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![An electronic Magnetic Tweezer setup in a microfluidics channel. In this initial version of the proposed device Hall Effect sensors [48] were proposed as electronic detectors of vertical bead movement. DNA strands are attached as single molecules above single Hall Effect sensors placed within a microfluidics channel. The DNA with digoxigenin incorporated at one end is surface attached through a dithiobis(succinimidyl propionate)(DSP) to link anti-digoxigenin antibodies on a 100nm2 gold patch, which was located above the sensor, while the paramagnetic bead is attached to the DNA though a biotin-streptavidin linkage. An external magnetic field (not shown) holds the attached paramagnetic beads in a vertical position slightly stretching the DNA. The EcoR124I molecular motors (shown in green) are introduced in two stages – the darker green DNA-binding MTase followed by the light green motor component, which attaches to DNA adjacent to the enzyme [49] and translocates the DNA through the bound complex producing supercoiled DNA [50]. This translocation results in vertical movement of the DNA-bound bead, which generates an electrical signal in the Hall Effect sensor (represented by the red colouring) and this output, from a single motor, can be measured in the device and displayed as shown. Random release of the DNA results in resetting of the vertical position of the paramagnetic bead.](profile/Keith-Firman/publication/227341872/figure/fig1/AS:269552130981916@1441277644936/An-electronic-Magnetic-Tweezer-setup-in-a-microfluidics-channel-In-this-initial-version.png)
An electronic Magnetic Tweezer setup in a microfluidics channel. In this initial version of the proposed device Hall Effect sensors [48] were proposed as electronic detectors of vertical bead movement. DNA strands are attached as single molecules above single Hall Effect sensors placed within a microfluidics channel. The DNA with digoxigenin incorporated at one end is surface attached through a dithiobis(succinimidyl propionate)(DSP) to link anti-digoxigenin antibodies on a 100nm2 gold patch, which was located above the sensor, while the paramagnetic bead is attached to the DNA though a biotin-streptavidin linkage. An external magnetic field (not shown) holds the attached paramagnetic beads in a vertical position slightly stretching the DNA. The EcoR124I molecular motors (shown in green) are introduced in two stages – the darker green DNA-binding MTase followed by the light green motor component, which attaches to DNA adjacent to the enzyme [49] and translocates the DNA through the bound complex producing supercoiled DNA [50]. This translocation results in vertical movement of the DNA-bound bead, which generates an electrical signal in the Hall Effect sensor (represented by the red colouring) and this output, from a single motor, can be measured in the device and displayed as shown. Random release of the DNA results in resetting of the vertical position of the paramagnetic bead.
Source publication
This review describes a European-funded project in the area of Synthetic Biology. The project seeks to demonstrate the application of engineering techniques and methodologies to the design and construction of a biosensor for detecting drug-target interactions at the single-molecule level. Production of the proteins required for the system followed...
Context in source publication
Context 1
... enzyme via motion of a paramagnetic bead attached to one end of the tethered DNA substrate. The original concept for a new device was an electronic version of this Magnetic Tweezer setup, which would be attached to a microfluidics system and allow real time detection of molecular motor activity in a highly parallel, semi-automatic system (Fig. 1). This system could then be used to study a variety of DNA modifying enzymes, many of which are potential targets for drug development (e.g. helicases, topoisomerases and ...Similar publications
This paper demonstrates that it is possible to trap and release a super paramagnetic micro bead by fixing three super paramagnetic micro beads in a triangular array at the sensitive end of a micro cantilever, and by simply switching on/off an external magnetic field. To provide evidence of this principle we trap a micro bead that is attached to the...
The various DNA-protein interactions associated with the expression of genetic information involve double-stranded DNA (dsDNA) bending. Due to the importance of the formation of the dsDNA bending structure, dsDNA bending properties have long been investigated in the biophysics field. Conventionally, DNA bendability is characterized by innate averag...
We review the current status of various aspects of biopolymertranslocation through nanopores and the challenges and opportunities it offers. Much of the interest generated by nanopores arises from their potential application to third-generation cheap and fast genome sequencing. Although the ultimate goal of single-nucleotide identification has not...
Significance
In single-molecule imaging of the F 1 - ATPase rotation by nanoprobes, there are jumps in the angular position. Their distribution is used in a method for detecting hidden states in the transitions during rotation steps. Complementing stalling experiments, this method reveals, now in free rotation without magnetic tweezers, that the mo...
