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1.  Structural and functional protein network analyses predict novel signaling functions for rhodopsin 
Proteomic analyses, literature mining, and structural data were combined to generate an extensive signaling network linked to the visual G protein-coupled receptor rhodopsin. Network analysis suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking.
Using a shotgun proteomic approach, we identified the protein inventory of the light sensing outer segment of the mammalian photoreceptor.These data, combined with literature mining, structural modeling, and computational analysis, offer a comprehensive view of signal transduction downstream of the visual G protein-coupled receptor rhodopsin.The network suggests novel signaling branches downstream of rhodopsin to cytoskeleton dynamics and vesicular trafficking.The network serves as a basis for elucidating physiological principles of photoreceptor function and suggests potential disease-associated proteins.
Photoreceptor cells are neurons capable of converting light into electrical signals. The rod outer segment (ROS) region of the photoreceptor cells is a cellular structure made of a stack of around 800 closed membrane disks loaded with rhodopsin (Liang et al, 2003; Nickell et al, 2007). In disc membranes, rhodopsin arranges itself into paracrystalline dimer arrays, enabling optimal association with the heterotrimeric G protein transducin as well as additional regulatory components (Ciarkowski et al, 2005). Disruption of these highly regulated structures and processes by germline mutations is the cause of severe blinding diseases such as retinitis pigmentosa, macular degeneration, or congenital stationary night blindness (Berger et al, 2010).
Traditionally, signal transduction networks have been studied by combining biochemical and genetic experiments addressing the relations among a small number of components. More recently, large throughput experiments using different techniques like two hybrid or co-immunoprecipitation coupled to mass spectrometry have added a new level of complexity (Ito et al, 2001; Gavin et al, 2002, 2006; Ho et al, 2002; Rual et al, 2005; Stelzl et al, 2005). However, in these studies, space, time, and the fact that many interactions detected for a particular protein are not compatible, are not taken into consideration. Structural information can help discriminate between direct and indirect interactions and more importantly it can determine if two or more predicted partners of any given protein or complex can simultaneously bind a target or rather compete for the same interaction surface (Kim et al, 2006).
In this work, we build a functional and dynamic interaction network centered on rhodopsin on a systems level, using six steps: In step 1, we experimentally identified the proteomic inventory of the porcine ROS, and we compared our data set with a recent proteomic study from bovine ROS (Kwok et al, 2008). The union of the two data sets was defined as the ‘initial experimental ROS proteome'. After removal of contaminants and applying filtering methods, a ‘core ROS proteome', consisting of 355 proteins, was defined.
In step 2, proteins of the core ROS proteome were assigned to six functional modules: (1) vision, signaling, transporters, and channels; (2) outer segment structure and morphogenesis; (3) housekeeping; (4) cytoskeleton and polarity; (5) vesicles formation and trafficking, and (6) metabolism.
In step 3, a protein-protein interaction network was constructed based on the literature mining. Since for most of the interactions experimental evidence was co-immunoprecipitation, or pull-down experiments, and in addition many of the edges in the network are supported by single experimental evidence, often derived from high-throughput approaches, we refer to this network, as ‘fuzzy ROS interactome'. Structural information was used to predict binary interactions, based on the finding that similar domain pairs are likely to interact in a similar way (‘nature repeats itself') (Aloy and Russell, 2002). To increase the confidence in the resulting network, edges supported by a single evidence not coming from yeast two-hybrid experiments were removed, exception being interactions where the evidence was the existence of a three-dimensional structure of the complex itself, or of a highly homologous complex. This curated static network (‘high-confidence ROS interactome') comprises 660 edges linking the majority of the nodes. By considering only edges supported by at least one evidence of direct binary interaction, we end up with a ‘high-confidence binary ROS interactome'. We next extended the published core pathway (Dell'Orco et al, 2009) using evidence from our high-confidence network. We find several new direct binary links to different cellular functional processes (Figure 4): the active rhodopsin interacts with Rac1 and the GTP form of Rho. There is also a connection between active rhodopsin and Arf4, as well as PDEδ with Rab13 and the GTP-bound form of Arl3 that links the vision cycle to vesicle trafficking and structure. We see a connection between PDEδ with prenyl-modified proteins, such as several small GTPases, as well as with rhodopsin kinase. Further, our network reveals several direct binary connections between Ca2+-regulated proteins and cytoskeleton proteins; these are CaMK2A with actinin, calmodulin with GAP43 and S1008, and PKC with 14-3-3 family members.
In step 4, part of the network was experimentally validated using three different approaches to identify physical protein associations that would occur under physiological conditions: (i) Co-segregation/co-sedimentation experiments, (ii) immunoprecipitations combined with mass spectrometry and/or subsequent immunoblotting, and (iii) utilizing the glycosylated N-terminus of rhodopsin to isolate its associated protein partners by Concanavalin A affinity purification. In total, 60 co-purification and co-elution experiments supported interactions that were already in our literature network, and new evidence from 175 co-IP experiments in this work was added. Next, we aimed to provide additional independent experimental confirmation for two of the novel networks and functional links proposed based on the network analysis: (i) the proposed complex between Rac1/RhoA/CRMP-2/tubulin/and ROCK II in ROS was investigated by culturing retinal explants in the presence of an ROCK II-specific inhibitor (Figure 6). While morphology of the retinas treated with ROCK II inhibitor appeared normal, immunohistochemistry analyses revealed several alterations on the protein level. (ii) We supported the hypothesis that PDEδ could function as a GDI for Rac1 in ROS, by demonstrating that PDEδ and Rac1 co localize in ROS and that PDEδ could dissociate Rac1 from ROS membranes in vitro.
