ABSTRACT
Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated genes. Using primer extension analysis and reporter assays, we show the importance of the DPE in transcriptional regulation of TRF2 target genes. It was previously shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, we discovered that short TRF2-bound DNA oligos are enriched for Inr and DPE motifs. Taken together, our findings highlight the role of short TRF2 as a preferential core promoter regulator.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation , Telomeric Repeat Binding Protein 2/metabolism , Amino Acid Motifs , Animals , Cell Line , Cells, Cultured , Drosophila Proteins/genetics , Protein Binding , TATA Box , Telomeric Repeat Binding Protein 2/geneticsABSTRACT
Interest in polydimethylsiloxane (PDMS) microfluidic devices has grown dramatically in recent years, particularly in the context of improved performance lab-on-a-chip devices with decreasing channel size enabling more devices on ever smaller chips. As channels become smaller, the resistance to flow increases and the device structure must be able to withstand higher internal pressures. We report herein the fabrication of microstructured surfaces that promote water mobility independent of surface static wetting properties. The key tool in this approach is the growth of ZnO nanorods on the bottom face of the microfluidic device. We show that water flow in these devices is similar whether the textured nanorod-bearing surface is hydrophilic or superhydrophobic; that is, the device tolerates a wide range of surface wetting properties without changing the water flow within the device. This is not the case for smooth surfaces with different wetting properties, wherein hydrophilic surfaces result in slower flow rates. The ability to create monolayer-coated ZnO nanorods in a PDMS microfluidic device also allows for a variety of surface modifications within standard mass-produced devices. The inorganic ZnO nanorods can be coated with alkyl phosphonate monolayers. These monolayers can be used to convert hydrophilic surfaces into hydrophobic and even superhydrophobic surfaces that provide a platform for further surface modification. We also report photopatterned biomolecule immobilization within the channels on the monolayer-coated ZnO rods.
ABSTRACT
The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.
Subject(s)
Microfluidic Analytical Techniques/methods , Protein Array Analysis/methods , Proteomics/methods , Receptors, Virus/metabolism , Simian virus 40/metabolism , HumansABSTRACT
Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.
Subject(s)
Activating Transcription Factor 1/chemistry , JNK Mitogen-Activated Protein Kinases/chemistry , Microfluidics/methods , Proto-Oncogene Proteins c-fos/chemistry , Response Elements , Transcription Factor AP-1/chemistry , Activating Transcription Factor 1/genetics , Activating Transcription Factor 1/metabolism , Binding Sites , DNA/chemistry , DNA/genetics , DNA/metabolism , Gene Expression Regulation , High-Throughput Screening Assays , Humans , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Kinetics , Microfluidics/instrumentation , Molecular Sequence Data , Nucleotide Motifs , Protein Binding , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolismABSTRACT
Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.
Subject(s)
Microfluidics/methods , Proteomics/methods , HEK293 Cells , HeLa Cells , Humans , Lab-On-A-Chip Devices , Microfluidics/instrumentation , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Proteomics/instrumentation , Tyrosine/metabolism , UbiquitinationABSTRACT
Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.
Subject(s)
Carrier Proteins/metabolism , Caveolin 2/metabolism , Cofilin 1/metabolism , Microfluidic Analytical Techniques/methods , Nuclear Proteins/metabolism , Respiratory Syncytial Viruses/physiology , Viral Matrix Proteins/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Chlorocebus aethiops , Gene Library , HEK293 Cells , Humans , Open Reading Frames , Vero Cells , Virus ReplicationABSTRACT
To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Signal Transduction , Transcription Factors/genetics , Acclimatization , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Gene Expression , Indoleacetic Acids/metabolism , Oxidative Stress , Plant Growth Regulators/metabolism , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sodium Chloride/metabolism , Stress, Physiological , Transcription Factors/metabolism , Two-Hybrid System Techniques , Zinc FingersABSTRACT
Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.