We present a statistical-mechanical model for stretched twisted double-helix DNA, where thermal fluctuations are treated explicitly from a Hamiltonian without using any scaling hypotheses. Our model applied to defect-free supercoiled DNA describes the coexistence of multiple plectoneme domains in long DNA molecules at physiological salt concentrati...
Citations
... Synthetic biology is helping to address previously unfeasible challenges the field of drug discovery. Progress on design of synthetic genetic circuits (Carbonell et al., 2014;Trosset and Carbonell, 2015) has opened the possibility of their use not only for production of drugs (Breitling and Takano, 2015) but also for the development of platforms for identification and validation of drug targets (Firman et al., 2012;Kasap et al., 2014) as well as for phenotypic cell-based screening approaches (Duportet et al., 2014) such as the screening for anti-cancer drugs presented in Gonzalez-Nicolini et al. (2004), that discriminates between proliferation competent and mitotically inert cells and eliminates preferentially neoplastic ones. With this purpose, (Gonzalez-Nicolini et al., 2004) engineered a transgenic CHO-K1-derived cell line to enable G1-specific growth arrest conditioned on the tetracycline responsive overexpression of the human cyclindependent kinase inhibitor p27. ...
Precision medicine in oncology needs to enhance its capabilities to match diagnostic and therapeutic technologies to individual patients. Synthetic biology streamlines the design and construction of functionalized devices through standardization and rational engineering of basic biological elements decoupled from their natural context. Remarkable improvements have opened the prospects for the availability of synthetic devices of enhanced mechanism clarity, robustness, sensitivity, as well as scalability and portability, which might bring new capabilities in precision cancer medicine implementations. In this review, we begin by presenting a brief overview of some of the major advances in the engineering of synthetic genetic circuits aimed to the control of gene expression and operating at the transcriptional, post-transcriptional/translational, and post-translational levels. We then focus on engineering synthetic circuits as an enabling methodology for the successful establishment of precision technologies in oncology. We describe significant advancements in our capabilities to tailor synthetic genetic circuits to specific applications in tumor diagnosis, tumor cell- and gene-based therapy, and drug delivery.
... Ligand discovery by REPSA [87] Glucose phosphate isomerase T. gondii Conceptual [100] T. brucei Functional assays with arabinose derivatives [101] Eimeria tenella In silico [102] Helicases Plasmodium sp. Functional assays [103] Hexose transporter Plasmodium sp. Functional assays with glucose derivatives [104][105][106] Babesia bovis Functional assays with glucose derivatives [107] Inosine monophosphate dehydrogenase C. parvum In vitro screening with transgenic T. gondii [108] Myosine A tail domain interacting protein ...
Introduction:
Despite the fact that diseases caused by protozoan parasites represent serious challenges for public health, animal production and welfare, only a limited panel of drugs has been marketed for clinical applications.
Areas covered:
Herein, the authors investigate two strategies, namely whole organism screening and target-based drug design. The present pharmacopoeia has resulted from whole organism screening, and the mode of action and targets of selected drugs are discussed. However, the more recent extensive genome sequencing efforts and the development of dry and wet lab genomics and proteomics that allow high-throughput screening of interactions between micromolecules and recombinant proteins has resulted in target-based drug design as the predominant focus in anti-parasitic drug development. Selected examples of target-based drug design studies are presented, and calcium-dependent protein kinases, important drug targets in apicomplexan parasites, are discussed in more detail.
Expert opinion:
Despite the enormous efforts in target-based drug development, this approach has not yet generated market-ready antiprotozoal drugs. However, whole-organism screening approaches, comprising of both in vitro and in vivo investigations, should not be disregarded. The repurposing of already approved and marketed drugs could be a suitable strategy to avoid fastidious approval procedures, especially in the case of neglected or veterinary parasitoses.
... Standard knock-out and knock-in experiments in cell or in mice are the common strategies for studying the influence of gene in cell-based or animal models. By using SB tools, expression of essential genes can be modulated by small molecules making synthetic cells a tool to identify drug-target interactions as presented, for example, by Firman et al. 143 144 in E. coli) enables us to insert mutations or incorporate a gene at any specified sites of the genome. CRISPR/Cas9 is a programmable DNA cleaving enzyme that uses guided RNA to identify the targeted region of the DNA. ...
