Patent Application
20190025297
A portion of the disclosure of this patent document may contain material, which is subject to copyright protection. Certain marks referenced herein may be common law or registered trademarks of the applicant, the assignee or third parties affiliated or unaffiliated with the applicant or the assignee. Use of these marks is for providing an enabling disclosure by way of example and shall not be construed to exclusively limit the scope of the disclosed subject matter to material associated with such marks.
The present disclosure pertains to next generation tools for the study of biological signaling processes and networks in living biological cells, and in particular to laboratory tools, methods, microscale sensors, microscale instrumentation, microfluidic, computerized instrumentation, computer simulation, and computer analysis tools relating to the study, analysis, and modeling of biological cell signaling. Aspects of the present application can also be readily used or adapted for the study, analysis, and modeling of biochemical processes and pathways for metabolism and gene regulation as well as laboratory or industrial activities pertaining to confined-environment chemistry, intercalation chemistry, chemical reactions in constrained systems, molecular encapsulation, and aspects of host-guest chemistry and the synthesis and study of meta-materials.
Material related to the topic of this patent application is provided in three earlier pending patent applications by one of the present inventors, specifically U.S. patent application Ser. No. 14/216,420, U.S. patent application Ser. No. 13/157,304, and paragraphs [00565] through [00569] of U.S. patent application Ser. No. 13/761,142. Pending patent application U.S. Ser. No. 13/761,142 additionally teaches throughout a variety of microscale sensors, microscale instrumentation, computer-controlled microfluidics, and computer-controlled instrumentation systems and methods useful to the present patent application. U.S. Pat. No. 8,594,848, also by the present inventor, teaches throughout additional computer-controlled microfluidics, microscale instrumentation, and computer-controlled instrumentation systems and methods useful to the present patent application. Allowed patent application U.S. Ser. No. 12/931,867, also by the present inventor, teaches throughout various microfluidic chemistry and further computer-controlled chemical processing systems and methods useful to the present patent application. U.S. Pat. Nos. 8,032,258 and 8,606,414 and pending U.S. patent application Ser. No. 13/251,288, also by the present inventor, teach controllable multichannel microfluidic chemical bus systems and methods useful to the present patent application.
Importance of Biochemical Signaling Pathways and Networks
Biochemical signaling networks play considerable roles in the cell cycle and most diseases, for example cancer. In addition to complicated multiple feedback loops and feedforward paths that regulate resulting dynamics, biochemical signaling networks include crosstalk among pathways, mechanical aspects of transport, conformation-dependent allosteric (state-dependent) reaction dynamics, enzyme recovery dynamics, exogenous regulatory controls, and many other exotic processes that are both critical to life processes and extremely complex. These render complex dynamics whose normal behavior, pathologies, and sensitivities are barely understood.
A more extensive analytical, quantitative, confirmative, and predictive understanding of biochemical signaling process, pathways, and networks is becoming increasingly indispensable. Comprehensive, accurate understanding and predictive modeling of biochemical signaling will play critical roles in future diagnostics and drug discovery, increasing replacing the roles of QSAR and other expensive and extensive approaches which, after a spectacular run of initial valuable productivity, have entirely failed to deliver new drugs. Further, the wide variability of side effects (both traceable and not traceable to the presence of undesirable enantiomers resultant from drug manufacturing processes) depends on the variability among patients' personal metabolic and signaling makeup. Additionally, many diseases such as cancer (lung cancer being a vibrant example) appear mechanistically to be optimally conquered via highly individualized analysis and therapies not unlike the notions of personalized or individualized medicine that are advocated for other sometimes controversial reasons. At the structural center of realistic approaches to all of these is again a comprehensive, accurate understanding and predictive modeling of biochemical signaling processes and signaling networks.
To date the approaches and results are at once both spectacular and primitive. A large number of signaling processes and pathways have been identified, with many new ones identified or conjectured every month. Many of these newly identified or conjectured signaling processes and pathways provide brand new understandings and explanations, and at times entirely unknown new phenomena. Experimental study of signaling processes and pathways have employed an impressive spectrum of technologies and methods but in many ways are limited to the introduction of instrumentation-observable markers into living cells, the testing of gross-effect biomarkers produced by living cells, laboratory-scale biochemical reaction studies, and probing of large molecule structure by biophysics and spectroscopic techniques. As powerful as these techniques have proven to be, they still suffer from immense limitation with regards to the types of behavior they can observe and characterize.
Examples of Current Biophysical Techniques Used to Study Protein-Protein Interactions
Some example techniques used to study protein-protein interactions include:
These are touched upon briefly in the subsections to follow.
Co-Immunoprecipitation (Co-IP)
Immunoprecipitation of intact protein also known as co-immunoprecipitation (Co-IP) is able to select an antibody that targets a known protein from a larger complex of proteins. The whole protein complex can be taken out which allows identification of unknown members of the complex. The proteins involved in the complex have to bind to each other tightly locking one member of the complex with an antibody. The concept of pulling protein complexes out of solution can also be referred to as “pull-down.” An example of the use of Co-IP in the study of the Hedgehog signaling pathway is provided in C. Tong; J. Jiang, “Using Immunoprecipitation to Study Protein—Protein Interactions in the Hedgehog-Signaling Pathway”. Methods In Molecular Biology Vol. 397, 2007, pp. 215-229 (available at http://link.springer.com/protocol/10.1007/978-1-59745-516-9_15).
Pull-Down Assay
Pull-down assay is a type of immunoprecipitation except that is the precipitation of target proteins instead of antibodies. It is an in vitro method capable of determining the physical interactions between proteins. This technique is usually used to confirm the existence of protein-protein interactions anticipated by other technique such as Co-IP but also to determine the existence of unknown protein-protein interactions. An example of the use of pull-down assay in the study of the Ras-Raf-MEK-ERK signaling pathway is provided in C. Song; W. Wang; M. Li; Y. Liu; D. Zheng, “Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway,” IUBMB Life. 2009 June; 61(6): pp. 685-92 (available at http://www.ncbi.nlm.nih.gov/pubmed/19472191).
