Systematic drug perturbations on cancer cells reveal diverse exit paths from proliferative state.

During a cell state transition, cells travel along trajectories in a gene expression state space. This dynamical systems framework complements the traditional concept of molecular pathways that drive cell phenotype switching. To expose the structure that hinders cancer cells from exiting robust proliferative state, we assessed the perturbation capacity of a drug library and identified 16 non-cytotoxic compounds that stimulate MCF7 breast cancer cells to exit from proliferative state to differentiated state. The transcriptome trajectories triggered by these drugs diverged, then converged. Chemical structures and drug targets of these compounds overlapped minimally. However, a network analysis of targeted pathways identified a core signaling pathway--indicating common stress-response and down-regulation of STAT1 before differentiation. This multi-trajectory analysis explores the cells' state transition with a multitude of perturbations in combination with traditional pathway analysis, leading to an encompassing picture of the dynamics of a therapeutically desired cell-state switching.

High-throughput and reliable protocols for animal microRNA library cloning.

MicroRNAs are short single-stranded RNA molecules (18-25 nucleotides). Because of their ability to silence gene expressions, they can be used to diagnose and treat tumors. Experimental construction of microRNA libraries was the most important step to identify microRNAs from animal tissues. Although there are many commercial kits with special protocols to construct microRNA libraries, this chapter provides the most reliable, high-throughput, and affordable protocols for microRNA library construction. The high-throughput capability of our protocols came from a double concentration (3 and 15%, thickness 1.5 mm) polyacrylamide gel electrophoresis (PAGE), which could directly extract microRNA-size RNAs from up to 400 µg total RNA (enough for two microRNA libraries). The reliability of our protocols was assured by a third PAGE, which selected PCR products of microRNA-size RNAs ligated with 5' and 3' linkers by a miRCat™ kit. Also, a MathCAD program was provided to automatically search short RNAs inserted between 5' and 3' linkers from thousands of sequencing text files.

Cloning and analysis of fetal ovary microRNAs in cattle.

Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation.

A simple mathematical model for electric cell-substrate impedance sensing with extended applications.

This paper presents a simple mathematical model to predict the impedance data acquired by electric cell-substrate impedance sensing (ECIS) at frequencies between 25 Hz and 60 kHz. With this model, the equivalent resistance (R) and capacitance (C) of biological samples adhered on gold surfaces could be more precisely measured at 4 kHz. ECIS applications were extended for real-time monitoring of living bacteria cultivated in Luria Bertani (LB) culture medium by two different approaches. In the former, we used a ferri/ferrocyanide redox couple in LB medium as an indicator for bacterial multiplication. Because the redox couple was toxic to some bacteria, we developed a second approach, in which l-cysteine self-assembled monolayers (SAM) on gold electrodes were used to detect living bacteria. The l-cysteine SAM could also be detected by ECIS. Unlike traditional impedance microbiological methods which need special culture media with low ions, our procedures significantly enhanced signal/noise ratios so bacteria could be detected in general purpose culture media. It was easy and convenient to obtain bacterial doubling times and evaluate the resistance of bacteria to antibiotics from ECIS spectra.

A microRNA repertoire for functional genome research in rainbow trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. miRNAs are most often identified through computational prediction from genome sequences. The rainbow trout genome sequence is not available yet, which does not allow miRNA prediction for this species which is of great economic interest for aquaculture and sport fisheries, and is a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. To identify miRNAs from rainbow trout, we constructed a miRNA library from a pool of nine somatic tissues. Analysis of the library identified 210 unique sequences representing 54 distinct miRNAs; 50 with conserved sequences matching previously identified miRNAs and four novel miRNAs. In addition, 13 miRNAs were computationally predicted from the rainbow trout transcriptome. Real-time PCR was used to measure miRNA expression patterns in adult somatic tissues and unfertilized eggs. The majority of the miRNAs showed characteristic tissue-specific expression patterns suggesting potential roles in maintaining tissue identity. Potential miRNA-target interactions were computationally predicted and single nucleotide polymorphisms (SNPs) were identified in the miRNAs and their target sites in the rainbow trout transcripts. The rainbow trout miRNAs identified and characterized in this study provide a new tool for functional genome research in salmonids. Tissue-specific miRNAs may serve as molecular markers, predictive of specific functional and diagnostic implications. The data on genetic polymorphisms in miRNA-target interactions is particularly useful for rainbow trout breeding programs.

Nonlabeled quartz crystal microbalance biosensor for bacterial detection using carbohydrate and lectin recognitions.