Subject(s)
High-Throughput Screening Assays , Microfluidic Analytical Techniques/instrumentation , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/metabolism , Cell Membrane/metabolism , Gene Library , HEK293 Cells , Humans , Phosphorylation , Protein Array Analysis , Protein-Tyrosine Kinases/geneticsABSTRACT
Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell-based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , Neoplasms , Precision Medicine , Humans , MCF-7 Cells , Neoplasms/diagnosis , Neoplasms/drug therapy , Neoplasms/metabolismABSTRACT
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is essential for HCV maturation. The NS3/4A protease is a target for several HCV treatments and is a well-known target for HCV drug discovery. The protein is membrane associated and thus probably interacts with other membrane proteins. However, the vast majority of known NS3 host partners are soluble proteins rather than membrane proteins, most likely due to lack of appropriate platforms for their discovery. Utilization of an integrated microfluidics platform enables analysis of membrane proteins in their native form. We screened over 2800 membrane proteins for interaction with NS3 and 90 previously unknown interactions were identified. Of these, several proteins were selected for validation by co-immunoprecipitation and for NS3 proteolytic activity. Bearing in mind the considerable number of interactions formed, together with the popularity of NS3/4A protease as a drug target, it was striking to note its lack of proteolytic activity. Only a single protein, Neuregulin1, was observed to be cleaved, adding to the 3 known NS3/4A cleavage targets. Neuregulin1 participates in neural proliferation. Recent studies have shown its involvement in HCV infection and hepatocellular carcinoma. We showed that NS3/4A triggers an increase in neuregulin1 mRNA levels in HCV infected cells. Despite this increase, its protein concentration is decreased due to proteolytic cleavage. Additionally, its EGF-like domain levels were increased, possibly explaining the ErbB2 and EGFR upregulation in HCV infected cells. The newly discovered protein interactions may provide insights into HCV infection mechanisms and potentially provide new therapeutic targets against HCV.
Subject(s)
Membrane Proteins/chemistry , Microfluidic Analytical Techniques , Neuregulin-1/metabolism , Oligonucleotide Array Sequence Analysis , Peptide Hydrolases/metabolism , Viral Nonstructural Proteins/metabolism , Cell Line , Humans , Membrane Proteins/metabolism , Neuregulin-1/genetics , Peptide LibraryABSTRACT
Realization of bioinspired molecular machines that can perform many and diverse operations in response to external chemical commands is a major goal in nanotechnology, but current molecular machines respond to only a few sequential commands. Lack of effective methods for introduction and removal of command compounds and low efficiencies of the reactions involved are major reasons for the limited performance. We introduce here a user interface based on a microfluidics device and single-molecule fluorescence spectroscopy that allows efficient introduction and removal of chemical commands and enables detailed study of the reaction mechanisms involved in the operation of synthetic molecular machines. The microfluidics provided 64 consecutive DNA strand commands to a DNA-based motor system immobilized inside the microfluidics, driving a bipedal walker to perform 32 steps on a DNA origami track. The microfluidics enabled removal of redundant strands, resulting in a 6-fold increase in processivity relative to an identical motor operated without strand removal and significantly more operations than previously reported for user-controlled DNA nanomachines. In the motor operated without strand removal, redundant strands interfere with motor operation and reduce its performance. The microfluidics also enabled computer control of motor direction and speed. Furthermore, analysis of the reaction kinetics and motor performance in the absence of redundant strands, made possible by the microfluidics, enabled accurate modeling of the walker processivity. This enabled identification of dynamic boundaries and provided an explanation, based on the "trap state" mechanism, for why the motor did not perform an even larger number of steps. This understanding is very important for the development of future motors with significantly improved performance. Our universal interface enables two-way communication between user and molecular machine and, relying on concepts similar to that of solid-phase synthesis, removes limitations on the number of external stimuli. This interface, therefore, is an important step toward realization of reliable, processive, reproducible, and useful externally controlled DNA nanomachines.
Subject(s)
DNA/chemistry , Immobilized Nucleic Acids/chemistry , Microfluidic Analytical Techniques/instrumentation , Nanostructures/chemistry , Biomechanical Phenomena , Fluorescence Resonance Energy Transfer , Kinetics , Lab-On-A-Chip Devices , Nanotechnology , Single Molecule Imaging , Surface PropertiesABSTRACT
Integrated microfluidics is a sophisticated three-dimensional (multi layer) solution for high complexity serial or parallel processes. Fabrication of integrated microfluidic devices requires soft lithography and the stacking of thin-patterned PDMS layers. Precise layer alignment and bonding is crucial. There are no previously reported standards for alignment of the layers, which is mostly performed using uncontrolled processes with very low alignment success. As a result, integrated microfluidics is mostly used in academia rather than in the many potential industrial applications. We have designed and manufactured a semiautomatic Microfluidic Device Assembly System (µDAS) for full device production. µDAS comprises an electrooptic mechanical system consisting of four main parts: optical system, smart media holder (for PDMS), a micropositioning xyzθ system and a macropositioning XY mechanism. The use of the µDAS yielded valuable information regarding PDMS as the material for device fabrication, revealed previously unidentified errors, and enabled optimization of a robust fabrication process. In addition, we have demonstrated the utilization of the µDAS technology for fabrication of a complex 3 layered device with over 12 000 micromechanical valves and an array of 64 × 64 DNA spots on a glass substrate with high yield and high accuracy. We increased fabrication yield from 25% to about 85% with an average layer alignment error of just â¼4 µm. It also increased our protein expression yields from 80% to over 90%, allowing us to investigate more proteins per experiment. The µDAS has great potential to become a valuable tool for both advancing integrated microfluidics in academia and producing and applying microfluidic devices in the industry.
Subject(s)
Automation/methods , Equipment Design/methods , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/instrumentation , DimethylpolysiloxanesABSTRACT
Endogenous peptide antibiotics (termed also host-defense or antimicrobial peptides) are known as evolutionarily old components of innate immunity. They were found initially in invertebrates, but later on also in vertebrates, including humans. This secondary, chemical immune system provides organisms with a repertoire of small peptides that act against invasion (for both offensive and defensive purposes) by occasional and obligate pathogens. Each antimicrobial peptide has a broad but not identical spectrum of antimicrobial activity, predominantly against bacteria, providing the host maximum coverage against a rather broad spectrum of microbial organisms. Many of these peptides interact with the target cell membranes and increase their permeability, which results in cell lysis. A second important family includes lipopeptides. They are produced in bacteria and fungi during cultivation on various carbon sources, and possess a strong antifungal activity. Unfortunately, native lipopeptides are non-cell selective and therefore extremely toxic to mammalian cells. Whereas extensive studies have emerged on the requirements for a peptide to be antibacterial, very little is known concerning the parameters that contribute to antifungal activity. This review summarizes recent studies aimed to understand how antimicrobial peptides and lipopeptides select their target cell. This includes a new group of lipopeptides highly potent against pathogenic fungi and yeast. They are composed of inert cationic peptides conjugated to aliphatic acids with different lengths. Deep understanding of the molecular mechanisms underlying the differential cells specificity of these families of host defense molecule is required to meet the challenges imposed by the life-threatening infections.
Subject(s)
Bacterial Infections/drug therapy , Mycoses/drug therapy , Peptides/pharmacology , Humans , Microbial Sensitivity Tests , Microscopy, Electron , Peptides/therapeutic useABSTRACT
We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host.
Subject(s)
Microfluidic Analytical Techniques/instrumentation , Protein Interaction Mapping/methods , Respiratory Syncytial Virus Infections/metabolism , Respiratory Syncytial Virus, Human/physiology , Viral Matrix Proteins/metabolism , Gene Library , HEK293 Cells , Host-Pathogen Interactions , Humans , Printing, Three-Dimensional , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Protein Interaction Mapping/instrumentation , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/metabolismABSTRACT
Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.
Subject(s)
Microfluidics/methods , SELEX Aptamer Technique/methods , Transcription Factors/metabolism , Base Sequence , Binding Sites , Gene Library , Microarray Analysis , Nucleotide Motifs/genetics , Protein Binding , Reproducibility of Results , Sample SizeABSTRACT
DNA methylation is an epigenetic modification essential for normal development and maintenance of somatic biological functions. DNA methylation provides heritable, long-term chromatin regulation and the aberrant methylation pattern is associated with complex diseases including cancer. Discovering novel therapeutic targets demands development of high-throughput, sensitive and inexpensive screening platforms for libraries of chemical or biological matter involved in DNA methylation establishment and maintenance. Here, we present a universal, high-throughput, microfluidic-based fluorometric assay for studying DNA methylation in vitro. The enzymatic activity of bacterial HPAII DNA methyltransferase and its kinetic properties are measured using the assay (K(m)(DNA) = 5.8 nM, K(m)(SAM) = 9.8 nM and Kcat = 0.04 s(-1)). Using the same platform, we then demonstrate a two-step approach for high-throughput in vitro identification and characterization of small molecule inhibitors of methylation. The approach is examined using known non-nucleoside inhibitors, SGI-1027 and RG108, for which we measured IC50 of 4.5 µM and 87.5 nM, respectively. The dual role of the microfluidic-based methylation assay both for the quantitative characterization of enzymatic activity and high-throughput screening of non-nucleoside inhibitors coupled with quantitative characterization of the inhibition potential highlights the advantages of our system for epigenetic studies.
Subject(s)
Bacterial Proteins/chemistry , DNA Methylation , DNA-Cytosine Methylases/chemistry , DNA/chemistry , Microfluidic Analytical Techniques , Aminoquinolines/chemistry , Bacterial Proteins/antagonists & inhibitors , DNA-Cytosine Methylases/antagonists & inhibitors , Epigenesis, Genetic , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Phthalimides/chemistry , Pyrimidines/chemistry , Tryptophan/analogs & derivatives , Tryptophan/chemistryABSTRACT
Viral-host interactions represent potential drug targets for novel antiviral strategies (Flisiak et al., Hepatology, 2008, 47, 817-26). Hence, it is important to establish an adequate platform for identifying and analyzing such interactions. In this review, we discuss bottlenecks in conventional protein-protein interaction methodologies and present the contribution of innovative microfluidic-based technologies towards a solution to these problems with respect to viral-host proteomics.
Subject(s)
Microfluidic Analytical Techniques/methods , Proteins/metabolism , Viruses/metabolism , Hepacivirus/metabolism , High-Throughput Screening Assays , Host-Pathogen Interactions , Humans , Microfluidic Analytical Techniques/instrumentation , Protein Interaction Maps , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolismABSTRACT
Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein, protein-RNA or protein-DNA interactions. The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity. Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins, DNA, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.
Subject(s)
High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Proteins/chemistry , Animals , DNA/genetics , DNA/metabolism , Oligonucleotide Array Sequence Analysis , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Proteins/chemical synthesis , Proteins/genetics , Proteins/metabolism , RabbitsABSTRACT
Host-defense cationic antimicrobial peptides ( approximately 12-50 aa long) play an essential protective role in the innate immune system of all organisms. Lipopeptides, however, are produced only in bacteria and fungi during cultivation, and they are composed of specific lipophilic moieties attached to anionic peptides (six to seven amino acids). Here we report the following. (i) The attachment of an aliphatic chain to otherwise inert, cationic D,L tetrapeptides endows them with potent activity against various microorganisms including antibiotic resistance strains. (ii) Cell specificity is determined by the sequence of the short peptidic chain and the length of the aliphatic moiety. (iii) Despite the fact that the peptidic chains are very short, their mode of action involves permeation and disintegration of membranes, similar to that of many long antimicrobial peptides. Besides adding important information on the parameters necessary for host-defense lipopeptides to kill microorganisms, the simple composition of these lipopeptides and their diverse specificities should make them economically available, innate immunity-mimicking antimicrobial and antifungal compounds for various applications.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Antifungal Agents/chemical synthesis , Antifungal Agents/pharmacology , Lipoproteins/chemistry , Lipoproteins/pharmacology , Peptides/pharmacology , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemistry , Candida albicans/drug effects , Candida albicans/ultrastructure , Cell Membrane Permeability/drug effects , Escherichia coli/drug effects , Escherichia coli/ultrastructure , Microscopy, Electron, Transmission , Peptides/chemical synthesis , Peptides/chemistry , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/ultrastructureABSTRACT
The dramatically increased frequency of opportunistic fungal infections has prompted research to diversify the arsenal of antifungal agents. Antimicrobial peptides constitute a promising family for future antibiotics with a new mode of action. However, only a few are effective against fungal pathogens because of their ability to self-assemble. Recently, we showed that the conjugation of fatty acids to the potent antibacterial peptide magainin endowed it with antifungal activity concomitant with an increase in its oligomeric state in solution. To investigate whether a high potency of the parental peptide is prerequisite for antifungal activity, we conjugated undecanoic acid (UA) and palmitic acid (PA) to inactive diastereomers of magainin containing four d-amino acids ([D]-4-magainin), as well as to a weakly active diastereomeric lytic peptide containing Lys and Leu ([D]-K(5)L(7)). All lipopeptides gained potent activity toward Cryptococcus neoformans. Most importantly, [D]-K(5)L(7)-UA was highly potent against all microorganisms tested, including bacteria, yeast, and opportunistic fungi. All lipopeptides increased the permeability of Escherichia coli spheroplasts and intact C. neoformans, as well as their corresponding membranes, phosphatidylethanol (PE)/phosphatidylglycerol (PG) and phosphatidylcholine (PC)/PE/phosphatidylinositol (PI)/ergosterol, respectively. The extent of membrane-permeating activity correlated with their biological function, suggesting that the plasma membrane was one of their major targets. Circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that their mode of oligomerization in solution, structure, and organization in membranes have important roles regarding their antibacterial and antifungal activities. Together with the advantage of using diastereomers versus all l-amino acid peptides, this study paves the way to the design of a new group of potent antifungal peptides urgently needed to combat opportunistic fungal infection.