Crosslinking Protein Interaction Analysis
Crosslinking reagents covalently bind protein-protein complexes together as they interact giving a method to measure them. Generally protein-protein interactions happen in short period of time. Crosslinking protein interaction analysis is able to freeze these brief contacts to study the proteins involved and the way they interact. An example of the use of crosslinking protein interaction analysis in the study of the MAPK signaling pathway is provided in W-K. Weng; L. Jarvis; T. W. LeBien, “Signaling through CD19 Activates Vav/Mitogen-Activated Protein Kinase Pathway and Induces Formation of a CD19/Vav/Phosphatidylinositol 3-Kinase Complex in Human B Cell Precursors,” Journal of Biological Chemistry 269:32514, 1994 (available at http://web.stanford.edu/˜wkweng/home.med/jbc/jbc.text.htm)
Label Transfer Protein Interaction Analysis
Label transfer tags proteins interacting with a protein of interest. Its technique blends with crosslinking technique to study protein-protein interactions. This method is enabling to uncover new interactions, endorse interactions proposed by other methods, as well as investigating the interface of interacting proteins. The label transfer technique can also detect weak or transient protein interactions regularly bypassing detection in co-immunoprecipitation and pull-down methods. An example of the use of label transfer protein interaction analysis in the study of the MAPK signaling pathway is provided in S. S. Andrews; Z. B. Hill; D. J. Maly, “Label Transfer Reagents to Probe p38 MAPK Binding Partners”, Chembiochem. Jan. 21, 2013; 14(2), pp. 209-216 (available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762675/#!po=86.1111)
Far-Western Blot Analysis
Far-Western blotting is a molecular biological method based on the technique of Western blotting to detect in vitro protein-protein interaction. In general, Western blotting identifies the protein of interest by using an antibody, while Far-Western blotting uses a non-antibody protein capable of binding to the protein of interest. Therefore, Western blotting is more useful for the detection of certain proteins, and Far-Western blotting to detect protein-protein interactions. An example of the use of far-western blot analysis in the study of the nuclear factor-kappaB signaling pathway is provided in X. Hu; O. Nesic-Taylor; J. Qiu; H. C. Rea; R. Fabian; D. K. Rassin; J. R. Perez-Polo, “Activation of nuclear factor-kappaB signaling pathway by interleukin-1 after hypoxia/ischemia in neonatal rat hippocampus and cortex,” J. Neurochem, 2005 April; 93(1), pp. 26-37 (available at http://www.ncbi.nlm.nih.gov/pubmed/15773902).
Analytical Ultracentrifugation (AUC)
Analytical Ultracentrifugation (AUC) applies the principles of centrifugal acceleration to separate components of samples given the shape and mass differences. This technique requires a rotor capable of spinning samples at speeds enough to create forces that are up to tens of thousands times greater than that of gravity. AUC is capable of performing analysis of the concentration of the samples during centrifugation by implementing light detection devices into the system. Using ultraviolet light absorption and/or interference optical refractive index sensitive system allows the operator to observe the sample concentration versus the axis of rotation. AUC primarily performs two types of analysis: sedimentation velocity and sedimentation equilibrium. An example of the use of AUC in the study of the Notch signaling pathway is provided in A. G. Allgood; D. Barrick, “Mapping the Deltex-binding surface on the notch ankyrin domain using analytical ultracentrifugation,” J Mol Biol. 2011 Nov. 25; 414(2), pp. 243-59, (available at http://www.ncbi.nlm.nih.gov/pubmed/22001695).
Flow Cytometry
Flow Cytometry is a technique in which cells are suspended in a fluid flowing through a focus of exciting light that is scattered. Usually labeled with fluorescent markers, the light is first absorbed then emitted at changed frequencies. The scattered or emitted light is measured by a sensor that is able to detect the size and molecular characteristics of individual cells. Flow cytometry enables tens of thousands of cells to be examined per minute and gathers the data to be processed by computer. An example of the use of flow cytometry in the study of the MAPK signaling pathway is provided in A. Mavropoulos; D. P. Bogdanos; C. Liaskos; T. Orfanidou; T. Simopoulou; E. Zafiriou; L. I. Sakkas; E. I. Rigopoulou, “Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases” Journal of Immunology Research Vol. 2014 (2014), p13 (available at http://www.hindawi.com/journals/jir/2014/671431/).
Fluorescence Spectroscopy
Fluorescence spectroscopy is an electromagnetic spectroscopy which analyzes fluorescence from a sample. It uses a beam of light (most commonly used is ultraviolet light), exciting the electrons in the molecules of compounds and causes emission of light. An example of the use of fluorescence spectroscopy in the study of the MAPK signaling pathway is provided in D. B. Slaughter; J. W. Schwartz; R. Li. “Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging,” Proc. Natl. Acad. Sci. Dec. 18, 2007; 104(51), pp. 20320-25. (available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2154429/#_ffn_sectitle).
Surface Plasmon Resonance (SPR)
Excited by incident light, surface plasmon resonance (SPR) is the accumulation of oscillation of electrons. It constitutes a powerful method and permits a real-time, label free detection of biomolecular interactions. SPR generates plasmons by hitting polarized light on an electrically conducting surface at the interface between two media causing reflected light's intensity diminution at a particular angle (resonance angle). Plasmons are proportional to the mass on a sensor surface. An example of the use of SPR in the study of the Hedgehog signaling pathway is provided in F. Zhang; J. S. McLellan; A. M. Ayala; D. J. Leahy; R. J. Linhardt, “Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway,” Biochemistry, 2007 Apr. 3; 46(13), pp. 3933-41 (available at http://www.ncbi.nlm.nih.gov/pubmed/17348690).