Synthetic biology (SB) is an emerging discipline, which is slowly reorienting the field of drug discovery. For thousands of years, living organisms such as plants were the major source of human medicines. The difficulty in resynthesizing natural products, however, often turned pharmaceutical industries away from this rich source for human medicine. More recently, progress on transformation through genetic manipulation of biosynthetic units in microorganisms has opened the possibility of in-depth exploration of the large chemical space of natural products derivatives. Success of SB in drug synthesis culminated with the bioproduction of artemisinin by microorganisms, a tour de force in protein and metabolic engineering. Today, synthetic cells are not only used as biofactories but also used as cell-based screening platforms for both target-based and phenotypic-based approaches. Engineered genetic circuits in synthetic cells are also used to decipher disease mechanisms or drug mechanism of actions and to study cell-cell communication within bacteria consortia. This review presents latest developments of SB in the field of drug discovery, including some challenging issues such as drug resistance and drug toxicity.
... Compounds interfering with DNA-modifying enzymes can be screened using a single molecule sensor as demonstrated for topoisomerase I (Koster et al., 2007). This method is investigated in more detail in a recent EU project developing a nanotechnology-based device for the study of drug-target interactions at the limit of sensitivity (Firman et al., 2012). ...
Antiparasitic chemotherapy is an important issue for drug development. Traditionally, novel compounds with antiprotozoan activities have been identified by screening of compound libraries in high-throughput systems. More recently developed approaches employ target-based drug design supported by genomics and proteomics of protozoan parasites. In this chapter, the drug targets in protozoan parasites are reviewed. The gene-expression machinery has been among the first targets for antiparasitic drugs and is still under investigation as a target for novel compounds. Other targets include cytoskeletal proteins, proteins involved in intracellular signaling, membranes, and enzymes participating in intermediary metabolism. In apicomplexan parasites, the apicoplast is a suitable target for established and novel drugs. Some drugs act on multiple subcellular targets. Drugs with nitro groups generate free radicals under anaerobic growth conditions, and drugs with peroxide groups generate radicals under aerobic growth conditions, both affecting multiple cellular pathways. Mefloquine and thiazolides are presented as examples for antiprotozoan compounds with multiple (side) effects. The classic approach of drug discovery employing high-throughput physiological screenings followed by identification of drug targets has yielded the mainstream of current antiprotozoal drugs. Target-based drug design supported by genomics and proteomics of protozoan parasites has not produced any antiparasitic drug so far. The reason for this is discussed and a synthesis of both methods is proposed.
... Compounds interfering with DNA-modifying enzymes can be screened using a single molecule sensor as demonstrated for topoisomerase I (Koster et al., 2007). This method is investigated in more detail in a recent EU project developing a nanotechnology-based device for the study of drug-target interactions at the limit of sensitivity (Firman et al., 2012). ...
The increasing demand for novel anti-parasitic drugs due to resistance formation to well-established chemotherapeutically
important compounds has increased the demands for a better understanding of the mechanism(s) of action
of existing drugs and of drugs in development. While different approaches have been developed to identify the targets
and thus mode of action of anti-parasitic compounds, it has become clear that many drugs act not only on one, but possibly
several parasite molecules or even pathways. Ideally, these targets are not present in any cells of the host. In the case
of apicomplexan parasites, the unique apicoplast, provides a suitable target for compounds binding to DNA or ribosomal
RNA of prokaryotic origin. In the case of intracellular pathogens, a given drug might not only affect the pathogen by directly
acting on parasite-associated targets, but also indirectly, by altering the host cell physiology. This in turn could affect
the parasite development and lead to parasite death. In this review, we provide an overview of strategies for target
identification, and present examples of selected drug targets, ranging from proteins to nucleic acids to intermediary metabolism.
Ethnopharmacological relevance:
There are many plants of genus Piper which have been reported to induce antidepressant-like effects, Piper sarmentosum (PS) is one of them. PS is a Chinese herbal medicine and a traditional edible vegetable.
Materials and methods:
In the present study, the antidepressant-like effects of PS extracts and the ethyl acetate fraction of PS extracts (PSY) were assessed using the open field test (OFT), forced swimming test (FST), and tail suspension test (TST) in mice. Furthermore, we applied a 4 consecutive weeks of chronic unpredictable mild stress (CUMS) as a model of depression in rats, followed by a sucrose preference test. Then we examined the possible mechanisms of this action. The activity of the hypothalamic-pituitary-adrenal (HPA) axis was evaluated by detecting the serum corticosterone (CORT) concentrations, and the protein expression levels of brain-derived neurotrophic factor (BDNF), the phosphorylated form CREB and ERK1/2 were detected by qRT-PCR or Western blot.
