DEBay: A computational tool for deconvolution of quantitative PCR data for estimation of cell type-specific gene expression in a mixed population.

The expression of a gene is commonly estimated by quantitative PCR (qPCR) using RNA isolated from a large number of pooled cells. Such pooled samples often have subpopulations of cells with different levels of expression of the target gene. Estimation of gene expression from an ensemble of cells obscures the pattern of expression in different subpopulations. Physical separation of various subpopulations is a demanding task. We have developed a computational tool, Deconvolution of Ensemble through Bayes-approach (DEBay), to estimate cell type-specific gene expression from qPCR data of a mixed population. DEBay estimates Normalized Gene Expression Coefficient (NGEC), which is a relative measure of the expression of the target gene in each cell type in a population. NGEC has a direct algebraic correspondence with the normalized fold change in gene expression measured by qPCR. DEBay can deconvolute both time-dependent and -independent gene expression profiles. It uses the Bayesian method of model selection and parameter estimation. We have evaluated DEBay using synthetic and real experimental data. DEBay is implemented in Python. A GUI of DEBay and its source code are available for download at SourceForge (https://sourceforge.net/projects/debay).

Percolation in a reduced equilibrium model of planar cell polarity.

Planar cell polarity (PCP) is a biological phenomenon where a large number of cells get polarized and coordinatedly align in a plane. PCP is an example of self-organization through local and global interactions between cells. In this work, we have used a lattice-based spin model for PCP that mimics the alignment of cells through local interactions. We have investigated the equilibrium behavior of this model. In this model, alignment of cells leads to the formation of clusters of aligned cells, and such clustering exhibits percolation transition. Even though the alignment of a cell in this model depends upon its neighbors, finite-size scaling analysis shows that this model belongs to the universality class of simple two-dimensional random percolation.

Peptides derived from a short stretch of diphtheria toxin bind to heparin-binding epidermal growth factor-like growth factor.

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10-7 to 10-8 M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.

Morphological State Transition Dynamics in EGF-Induced Epithelial to Mesenchymal Transition.

Epithelial to Mesenchymal Transition (EMT) is a multi-state process. Here, we investigated phenotypic state transition dynamics of Epidermal Growth Factor (EGF)-induced EMT in a breast cancer cell line MDA-MB-468. We have defined phenotypic states of these cells in terms of their morphologies and have shown that these cells have three distinct morphological states-cobble, spindle, and circular. The spindle and circular states are the migratory phenotypes. Using quantitative image analysis and mathematical modeling, we have deciphered state transition trajectories in different experimental conditions. This analysis shows that the phenotypic state transition during EGF-induced EMT in these cells is reversible, and depends upon the dose of EGF and level of phosphorylation of the EGF receptor (EGFR). The dominant reversible state transition trajectory in this system was cobble to circular to spindle to cobble. We have observed that there exists an ultrasensitive on/off switch involving phospho-EGFR that decides the transition of cells in and out of the circular state. In general, our observations can be explained by the conventional quasi-potential landscape model for phenotypic state transition. As an alternative to this model, we have proposed a simpler discretized energy-level model to explain the observed state transition dynamics.

Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin.

Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells.

Autoregulation and heterogeneity in expression of human Cripto-1.

Cripto-1 (CR-1) is involved in various processes in embryonic development and cancer. Multiple pathways regulate CR-1 expression. Our present work demonstrates a possible positive feedback circuit where CR-1 induces its own expression. Using U-87 MG cells treated with exogenous CR-1, we show that such induction involves ALK4/SMAD2/3 pathway. Stochasticity in gene expression gives rise to heterogeneity in expression in genetically identical cells. Positive feedback increases such heterogeneity and often gives rise to two subpopulations of cells, having higher and lower expression of a gene. Using flow cytometry, we show that U-87 MG cells have a minuscule subpopulation with detectable expression of CR-1. Induction of CR-1 expression, by exogenous CR-1, increases the size of this CR-1 positive subpopulation. However, even at very high dose, most of the cells remain CR-1 negative. We show that population behavior of CR-1 induction has a signature similar to bimodal expression expected in a transcriptional circuit with positive feedback. We further show that treatment of U-87 MG cells with CR-1 leads to higher expression of drug efflux protein MDR-1 in the CR-1 positive subpopulation, indicating correlated induction of these two proteins. Positive feedback driven heterogeneity in expression of CR-1 may play crucial role in phenotypic diversification of cancer cells.