Calorimetry
Calorimetry is the amount of heat measurement associated with chemical reaction changes such as physical changes and phase transitions which are accompanied by heat transfer. Heat can be generated (exothermic), consumed (endothermic), or dissipated. An example of the use of calorimetry in the study of the RTK signaling pathway is provided in A. M. Spurches; H. J. Argiros; K. H. Lee; L. L. Haas; S. C. Pero; D. N. Krag; P. P. Roller; D. E. Wilcox; B. a. Lyions, “Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain,” J Mol Recognit. 2007 July-August; 20(4), pp. 245-52. (available at http://www.ncbi.nlm.nih.gov/pubmed/17705331)
Modeling of Signaling Networks
The scale, nonlinearities, and interconnected complexity of biochemical signaling networks have been initially addressed with attempts to modularize. A newly popular approach is the partition of complex signaling networks into small sections that are characterized as behaving like combinational and state-retaining logic circuits, i.e., so-called signaling “motifs.” However, the scale, interconnected complexity, and adaptively of biochemical signaling networks exceeds human comprehension and defies attempts to modularize. In many cases, conceptual modeling of biological signaling network has proven ineffective and at time deceptive as it is mentally impossible to juggle large pathways involving many components and because the mathematical behavior is too hard to intuitively characterize. In many ways the predictive modeling of signaling networks has exceeded what can be done without computer support.
Further, the explosion of genomic and proteomic laboratory analysis, bioinformatics, research publications, and “big data” analysis has created vast tomes of unverified signaling network models. Many of these are the result of automated statistical analysis of the results of automatic inferences drawn from automatic word searches on research publications text, be they speculative, unconfirmed, or robust verified.
Accurate Measurement of Rate Constants
To the extent that there is some valid degree of topological accuracy, reaction rates and many other parameters required for accurate analytical modeling are often unavailable. Further, the measurements of reaction rates and other parameters required for accurate analytical modeling is often made under artificial and inaccurate circumstances, for example not including the profound effects of molecular crowding, localized and confined reaction environments, or even being able to accurately control for other potentially interfering processes.
Summarizing
Summarizing some of the points thus far together with some additional remarks:
As to some of the powerful tools and methods employed to date for characterizing biochemical signaling processes, signaling network topologies, and signaling process modeling parameters,
In mechanical, electrical, optical, chemical, material science, and other forms of engineering there has been great success in creating abstract mathematical models (“CAD tools”) that accurate provide predictive analytical and design tools. These tools incorporate vast amounts of confirmed physical science, accurate physical measurements, and confirmed analytical models combined into a comprehensive framework, and virtually no contemporary suspension bridge, transportation vehicle, integrated circuit, consumer product, or chemical plant is designed without such tools. However, such tools have not yet become possible because of the shortcomings and shortages of needed confirmed physical science, needed accurate physical measurements, needed confirmed analytical models, and needed comprehensive framework.
Each of the five examples cited in the example spectrum of tools used in the study of biochemical signaling networks shown in
The present application is directed to creating new tools and methods of research in the areas of biochemical signaling, rate constant determination, protein interaction, modeling, disease processes, drug discovery, cell biology, and other applications.
For purposes of summarizing, certain aspects, advantages, and novel features are described herein. Not all such advantages may be achieved in accordance with any one particular embodiment. Thus, the disclosed subject matter may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages without achieving all advantages as may be taught or suggested herein.
The approach adapts the concept of a “breadboard” such as the electronic breadboards used in electronic circuitry prototyping and optical breadboards used in optical system R&D. The term breadboard comes from early experimental engineering practice where experimental electronics circuits were literally built on refined pieces of wood used for slicing bread, or pieces of wood suggestive of such, spreading the locations of electronic components across the surface of the wood and interconnecting the leads of the electronic components by connecting them to screws or nails put into the wood where needed. Often such a “breadboarded” electronic circuit was only a portion of a larger system. Today more formalized and structured breadboarding environments are available for electronics and optics, and typically these are richly interconnected to a number of external measurement devices, signal sources, and controlling devices. It is an analogy with many of these aspects that the term “breadboard” is used here. Other analogies could be made by viewing the present invention as a controlled (and/or instrumented) fluidics-based assay plate or a controlled (and/or instrumented) microfluidics version of a test tube.
The present invention includes approaches for the selective piecewise construction of replicas of portions of naturally-occurring biochemical processes and pathways for signaling, metabolism, and gene regulation.
These replicas can for example be implemented in microscale and nanoscale fluidic environments, can internally comprise one or more microscale and nanoscale fluidic environments, can be computer-controlled, and can comprise extensive monitoring via internal sensors, external sensors, and other types of instrumentation.
The replicas can for example additionally be arranged to include a variety of constituent species such as enzymes, other proteins, lipids, ions, peptides, and other materials, and the introduction of such constituent species can be provided under controlled conditions under controlled timing.
The replicas can for example additionally be arranged to include the controlled presence and controlled introduction of varying degrees of competitive species, drugs, and environmental influences (hormone, temperature, chemical, etc.).
Further, and in many cases importantly, the replicas can be arranged to include controlled degrees of substitute or representative molecular crowding.
The embodiments of the present application are directed to address problems in the study, analysis, and modeling of biological signaling processes and networks in living biological cells. Because of this and the “breadboard” abstraction adopted from electronics prototyping and optical R&D, for convenience embodiments of the present application will be referred to as a “biological signaling breadboard.” Embodiments of the present application can be readily used or adapted for the study, analysis, and modeling of biochemical processes and pathways for metabolism and gene regulation.