High percentages of harmful microbes or their secreting toxins bind to specific carbohydrate sequences on human cells at the recognition and attachment sites. A number of studies also show that lectins react with specific structures of bacteria and fungi. In this report, we take advantage of the fact that a high percentage of microorganisms have both carbohydrate and lectin binding pockets at their surface. We demonstrate here for the first time that a carbohydrate nonlabeled mass sensor in combination with lectin-bacterial O-antigen recognition can be used for detection of high molecular weight bacterial targets with remarkably high sensitivity and enhanced specificity. A functional mannose self-assembled monolayer in combination with lectin concanavalin A (Con A) was used as molecular recognition elements for the detection of Escherichia coli W1485 using a quartz crytsal microbalance (QCM) as a transducer. The multivalent binding of Con A to the E. coli surface O-antigen favors the strong adhesion of E. coli to the mannose-modified QCM surface by forming bridges between these two. As a result, the contact area between cell and QCM surface that increases leads to rigid and strong attachment. Therefore, it enhances the binding between E. coli and the mannose. Our results show a significant improvement of the sensitivity and specificity of the carbohydrate QCM biosensor with a experimental detection limit of a few hundred bacterial cells. The linear range is from 7.5 x 10(2) to 7.5 x 10(7) cells/mL, which is four decades wider than the mannose-alone QCM sensor. The change of damping resistances for E. coli adhesion experiments was no more than 1.4%, suggesting that the bacterial attachment was rigid, rather than a viscoelastic behavior. Little nonspecific binding was observed for Staphylococcus aureus and other proteins (fetal bovine serum, Erythrina cristagalli lectin). Our approach not only overcomes the challenges of applying QCM technology for bacterial detection but also increases the binding of bacteria to their carbohydrate receptor through bacterial surface binding lectins that significantly enhanced specificity and sensitivity of QCM biosensors. Combining carbohydrate and lectin recognition events with an appropriate QCM transducer can yield sensor devices highly suitable for the fast, reversible, and straightforward on-line screening and detection of bacteria in food, water, and clinical and biodefense areas.

Assessment of cytotoxicity by emerging impedance spectroscopy.

An on-line and continuous technique based on electric cell substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to toxicants. Mercury chloride (HgCl(2)), cadmium chloride (CdCl(2)), benzalkonium chloride (BAK), sodium arsenate (Na(2)HAsO(4)), and trinitrobenzene (TNB) were used as five test models. The first four chemicals serve as a model for acute toxicants, and TNB represents a model for long-term cytotoxicity effects. Adhesion, spreading, and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to toxicants led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide dynamic information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the adhesion, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.

Thermodynamic instability in supersaturated lysozyme solutions: effect of salt and role of concentration fluctuations.

Experimental and theoretical work has suggested that protein crystal nucleation can be affected by the separation of two metastable liquid phases with different local concentrations, or more specifically by critical density fluctuations. We measure the amplitude and correlation length of local concentration fluctuations by light scattering for supersaturated solutions of hen egg-white lysozyme (at pH 4.5 and at different NaCl concentrations, up to 7% w/v). By extrapolating the critical divergent behavior of concentration fluctuation amplitude versus temperature, we determine the spinodal line, that is the limit of stability. Cloud-point measurements are used to determine liquid-liquid coexistence, consistent with previous work. In the present work, which is an extensive study of off-critical fluctuations in supersaturated protein solution, we observe a nonclassical scaling divergent behavior of the correlation length of concentration fluctuations, thus suggesting that off-critical fluctuations may have a role in crystallization kinetics. To appropriately fit the spinodal data, an entropic term must be added to the van der Waals or to the adhesive hard-sphere model. We interpret this contribution as due to the salt-induced modulation of protein hydration.

On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS).

An on-line and continuous technique based on electric cell-substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to mercury chloride and 1,3,5-trinitrobenzene (TNB). Attachment, spreading and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to mercury chloride or TNB led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the attachment, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.

Assessment of cytotoxicity using electric cell-substrate impedance sensing: concentration and time response function approach.

This paper describes a simple and convenient method to measure the concentration and time response function f (C,t) of cells exposed to a toxicant by electric cell-substrate impedance sensing. Attachment and spreading of fibroblastic V79 cells cultured on small gold electrodes precoated with fibronectin were detected as electrical resistance changes. With this method, chemical cytotoxicity was easily screened by observing the response function of attached cells in the presence of inhibitor. The cytotoxicities of three test models, cadmium chloride, sodium arsenate, and benzalkonium chloride, were quantified by measuring the percentage inhibition as a function of the inhibitor concentration. The half-inhibition concentration, the required concentration to achieve 50% inhibition, derived from the response function agreed well with the results obtained using the standard neutral red assay.

An in-depth analysis of electric cell-substrate impedance sensing to study the attachment and spreading of mammalian cells.

The attachment and spreading of fibroblast cells on a gold surface coated with fibronectin or ovalbumin were studied by a modified electric cell-substrate impedance sensor. In this system, cells were cultured in a well, equipped with a detecting gold electrode (surface area of 0.057 mm2) and a gold counter electrode (18 mm2). Based on a comprehensive theoretical framework, the impedance of the electrode-electrolyte interface and a cell layer was precisely obtained for frequencies ranging from 1 to 10 kHz. Surface concentrations of the protein adsorbed on the gold surface were determined by a surface plasmon resonance biosensor. The resistance change of the electrode-electrolyte interface at 4 kHz increased linearly with the number of fibroblast cells attached on the detecting electrode. The slope of the linear relationship appeared to depend on the type of coating protein. As the surface area occupied by the cells was also proportional to the cell number, the resistance change was in turn proportional to the area covered by the cells.