Results:
The results showed that PS extracts (100, 200mg/kg) and PSY (12.5, 25, 50mg/kg) treatment produced antidepressant-like effects in mice similar to fluoxetine (20mg/kg), indicated by the reduced immobility time in the FST and TST, while both had no influence on the locomotor activity in the OFT. PSY treatment significantly increased sucrose preference and reduced serum CORT levels in CUMS rats. Moreover, PSY up-regulated BDNF protein levels, and increased CREB and ERK phosphorylation levels in the hippocampus on CUMS rats.
Conclusions:
These findings suggest that the antidepressant-like effects of PS extracts and PSY are mediated, at least in part, by modulating HPA axis, BDNF, CREB and ERK phosphorylation and expression in the hippocampus.
In past decades, nanostructured materials have shown promise of revolutionizing a wide number of areas of needed development, including theranostic, electronic, and photonic materials. Applications of these fields include yet wider range of specialties, all of which can be incorporated into sensor material design. This shall be the focus of my research laboratory; fundamental material properties and applications of nanomaterials to sensor design. I believe that my background in inorganic nanomaterial synthesis (for photovoltaics and catalysis), cell membrane-nanomaterial interactions, materials & surface science, sensor design, and management have prepared me for a promising career heading a research laboratory. This research laboratory shall span a spectrum of expertise, all of which shall fall under the umbrella of sensor design.
Teaching
In my years of being a graduate student, I have assisted teaching a number of courses and labs, and this fall will be assisting in the development of a chemical & biomolecular engineering elective course. Being the recipient of the University of Akron's Department of Chemical & Biomolecular Engineering Outstanding Teaching Assistant Award (Fall 2011), I understand that teaching graduate and undergraduate courses requires a certain level of dedication, which I plan to show through my outreach, elective development, and general teaching philosophy of “talking with” students instead of “talking at” students. I will apply this doctrine to both my course and research instructing, which I shall be presenting in further detail in this poster session.
Evan K. Wujcik, M.S., M.B.A. (LinkedIn Profile: http://www.linkedin.com/in/evankwujcik)
Advisor:Assistant Professor of Chemical & Biomolecular Engineering Chelsea N. Monty
Be on the lookout for my other 2012 AIChE oral/poster presentations:
* Oral presentation at the 2012 AIChE National Conference (November) in Pittsburgh, PA, U.S.A., Title: Diagnostic Sodium Ion Sensor for the Real-Time Screening and Diagnosis of Cystic Fibrosis
* Oral presentation at the 2012 AIChE National Conference (November) in Pittsburgh, PA, U.S.A., Title: Membrane Preconcentration Technique for the Colorimetric Detection of Trihalomethanes in Water
* Poster presentation at the 2012 AIChE National Conference (November) in Pittsburgh, PA, U.S.A., Title: Diagnostic Na+ Sensor for the Real-Time Screening and Diagnosis of Cystic Fibrosis
One fascinating recent avenue of study in the field of synthetic biology is the creation of biomolecule-based computers. The main components of a computing device consist of an arithmetic logic unit, the control unit, memory, and the input and output devices. Boolean logic gates are at the core of the operational machinery of these parts, hence to make biocomputers a reality, biomolecular logic gates become a necessity. Indeed, with the advent of more sophisticated biological tools, both nucleic acid- and protein-based logic systems have been generated. These devices function in the context of either test tubes or living cells and yield highly specific outputs given a set of inputs. In this review, we discuss various types of biomolecular logic gates that have been synthesized, with particular emphasis on recent developments that promise increased complexity of logic gate circuitry, improved computational speed, and potential clinical applications.
A simple and dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with dual-bioactive [nuclear factor-κB (NF-κB) and β2 -adrenergic receptor] luciferase reporter assay systems was developed to rapidly characterize the chemical structure of various bioactive compounds of TCM preparations. Chuanbeipipa dropping pills, a traditional Chinese medicine preparation used for the clinical therapy of chronic obstructive lung disease and cough caused by bronchial catarrh, was analyzed with this method. Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and β2 -adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time-of-flight mass spectrometry data. One β2 -adrenergic receptor agonist (ephedrine) and two structural types of NF-κB inhibitors (platycosides derivatives and ursolic acid derivatives) were characterized. Platycodin D3 and E were considered new NF-κB inhibitors. Further cytokine and chemokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. Compared with conventional fingerprints, activity-integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that multiple bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential multi-target lead compounds in complex herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.