Embodiments of the biological signaling breadboard or features therein can comprise one or more microscale or nanoscale chemical reaction environments, each for example chemical reaction environment arranged to:
Embodiments of the biochemical signaling breadboard can further be configured to interface with a computing system performing one or more of the following functions:
In embodiments where there are more than one microscale or nanoscale chemical reaction environments, where advantageous, two or more of these microscale or nanoscale chemical reaction environments can be configured to comprise linking algorithms or other arrangements wherein received measurement information associated with one of the microscale or nanoscale chemical reaction environments is used for creating and timing the transmitting of control information directed to at least one other of the microscale or nanoscale chemical reaction environments.
In embodiments comprising at least one such linking algorithm, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a signaling cascade or portion of a signaling network.
In embodiments comprising a plurality of such linking algorithm, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a larger signaling cascade, portion of a signaling network, or entire signaling network.
In embodiments comprising a plurality of such linking algorithms, and wherein the linking introduces a feedback loop involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedback, a signaling cascade with feedback, a portion of a signaling network comprising feedback, or entire signaling network comprising feedback.
In embodiments comprising a plurality of such linking algorithms, and wherein the linking introduces a feedforward path involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedforward, a signaling cascade with feedforward, a portion of a signaling network comprising feedforward, or entire signaling network comprising feedforward paths.
Embodiments of the biochemical signaling breadboard can further be configured to include at least one fluidic interconnection between at least two of the microscale or nanoscale chemical reaction environments. In some embodiments the fluidic interconnection is simply gated on and off under computer control. In other embodiments, the fluidic interconnection is realized as one possible configuration of a computer-controlled reconfigurable fluidic interconnection network. In some embodiments the computer-controlled reconfigurable fluidic interconnection network can be implemented as or comprise aspects of a controllable multichannel microfluidic chemical bus such as that taught in pending U.S. Pat. Nos. 8,032,258 and 8,606,414 and pending U.S. patent application Ser. No. 13/251,288.
In embodiments comprising at least one such fluidic interconnection, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a signaling cascade or portion of a signaling network.
In embodiments comprising a plurality of such fluidic interconnections, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a larger signaling cascade, portion of a signaling network, or entire signaling network.
In embodiments comprising a plurality of such fluidic interconnections, and wherein the linking introduces a feedback loop involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedback, a signaling cascade with feedback, a portion of a signaling network comprising feedback, or entire signaling network comprising feedback.
In embodiments comprising a plurality of such fluidic interconnections, and wherein the linking introduces a feedforward path involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedforward, a signaling cascade with feedforward, a portion of a signaling network comprising feedforward, or entire signaling network comprising feedforward paths.
The above and other aspects, features and/or advantages of the present application may become more apparent upon consideration of the following description of embodiments taken in conjunction with the accompanying drawing figures, wherein:
In the following description, reference is made to the accompanying drawing figures which form a part hereof, and which show by way of illustration specific embodiments of the present application. It is to be understood by those of ordinary skill in this technological field that other embodiments may be utilized, and structural, electrical, as well as procedural changes may be made without departing from the scope of the present application.
In the following description, numerous specific details are set forth to provide a thorough description of various embodiments. Certain embodiments may be practiced without these specific details or with some variations in detail. In some instances, certain features are described in less detail so as not to obscure other aspects. The level of detail associated with each of the elements or features should not be construed to qualify the novelty or importance of one feature over the others.
The present disclosure describes approaches to additional tools for the study of biological cell signaling employing methods, microscale sensors, microscale instrumentation, microfluidic, and computerized instrumentation. The approach adapts the concept of a “breadboard” such as the electronic breadboards used in electronic circuitry prototyping and optical breadboards used in optical system R&D. Embodiments of the present application will be referred to as “biological signaling breadboards” for convenience. Results from and the environments comprised by embodiments of the present application can be used by and combined with computer simulation and computer analysis tools relating to the study, analysis, and modeling of biological cell signaling, for example the modeling environment described in U.S. Pat. No. 8,660,823 by the present inventor. For example, results from and the environments comprised by embodiments of the present application can be used by and combined with computer simulation and computer analysis tools in manners such as those described in pending U.S. patent application Ser. No. 13/157,304.
Among other things, the present disclosure describes approaches for selective piecewise construction of replicas of portions of naturally-occurring biochemical processes and pathways for signaling, metabolism, and gene regulation. These replicas can for example be implemented in microscale and nanoscale fluidic environments, can internally comprise one or more microscale and nanoscale fluidic environments, can be computer-controlled, and can comprise extensive monitoring via internal sensors, external sensors, and other types of instrumentation. The replicas can for example additionally be arranged to include a variety of constituent species such as enzymes, other proteins, lipids, ions, peptides, and other materials, and the introduction of such constituent species can be provided under controlled conditions under controlled timing. The replicas can for example additionally be arranged to include the controlled presence and controlled introduction of varying degrees of competitive species, drugs, and environmental influences (hormone, temperature, chemical, etc.). Further, the replicas can be arranged to include controlled degrees of substitute or representative molecular crowding.
In embodiments where there are more than one microscale or nanoscale chemical reaction environments, where advantageous, two or more of these microscale or nanoscale chemical reaction environments can be configured to comprise linking algorithms or other arrangements wherein received measurement information associated with one of the microscale or nanoscale chemical reaction environments is used for creating and timing the transmitting of control information directed to at least one other of the microscale or nanoscale chemical reaction environments.
The embodiments of the present application are directed to the study, analysis, and modeling of biological cell signaling, and also could be used for the study, analysis, and modeling of biochemical processes and pathways for metabolism and gene regulation. In that it can combine physical measurements with computer control driving by mathematical models,
Additionally, aspects of the present application can also be readily used or adapted for laboratory or industrial activities pertaining to confined-environment chemistry, intercalation chemistry, chemical reactions in constrained systems, molecular encapsulation, and aspects of host-guest chemistry and the synthesis and study of meta-materials.