A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 µM and 0.46 µM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 µM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising.

Recombinant receptor-binding domain of diphtheria toxin increases the potency of curcumin by enhancing cellular uptake.

Diphtheria toxin (DT) binds to a specific cell surface receptor, gets internalized, and causes cytotoxicity through its catalytic domain. The toxicity of DT is used in several therapeutic molecules. Here, we have exploited the receptor-binding ability of DT to increase cellular uptake of curcumin, a hydrophobic molecule with low bioavailability and cellular uptake. We have expressed only the receptor-binding domain of DT (RDT) in Escherichia coli. Purified RDT binds to the receptor with an affinity equivalent to that of full-length DT. It also binds to curcumin forming a curcumin-RDT complex, and this increases the fluorescence intensity and fluorescence lifetime of curcumin. The curcumin-RDT complex binds to the receptor and associates with human glioblastoma cells (U-87 MG) expressing the receptor. The cellular uptake of curcumin is higher for the curcumin-RDT complex than curcumin alone. This increase in uptake enhances the antiproliferative effect of curcumin and induces apoptosis of these cells even at a lower dose.

Systems biology: a biologist's viewpoint.

The debate over reductionism and antireductionism in biology is very old. Even the systems approach in biology is more than five decades old. However, mainstream biology, particularly experimental biology, has broadly sidestepped those debates and ideas. Post-genome data explosion and development of high-throughput techniques led to resurfacing of those ideas and debates as a new incarnation called Systems Biology. Though experimental biologists have co-opted systems biology and hailed it as a paradigm shift, it is practiced in different shades and understood with divergent meanings. Biology has certain questions linked with organization of multiple components and processes. Often such questions involve multilevel systems. Here in this essay we argue that systems theory provides required framework and abstractions to explore those questions. We argue that systems biology should follow the logical and mathematical approach of systems theory and transmogrification of systems biology to mere collection of higher dimensional data must be avoided. Therefore, the questions that we ask and the priority of those questions should also change. Systems biology should focus on system-level properties and investigate complexity without shying away from it.

Human recombinant Cripto-1 increases doubling time and reduces proliferation of HeLa cells independent of pro-proliferation pathways.

Human oncofetal protein Cripto-1 (CR-1) is overexpressed in many types of cancers. CR-1 binds to cell surface Glypican-1 to activate Erk1/2 MAPK and Akt pathways leading to cell proliferation. However, we show that treatment with recombinant CR-1 reduces proliferation of HeLa cells by increasing the doubling time without triggering cell death or cell cycle arrest. Using a comparative study with U-87 MG cells, we show that the pro-proliferative pathway of CR-1 is not effective in HeLa cells due to lower expression of Glypican-1. Further we show that treatment with recombinant CR-1 increases PTEN in HeLa cells leading to downregulation of PI3K/Akt pathway. The anti-proliferative effect gets potentiated when the pro-proliferative pathway is blocked.

Photodynamic efficacy of chlorin p6: a pH dependent study in aqueous and lipid environment.

Photodynamic efficacy of chlorin p6, a potential candidate of photodynamic therapy (PDT), has been studied at pH 5.0, 6.0 and 7.6 in aqueous and lipid environment. Increased chlorin p6 mediated photodynamic bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a measure of singlet oxygen yield, was obtained at higher pH. Rate of photodynamic bleaching of RNO was also higher at higher pH and the rate decreased with lowering in pH of irradiated solution. Photodynamic oxidation of tryptophan was also found to be higher at higher pH. Diminished oxidation of RNO was obtained with decrease in pH of irradiated solution. Both, RNO bleaching and tryptophan oxidation was significantly reduced by sodium azide, a known quencher of singlet oxygen. At lower pH, chlorin p6 mediated photodynamic malondialdehyde (MDA) and lipid hydroperoxide formation in egg lecithin liposome was higher. At higher pH chlorin p6 was found to be photodynamically more effective in aqueous environment whereas at lower pH chlorin p6 was photodynamically more effective in hydrophobic environment.

Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen.

BACKGROUND: Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. RESULTS: Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. CONCLUSION: Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization.

Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein.

The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.

Pharmacokinetics and phototoxicity of purpurin-18 in human colon carcinoma cells using liposomes as delivery vehicles.

Pharmacokinetics and phototoxicity of purpurin-18 (Pp18) in human colon carcinoma cells (Colo-205) was studied using liposomes as delivery vehicles. Cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and neutral red uptake assay, and mode of cell death was assessed by the study of cell morphology and nuclear staining with Hoechst 33342-propidium iodide. Pp18 solubilized in dimethyl sulfoxide saline solution was observed to aggregate (Q-band absorption 740 nm), resulting in very poor cellular uptake. Pp18 incorporated in liposome remained in monomeric form (Q-band absorption 695 nm), but due to the presence of an anhydride ring in the molecule it readily yielded another photosensitizer, chlorin p6 (Q-band absorption 662 nm). Measurements at various pH showed that Pp18 in liposome was stable at acidic pH (6.5). Incubation of cells with 6.0 microM Pp18 in liposome at pH 6.5 showed a rapid cellular uptake. Spectrofluorometric measurements showed the presence of both Pp18 and chlorin p6, indicating conversion of some amount of Pp18 into chlorin p6 in the cells. Fluorescence microscopy revealed that the fluorescence was localized mainly in the cytoplasm, sparing the nucleus. Illumination of cells to white light after 4-h incubation with Pp18 liposome preparation was observed to lead to dose-dependent decrease in cell viability. At low irradiation time, cells displayed formation of plasma membrane blebs and micronuclei typical of apoptotic cell death. In contrast, at higher irradiation time, cell swelling and vacuolization in nucleus was observed, suggesting cell death due to necrosis. Irradiation with narrow bandwidth light showed that at low pH, the relative phototoxicity due to pp18 was higher than that due to chlorin p6. It is suggested that the pH-dependent conversion of pp18 to chlorin p6 can be exploited to increase PDT selectivity.

Generation and characterization of a high-affinity chimaeric antibody against hepatitis B surface antigen.

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V(H) (heavy-chain variable region) and V(L) (light-chain variable region) genes of this mouse antibody, and fused them with C(H)1 (heavy-chain constant domain 1) of human IgG1 and C(L) (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C(H)2 and C(H)3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate ( approximately 5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection.

The analysis of molecular signatures of antigen-driven affinity selection of B cells is of immense use in studies on normal and abnormal B cell development. Most of the published literature compares the expected and observed frequencies of replacement (R) and silent (S) mutations in the complementarity-determining regions (CDRs) and the framework regions (FRs) of antibody genes to identify the signature of antigenic selection. The basic assumption of this statistical method is that antigenic selection creates a bias for R mutations in the CDRs and for S mutations in the FRs. However, it has been argued that the differences in intrinsic mutability among different regions of an antibody gene can generate a statistically significant bias even in the absence of any antigenic selection. We have modified the existing statistical method to include the effects of intrinsic mutability of different regions of an antibody gene. We used this method to analyse sequences of several B cell-derived monoclonals against T-dependent antigens, T-independent antigens, clones derived from lymphoma and amyloidogenic clones. Our sequence analysis indicates that even after correcting for the intrinsic mutability of antibody genes, statistical parameters fail to reflect the role of antigen-driven affinity selection in maturation of many clones. We suggest that, contrary to the basic assumption of such statistical methods, selection can act both for and against R mutations in the CDR as well as in the FR regions. In addition we have identified different methodological difficulties in the current uses of such statistical analysis of antibody genes.

High affinity mouse-human chimeric Fab against hepatitis B surface antigen.

AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.

Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv.

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1 M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precursor revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation.