Employee of: Pfizer, T. Hirose Shareholder of: Pfizer, Employee of: Pfizer Japan, S. Toyoizumi Employee of: Pfizer Japan, A. Hazra Shareholder of: Pfizer, Employee of: Pfizer, S. Krishnaswami Shareholder of: Pfizer, Employee of: Pfizer

Emerging areas of research in the assessment of pharmacokinetics in patients with chronic kidney disease

Chronic kidney disease (CKD) has been shown to alter the pharmacokinetics of drugs that are eliminated not only via the renal pathway but also by nonrenal clearance and transport. Dosing recommendations in subjects with CKD have historically come from small pharmacokinetic (PK) studies, which have been insulated from the broader clinical development strategy. Opportunities for prospective strategic integration of both preclinical and clinical data on drug clearance mechanisms, model‐based approaches, and clinical knowledge of therapeutic index are therefore often missed in designing and analyzing the results of PK studies in subjects with CKD, and eventually providing dosing recommendations. These considerations are valuable in designing informative PK studies in subjects with CKD, as well as for guiding kidney function‐related inclusion/exclusion criteria in the broader clinical program and ultimately defining dosing guidelines that optimize benefit‐risk balance for these special patient populations based on all available data. This paper offers points to consider for drug developers to increase adoption of a contemporary multidisciplinary approach, which includes key considerations on study design and conduct, methodologies for analysis (population PK and physiologically based PK modeling), and a roadmap to interpret the effect of kidney function on the overall benefit‐risk profile of drugs in development.

THU0143 Pharmacokinetics, Bioavailability and Safety of A Modified Release Once Daily Formulation of Tofacitinib in Healthy Volunteers

Background Tofacitinib is a novel, oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The efficacy and safety of an immediate-release (IR) formulation of tofacitinib, dosed twice daily (BID), has been assessed in patients with active moderate to severe RA. To facilitate once daily (QD) dosing, a novel modified-release (MR) formulation has been designed to achieve comparability of key systemic exposure parameters. Objectives To compare the extent of exposure between a single dose of tofacitinib MR 11 mg vs an IR 2x5 mg dose in healthy volunteers (HV). Methods This was a randomised, open label, 2-way cross-over study conducted in 26 HV. Following an overnight fast, HV were randomised to receive either a single dose of MR 11 mg (MR; test) or IR 2 x 5 mg (IR; reference). Treatments were separated by a 72-hour (h) washout. Pharmacokinetic (PK) parameters were calculated using non-compartmental analyses. The primary endpoint was extent of tofacitinib exposure, measured as area under the concentration-time curve from time zero extrapolated to infinite time (AUCinf). A mixed-effects model was used to generate adjusted geometric mean ratios (MR/IR) and 90% confidence intervals (CIs). The steady-state (SS) profiles of tofacitinib MR and IR were predicted using single-dose data from this study. Results All 26 HV completed the study and were included in the analyses. The study population had a mean age of 33.6 years, a mean body weight of 77.5 kg, and was 19% female. For the MR and IR formulations, geometric mean AUCinf (ng*h/mL) was 297.5 and 286.3, respectively, resulting in an MR/IR ratio of 103.91% (90% CI: 100.49%, 107.45%). Maximum plasma concentration (Cmax; ng/mL) adjusted for formulation was 40.75 and 44.10 for MR and IR, respectively, resulting in an MR/IR ratio of 92.40% (90% CI: 84.99%, 100.45%). For both parameters, 90% CI values were wholly contained within the 80–125% range of bioequivalence. Mean terminal half-life was 5.71 h and 3.41 h for MR and IR formulations, respectively. The most common adverse events (AEs) were nausea, abdominal pain, back pain and headache. The incidence of AEs was similar between treatment groups and no serious AEs were reported. Predictions following SS dosing indicate similar time above JAK1/3 half maximal inhibitory concentration signalling thresholds and similar AUC, peak concentration and minimum concentration values between MR and IR formulations. Conclusions This study demonstrates the single dose equivalence of AUCinf and Cmax of the MR and IR formulations of tofacitinib. Single doses of both formulations were well tolerated. This novel MR formulation of tofacitinib facilitates an opportunity to enable QD dosing, while maintaining systemic drug concentrations similar to the IR formulation (administered BID). Multiple-dose studies will be conducted to confirm the predictions of the SS PK profile and demonstrate equivalence between formulations following SS dosing. Acknowledgements This study was sponsored by Pfizer Inc. Pfizer personnel were involved in protocol development and data analysis. Editorial support was provided by Claire Cridland of CMC and funded by Pfizer Inc. Disclosure of Interest : M. Lamba Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, R. Wang Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, T. Fletcher Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, C. Alvey Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, A. Hazra Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, J. Kushner Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, J. Larmann Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, T. Stock Shareholder of: Pfizer Inc, Employee of: Pfizer Inc DOI 10.1136/annrheumdis-2014-eular.1521

Reversibility of peripheral blood leukocyte phenotypic and functional changes after exposure to and withdrawal from tofacitinib, a Janus kinase inhibitor, in healthy volunteers

This study evaluated the short-term effects of tofacitinib treatment on peripheral blood leukocyte phenotype and function, and the reversibility of any such effects following treatment withdrawal in healthy volunteers. Cytomegalovirus (CMV)-seropositive subjects received oral tofacitinib 10 mg twice daily for 4 weeks and were followed for 4 weeks after drug withdrawal. There were slight increases in total lymphocyte and total T-cell counts during tofacitinib treatment, and B-cell counts increased by up to 26%. There were no significant changes in granulocyte or monocyte counts, or granulocyte function. Naïve and central memory T-cell counts increased during treatment, while all subsets of activated T cells were decreased by up to 69%. T-cell subsets other than effector memory cluster of differentiation (CD)4+, activated naïve CD4+ and effector CD8+ T-cell counts and B-cell counts, normalized 4 weeks after withdrawal. Following ex vivo activation, measures of CMV-specific T-cell responses, and antigen non-specific T-cell-mediated cytotoxicity and interferon (IFN)-γ production, decreased slightly. These T-cell functional changes were most pronounced at Day 15, partially normalized while still on tofacitinib and returned to baseline after drug withdrawal. Total natural killer (NK)-cell counts decreased by 33%, returning towards baseline after drug withdrawal. NK-cell function decreased during tofacitinib treatment, but without a consistent time course across measured parameters. However, markers of NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity and IFN-γ production were decreased up to 42% 1 month after drug withdrawal. CMV DNA was not detectable in whole blood, and there were no cases of herpes zoster reactivation. No new safety concerns arose. In conclusion, the effect of short-term tofacitinib treatment on leukocyte composition and function in healthy CMV+ volunteers is modest and largely reversible 4 weeks after withdrawal.

Exposure–response characterization of tofacitinib efficacy in moderate to severe ulcerative colitis: Results from a dose‐ranging phase 2 trial

Tofacitinib is an oral, small molecule JAK inhibitor being investigated for ulcerative colitis (UC). In a phase 2 dose‐ranging study, tofacitinib demonstrated efficacy vs. placebo as UC induction therapy. In this posthoc analysis, we aimed to compare tofacitinib dose and plasma concentration as predictors of efficacy and identify covariates that determined efficacy in patients with UC.