Naturally-Occurring Biochemical Signaling Pathways
As described above, network topology graphs (the term “graph” here being the mathematical term meaning a collection of nodes and a collection of links or “edges” interconnecting these nodes) for naturally-occurring biochemical signaling pathways can be quite large and are always subject to change subject to new findings and their acceptance. As a representative example,
This and other pathways could be separated or segmented into interconnected smaller portions of the overall pathway with, for example, pair-wise interfaces among smaller portions of the overall pathway, these pair-wise interfaces implementing or supporting the interconnection. Several additional example pathways implicated in many human diseases, and detailed partition of these into smaller portions or segments, are provided in later in the discussion.
Leveraging this general approach provides a basis for creating a “biological signaling breadboard” as will be described. A larger pathway can be separated or segmented into interconnected smaller portions, at least one of which can be to some degree of controlled approximation be accurately emulated with some form of replica microscale and/or nanoscale fluidic implementation whose constituent species can be closely controlled and at least one aspect of whose biochemical behavior can be closely measured by some means with adequate accuracy. The control and measurement information can be interfaced with a computer that executes algorithms comprising for example one or more of a control process, control event-script, experiment, data recording, and mathematical model.
Example Partitions of Natural Biochemical Signaling Pathways
As an abstract example,
In an embodiment of the present invention or extensions of the present invention, the entire example representative pathway segment depicted in
In another embodiment of the present invention or extensions of the present invention, the entire example representative pathway segment depicted in
In yet another embodiment of the present invention or extensions of the present invention, the entire example representative pathway segment depicted in
Various steps in biochemical pathways such as those depicted in
Accordingly, implementation of reaction chambers provided with continent materials in (fluid and/or gas) media and supporting reactions of these constituents thus provide a means of creating “replica reaction” steps that can emulate steps in natural biochemical pathways. Further, with increasing levels of precision made possible by various aspects of the invention and its implementation, steps in biochemical pathways can be increasingly accurately emulated by these reaction chambers.
Measurement of the concentration or other properties of product or other target constituent material(s) created or affected in the reaction chamber can be used to monitor the “replica reaction” and serve as output of emulations of one or more steps in biochemical pathways emulated by these reaction chambers. Although in principle biochemical pathway reaction steps can be freely defined and reorganized, when the invention is configured so that a reaction chamber implements a single reaction step (which can involve one or more constituents and can, as appropriate, include inhibitors and catalysis as well as reactants), the invention will be said to be implementing a “stepwise” emulation, and when the invention is configured so that a reaction chamber implements a plurality of reaction steps in the same reaction chamber, the invention will be said to be implementing a “stepwise” emulation.
By implementing the reaction chambers and associated material transport with microfluidic and microfluidic-like technology, or with nanofluidic and nanofluidic-like technology, the quantities of constituent material (which can be rare, expensive, or require custom derivation or synthesis) can be kept small and a wide variety of microfluidic and nanofluidic sensing methods can be used. For example, sensing of reaction products, binding, molecular states, etc. in the recation chambers can be performed using optical, spectroscopic, and/or electrical techniques such as fluorescent markers, spin traps, chromaphores, antibodies markers, antibody-based bioFETs, enzyme-based bioFETs, chemFETs, other electrochemical sensors, etc., as well as a wide range of other current, evolving, emergent, and future techniques.
Further, with the proper use of for example, structured polymers, graphene and graphite structures, pillared clays and other types of structured clays, structured synthetic zeolites, inclusion compounds and cavity-containing supra-molecular compounds (for example cyclodextrins, calixarenes, and other organic host lattices), clathrates, liposomes, and various types of self-assembled supra-molecular structures, the simple reaction chambers can be structured to emulate various types of micro-reaction environments, for example as occur in cell mitochondria.
Regarding the example additional classes of processes depicted in the right side of
The present invention provides for two replica stoichiometric reactions that are separated by a shared non-stoichiometric process to simulate the effect of the non-stoichiometric process, for example employing an emulation-simulation hybrid as defined above.
Turning attention now largely to the implementation of stoichiometric replica reactions,
Regarding measurement, although some measurement techniques do not affect reaction products, constituents, or other reaction agents, many useful measurement methods will consume reaction products, constituents, or other reaction agents—for example sensing methods involving antibodies that bind to reaction products, constituents, or other reaction agents in order to detect them.
With the invention thus somewhat established, it is possible to compare it somewhat with in vitro and in vivo techniques used in studying the biochemistry and phenomenology of biochemical pathways as suggested in
Regarding the controlled application of constituents,
Implementing Replicas of Partitioned Biochemical Pathways
Embodiments of the biological signaling breadboard or features therein can comprise one or more microscale or nanoscale chemical reaction environments that can be used to implement a replica partitioned portion of larger biochemical processes and pathways for signaling, metabolism, and gene regulation. For example, such replica partitioned chemical reaction environments can be arranged to:
In many embodiments, replica partitioned chemical reaction environments can be arranged to provide outlets for removing the reaction products.
In some embodiments, replica partitioned chemical reaction environments can comprise at least one membrane.
Functionally Interconnecting Replica Partition Chemical Reaction Environments with a Computer and Algorithms for Chemical Reaction Environment Control and Chemical Reaction Environment Measurements
Embodiments of the biochemical signaling breadboard can further be configured to interface with a computing system performing one or more of the following functions:
Functionally Interconnecting Replica Partition Chemical Reaction Environments Using Computer Algorithms Invoking Chemical Reaction Environment Control Responsive to Chemical Reaction Environment Measurements
In embodiments where there are more than one microscale or nanoscale chemical reaction environments can be configured to comprise linking algorithms or other arrangements wherein received measurement information associated with one of the microscale or nanoscale chemical reaction environments is used for creating and timing the transmitting of control information directed to at least one other of the microscale or nanoscale chemical reaction environments.
In embodiments comprising at least one such linking algorithm, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a signaling cascade or portion of a signaling network.
In embodiments comprising a plurality of such linking algorithm, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a larger signaling cascade, portion of a signaling network, or entire signaling network.
In embodiments comprising a plurality of such linking algorithms, and wherein the linking introduces a feedback loop involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedback, a signaling cascade with feedback, a portion of a signaling network comprising feedback, or entire signaling network comprising feedback.
In embodiments comprising a plurality of such linking algorithms, and wherein the linking introduces a feedforward path involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedforward, a signaling cascade with feedforward, a portion of a signaling network comprising feedforward, or entire signaling network comprising feedforward paths.
In an embodiment, the two partitions depicted in
In an embodiment, the two partitions depicted in
Fluidically Interconnecting Replica Partition Chemical Reaction Environments
Embodiments of the biochemical signaling breadboard can further be configured to include at least one fluidic interconnection between at least two of the microscale or nanoscale chemical reaction environments. In some embodiments the fluidic interconnection is simply gated on and off under computer control. In other embodiments the fluidic interconnection is realized as one possible configuration of a computer-controlled reconfigurable fluidic interconnection network. In some embodiments the computer-controlled reconfigurable fluidic interconnection network can be implemented as, or comprise aspects of, a controllable multichannel microfluidic chemical bus such as that taught in pending U.S. Pat. Nos. 8,032,258 and 8,606,414 and pending U.S. patent application Ser. No. 13/251,288.
In embodiments comprising at least one such fluidic interconnection, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a signaling cascade or portion of a signaling network.
In embodiments comprising a plurality of such fluidic interconnection, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate a larger signaling cascade, portion of a signaling network, or entire signaling network.
In embodiments comprising a plurality of such fluidic interconnections, and wherein the linking introduces a feedback loop involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedback, a signaling cascade with feedback, a portion of a signaling network comprising feedback, or entire signaling network comprising feedback.
In embodiments comprising a plurality of such fluidic interconnections, and wherein the linking introduces a feedforward path involving two or more of the microscale or nanoscale chemical reaction environments, the combination of controlled and (sensor or instrument) monitored microscale or nanoscale chemical reaction environments together with the linking algorithms can be used to emulate signaling feedforward, a signaling cascade with feedforward, a portion of a signaling network comprising feedforward, or entire signaling network comprising feedforward paths.
Anomalous Diffusion Processes Resulting from Molecular Crowding and Confined/Constrained Biochemical Reaction Environments
An important aspect in a faithfully rendered replica chemical reaction environment is provision for the almost universally ignored need for the inclusion of the effects if not accurate emulation molecular crowding. Formulations, models, simulations, and emulations that do not include accurate provisions for molecular crowding provide incorrect molecular-transport statistical thermodynamics and can completely omit reaction processes that naturally occur in living cells. For example, the statistical thermodynamics for molecular-transport without consideration of molecular crowding are those of classical Brownian motion, which in turn provides statistically reproducibility of time-observables, ensemble averaging, long-time convergences, the interchangeable equivalence of time-averaging and ensemble-averaging, and other ergodic and related properties. In contrast, the dense macromolecular environment inside living cells induces molecular crowding that dramatically shifts the statistical thermodynamics for molecular-transport to at least two gross types of “anomalous diffusion” processes that absolutely do not share the many singular privileges of classical Brownian motion. For these, time-averaged observables are not reproducible, time-translation invariance are not respected, molecular-transport is not regularized, diffusion is jumpier and slower, and diffusion exponents differing significantly from the value of 1 provided by classical Brownian motion result, with implications as to whether molecular interactions involving reaction barriers and restricted alignments have sufficient time to occur. Anomalous diffusion processes for molecules traveling throughout the crowded molecular environment of a living cell can be more accuracy modeled with the Continuous Time Random Walk (“CTRW”), a non-ergodic random process demonstrating excellent agreement with a wide range of measurement subjects. Lattice models are employed for modeling diffusion-limited bimolecular reactions where a small number of reactants diffuse in a crowded environment among a much larger number of inert particles; these provide confirming related results from a colloidal physical chemistry viewpoint. Anomalous diffusion processes for molecules confined within a smaller spatial region, for example in the telomeres of a chromosome, a monomer in a polymer chain, or a molecule embedded in a membrane, is the Fractional Brownian Motion (“FBM”) process. In contrast to classical Brownian motion, diffusion exponents for appropriate CTRM models have values of ˜0.7 while diffusion exponents for appropriate FMB models can have value of ˜0.3. Accessible well-written accounts describing these and other recent appreciations of molecular crowding can be found in the article by E. Barkai, Y. Garini, and R. Metzler entitled “Strange Kinetics of Single Molecules in Living Cells” published in Physics Today, Vol. 65 No. 8, August 2012, pp. 29-35 and the chapter by A. Minton and G. Rivas entitled “Biochemical Reactions in the Crowded and Confined Physiological Environment: Physical Chemistry Meets Synthetic Biology” in The Minimal Cell: The Biophysics of Cell Compartment and the Origin of Cell Functionality, P. Luisi and P. Stano (eds.) 2011, ISBN 9048199433, pp. 73-89.
Controlled Molecular Crowding Emulation
Thus not only would a model or simulation that does not account for molecular crowding encounter accuracy shortcomings, but an emulated or other experimental environment for studying the existence of and rate constants for biochemical reactions that occur in cells which does not account for molecular crowding can be expected to have significant, if not profound, accuracy shortcomings.
Accordingly, the present application provides for emulating molecular crowding through the controlled introduction of molecular crowding constituents into a monitored microscale or nanoscale chemical reaction environment. Replica partition chemical reaction environments can be arranged to include controlled degrees of substitute or representative molecular crowding. For example,
Confined and Constrained Reaction Environments for Replica Partition Chemical Reaction Environments
Similarly, not only would a model or simulation that does not account for confined and constrained reaction environments encounter accuracy shortcomings, but an emulated or other experimental environment for studying the existence of and rate constants for biochemical reactions that occur in cells which does not account for confined and constrained reaction environments can be expected to have significant, if not profound, accuracy shortcomings.
Aforementioned examples of confined and constrained reaction environments inside living cells include telomeres of a chromosome, a monomer in a polymer chain, or a molecule embedded in a membrane, but many other examples are comprised in cell organelles. As mentioned earlier, these diffusion processes (exhibiting FBM behavior and having diffusion exponent value of for example ˜0.3) differ profoundly from classical Brownian motion (having diffusion exponent value of 1) and also differ significantly from the types of anomalous diffusion processes for molecules traveling throughout the crowded molecular environment of a living cell (exhibiting CTRW behavior and having diffusion exponent value of for example ˜0.7 and other relevant statistical differences from FBM behavior).
Accordingly, the present application provides for replica partition chemical reaction environments to internally comprise one or more confined or constrained reaction environments for emulation of processes occurring within confined reaction environments inside living cells.
Confined and constrained reaction environments can be applicably implemented in a wide variety of ways including but not restricted to structured polymers, graphene and graphite structures, pillared clays and other types of structured clays, structured synthetic zeolites, inclusion compounds and cavity-containing supra-molecular compounds (for example cyclodextrins, calixarenes, and other organic host lattices), clathrates, liposomes, and various types of self-assembled supra-molecular structures. The present application also provides for the use of ordinary and controlled micelles where practical, advantageous, and feasible.
Controlled Dispensing of Drug Constituents into Replica Partition Chemical Reaction Environments
The present application provides for the controlled dispensing of drug constituents into replica partition chemical reaction environments using the same general system architecture. For example,
Example Measurement Implementations
The present application provides for replica partition chemical reaction environments to internally comprise one or more of monitoring via internal sensors, monitoring via external sensors, and monitoring via other types of instrumentation such a microscopes, NMR systems, lensless optical microscopy and/or optical tomography such as that taught in pending U.S. patent application Ser. Nos. 12/817,107 and 14/105,108 by the present inventor, as well as a wide variety of other types of instruments.
As discussed earlier, internal sensing methods for the sensing of activities in the reaction chambers with internal sensors can be implemented in a wide variety of ways, for example including but not restricted to optical methods involving limited-influence fluorophore and chromophore probes on reactant molecules, bioFET sensing of reaction product concentrations, and a wide range of other current, evolving, emergent, and future techniques.
The Wnt Signaling Pathway plays an important role in the development of the embryo in many organisms, including humans. During embryonic development, the Wnt pathway plays a key role in body axis formation, especially the anteroposterior and dorsoventral axes. The formation of the body axes during early embryonic development is crucial in determining the overall outcome of the organism. Wnt is also involved in the formation of the dorsal-ventral of the central nervous system through axon guidance. Wnt proteins guide the axons of the spinal cord in an anterior-posterior direction.
The Wnt pathway is also involved in cell differentiation, cell proliferation, and cell migration. In cell differentiation, Wnt signaling promotes differentiation of stem cells into mesoderm and endoderm progenitor cells. These cells are further promoted to differentiate into even more specific cell types. Wnt signaling has been shown to a role in germ cell determination, hair follicle development, lung tissue development, ovary development, nephron development, gut tissue specification, and sex determination. In cell proliferation, the increased levels of β-catenin can initiate transcription of proteins such as cyclin D1, and cmyc, in which both controls the G1 to S phase in the cell cycle. During S phase, DNA replicates and leads to mitosis, which is responsible for cell proliferation. In cell migration, particularly during convergent extension, Wnt signaling aids to mediate the establishment of body axes, tissue formation, limb induction, and other processes during embryonic development.
The canonical Wnt signaling plays a role in the development of benign and malignant breast tumors. It was indicated due to the high levels of β-catenin in the nucleus and cytoplasm. Increased β-catenin is strongly correlated with poor prognosis in breast cancer patients. The over accumulation of β-catenin may be due to mutations in β-catenin, deficiencies in the destruction complex, overexpression of Wnt ligands, or loss of inhibitors.
Wnt signaling can also be involved in type II diabetes, due to its involvement in insulin sensitivity. Overexpression of Wnt5b could lead to increased rate of type II diabetes due to its involvement in adipogenesis, and since type II diabetes has high co-morbidity with obesity. Wnt signaling also activates mitochondrial biogenesis, which leads to an increase production of reactive oxygen species, known to induce DNA and cellular damage. This can cause the development of acute hepatic insulin resistance.
while the following table describes the “on” case.
The 5th column of the above table lists constituents of the pathway step described in that row of the table. These constituents would be used in a replica reaction executed by the present invention corresponding to the pathway step described in the associated row of the above table.
The named constituents involved the example contemporary understanding of the Wnt pathway represented by the above table can be produced in a laboratory or in some cases are commercially available or can be produced commercially in the future for use in the present invention and extensions of the present invention. As an example, the table below lists example sources for the constituents identified in the table above.
The Sonic Hedgehog signaling pathway plays a crucial role during development of the vertebrate limb. Sonic Hedgehog signaling promotes proliferation of adult stem cells from tissues, including hematopoietic cells, mammary, and neural stem cells. Mutation or disruption of hedgehog signaling during embryonic development can lead to severe developmental abnormalities, such as Holoprosencephaly, and Cyclopia.
Hedgehog pathway activation has been indicated and implicated in the development of cancers in organs, including the brain, lungs, mammary gland, prostate and skin. The most common form of cancer, Basal cell carcinoma, has the closest association with hedgehog signaling. Patients with this form of cancer have been identified to have mutations in Patched and Smoothened. Unusual activation of the hedgehog pathway leads to development of cancer through transformation of adult stem cells into cancer stem cells, which forms tumors.
while the following table describes the “on” case.
The 5th column of the above table lists constituents of the pathway step described in that row of the table. These constituents would be used in a replica reaction executed by the present invention corresponding to the pathway step described in the associated row of the above table.
The named constituents involved the example contemporary understanding of the Hedgehog pathway represented by the above table can be produced in a laboratory or in some cases are commercially available or can be produced commercially in the future for use in the present invention and extensions of the present invention. As an example, the table below lists example sources for the constituents identified in the table above.
The MAPK signaling pathway regulate many cellular activities, including proliferation, differentiation, survival, and death. MAPK signaling pathways have also been implemented in developing many human diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral schlerosis.
The ERK signaling pathway contributes in several tumor developments. The phosphorylation by ERK of proteins, including myosin light chain kinase, focal adhesion kinase, and paxillin induces cancer cell migration. In addition, the ERK pathway also promotes activation of matrix metalloproteinases, which in turn promotes degradation of extracellular matrix proteins followed up with tumor invasion. ERK signaling regulates activities and levels of the proapoptotic protein BIM, and anti-apoptotic protein, MCL-1, which in turn promotes the survival of cancer cells. Increased MCL-1 is also associated with poor prognosis and resistance to anticancer drugs.
Mutations taken place in the EGFR, the activator of the ERK pathway, occur in the lung and colorectal cancers.
The 5th column of the above table lists constituents of the pathway step described in that row of the table. These constituents would be used in a replica reaction executed by the present invention corresponding to the pathway step described in the associated row of the above table.
The named constituents involved the example contemporary understanding of the RTK pathway represented by the above table can be produced in a laboratory or in some cases are commercially available or can be produced commercially in the future for use in the present invention and extensions of the present invention. As an example, the table below lists example sources for the constituents identified in the table above.
The JAK-STAT signaling pathway mediates cellular responses to many of cytokines and growth factors, such as proliferation, differentiation, migration, and apoptosis. All of these responses are crucial for the development and homeostasis of hematopoietic cells. In mammalian systems, JAK/STAT signaling plays a key role in controlling organ or tissue size.
Disruption or mutation of the homeostatic process of JAK-STAT activation may lead to oncogenic results. Genetic abnormalities that induce active JAK-STAT signaling have been demonstrated in many hematologic malignancies. In addition, somatic JAK2 mutations have been identified in many patients with myeloproliferative neoplasm.
The 5th column of the above table lists constituents of the pathway step described in that row of the table. These constituents would be used in a replica reaction executed by the present invention corresponding to the pathway step described in the associated row of the above table.
The named constituents involved the example contemporary understanding of the JAK-STAT pathway represented by the above table can be produced in a laboratory or in some cases are commercially available or can be produced commercially in the future for use in the present invention and extensions of the present invention. As an example, the table below lists example sources for some of the constituents identified in the table above.
The Notch signaling pathway plays a crucial role in cell-cell communication, and regulation of embryonic development. Notch signaling is essential in the regulation of polarity and during left-right asymmetry determination. Mutation experiments have shown that, in the absence of Notch signaling, abnormal anterior-posterior polarity in somites occurs.
The Notch signaling is essential for sustaining NPCs in the developing brain. Loss-of-function mutations in the pathway cause precocious neuronal differentiation and NPC depletion. The NUMB protein is capable of antagonizing Notch effects, hindering the cell cycle and differentiation of NPCs. Notch signaling controls NPC self-renewal and cell fate specification.
Notch also plays an important role in cardiac development. Notch signaling regulates the atrioventricular boundary formation between the atrioventricular and the myocardium. Notch signaling is also involved in the ventricular endocardium, and is required for proper trabeculae development after myocardial specification. Notch signaling is also found to be involved in stages of pancreatic development, intestinal development, and bone development.
The 5th column of the above table lists constituents of the pathway step described in that row of the table. These constituents would be used in a replica reaction executed by the present invention corresponding to the pathway step described in the associated row of the above table.
The named constituents involved the example contemporary understanding of the Notch pathway represented by the above table can be produced in a laboratory or in some cases are commercially available or can be produced commercially in the future for use in the present invention and extensions of the present invention. As an example, the table below lists example sources for some of the constituents identified in the table above.
The terms “certain embodiments”, “an embodiment”, “embodiment”, “embodiments”, “the embodiment”, “the embodiments”, “one or more embodiments”, “some embodiments”, and “one embodiment” mean one or more (but not all) embodiments unless expressly specified otherwise. The terms “including”, “comprising”, “having” and variations thereof mean “including but not limited to”, unless expressly specified otherwise. The enumerated listing of items does not imply that any or all of the items are mutually exclusive, unless expressly specified otherwise. The terms “a”, “an” and “the” mean “one or more”, unless expressly specified otherwise.
The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the present application to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the present application and its practical applications, to thereby enable others skilled in the art to best utilize embodiments of the present application and various embodiments with various modifications as are suited to the particular use contemplated.
While aspects of the present application has been described in detail with reference to disclosed embodiments, various modifications within the scope of the present application will be apparent to those of ordinary skill in this technological field. It is to be appreciated that features described with respect to one embodiment typically can be applied to other embodiments.
The present application can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the present application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Although exemplary embodiments have been provided in detail, various changes, substitutions and alternations could be made thereto without departing from spirit and scope of the disclosed subject matter as defined by the appended claims. Variations described for the embodiments may be realized in any combination desirable for each particular application. Thus particular limitations and embodiment enhancements described herein, which may have particular advantages to a particular application, need not be used for all applications. Also, not all limitations need be implemented in methods, systems, and apparatuses including one or more concepts described with relation to the provided embodiments. Therefore, the present application properly is to be construed with reference to the claims.
This application is a Continuation-In-Part of pending U.S. patent application Ser. No. 14/216,420, filed Mar. 17, 2014, which claims benefit of priority from Provisional U.S. Patent application Ser. No. 61/802,127, filed Mar. 15, 2013, the contents of which are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61802127 | Mar 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14216420 | Mar 2014 | US |
| Child | 14485791 | US |