In step 5, we use structural information to distinguish between mutually compatible (‘AND') or excluded (‘XOR') interactions. This enables breaking a network of nodes and edges into functional machines or sub-networks/modules. In the vision branch, both ‘AND' and ‘XOR' gates synergize. This may allow dynamic tuning of light and dark states. However, all connections from the vision module to other modules are ‘XOR' connections suggesting that competition, in connection with local protein concentration changes, could be important for transmitting signals from the core vision module.
In the last step, we map and functionally characterize the known mutations that produce blindness.
In summary, this represents the first comprehensive, dynamic, and integrative rhodopsin signaling network, which can be the basis for integrating and mapping newly discovered disease mutants, to guide protein or signaling branch-specific therapies.
Orchestration of signaling, photoreceptor structural integrity, and maintenance needed for mammalian vision remain enigmatic. By integrating three proteomic data sets, literature mining, computational analyses, and structural information, we have generated a multiscale signal transduction network linked to the visual G protein-coupled receptor (GPCR) rhodopsin, the major protein component of rod outer segments. This network was complemented by domain decomposition of protein–protein interactions and then qualified for mutually exclusive or mutually compatible interactions and ternary complex formation using structural data. The resulting information not only offers a comprehensive view of signal transduction induced by this GPCR but also suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking, predicting an important level of regulation through small GTPases. Further, it demonstrates a specific disease susceptibility of the core visual pathway due to the uniqueness of its components present mainly in the eye. As a comprehensive multiscale network, it can serve as a basis to elucidate the physiological principles of photoreceptor function, identify potential disease-associated genes and proteins, and guide the development of therapies that target specific branches of the signaling pathway.
doi:10.1038/msb.2011.83
PMCID: PMC3261702  PMID: 22108793
protein interaction network; rhodopsin signaling; structural modeling
2.  A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals 
Biosensors & Bioelectronics  2013;40(1):368-376.
Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas.
Graphical abstract
Highlights
► Modular cellular biosensors comprising exchangeable genetic sensors, logic circuits and actuators. ► A set of biosensors for toxic metals and bacterial signalling molecules. ► Genetic logic circuits functioning as biological filters and amplifiers to enhance sensing selectivity and sensitivity. ► A triple-input AND logic gated sensor using multiple cellular consortia.
doi:10.1016/j.bios.2012.08.011
PMCID: PMC3507625  PMID: 22981411
Cellular biosensor; Genetic circuits; Logic gates; Cell programming; Synthetic biology
3.  Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch 
We designed and constructed a genetic sequential logic circuit that can function as a push-on push-off switch. The circuit consists of a bistable switch module and a NOR gate module.The bistable switch module and NOR gate module were rationally designed and constructed.The two above modules were coupled by two interconnecting parts, cIind- and lacI. When optimizing the defined function, we fine-tuned the expression of the two interconnecting parts by directed evolution.Three control circuits were constructed to show the interconnecting parts are essential for achieving the defined function.
Design and synthesis of basic functional circuits are the fundamental tasks of synthetic biologists. Before it is possible to engineer higher-order genetic networks that can perform complex functions, a toolkit of basic devices must be developed. Among those devices, sequential logic circuits are expected to be the foundation of genetic information-processing systems.
As in electronics, combinational and sequential logic circuits are two kinds of fundamental processors in cells. In a combinational logic circuit, the output depends only on the present inputs, whereas in a sequential logic circuit, the output also depends on the history of the input due to its own memory. If we can successfully construct the two kinds of basic logic circuits in a cell, they can serve as building blocks to be assembled into high-order genetic circuits and implement more sophisticated computation.
Construction of genetic combinational logic circuits (GSLCs), such as AND, OR, and NOR gates, has been frequently reported in the last decade (Guet et al, 2002; Dueber et al, 2003; Anderson et al, 2007; Win and Smolke, 2008). Meanwhile toggle switches, which can function as memory modules, have been implemented in prokaryotic and eukaryotic cells (Becskei et al, 2001; Kramer et al, 2004; Ajo-Franklin et al, 2007).
Here, we constructed a novel GSLC that functions as a push-on push-off switch by coupling a combinational logic module with a bistable switch module (Figure 1A). When the internal state of the memory is in the ‘ON' state, the external UV input makes the circuit's output promoter PNOR generate an ‘OFF' pulse signal and register the ‘OFF' state into the memory; when the internal state is in the ‘OFF' state, the same external UV input induces the circuit's output promoter PNOR to generate an ‘ON' pulse signal and register the ‘ON' state into the memory.
In our design, the combinatorial logic gate is a NOR gate and the switch module is a clearable bistable switch (Figure 1C). Two interconnecting parts are designed to connect the NOR gate and the bistable switch (Figure 1D). UV irradiation was used as both an external input signal and a reset signal for the clearable bistable switch (Figure 1B).
Before implementing the experimental construction, we used a set of ordinary differential equations to simulate the dynamic process. With a set of reasonable parameters, the simulation results showed that the circuit could function as a push-on push-off switch (Figure 1E). Then the bistable switch module and NOR gate module were rationally designed and constructed. Our experimental results showed that the corresponding functions were implemented very well.
After the construction of the memory and the NOR gate module, we coupled the two modules together by fine-tuning the expression of two interconnecting parts lacI and cIind−. The two libraries for the ribosome-binding sites (RBSs) of lacI and cIind− were simultaneously transformed into Escherichia coli cells harboring the memory module plasmid. After growth on agar plates with appropriate antibiotics, colonies containing all three plasmids were selected.
With efficient mutation libraries, we developed a new screening method to select the functional circuits. The experimental process is described in Figure 4A. It consists of two rounds of selection. In the first round of selection, approximatelybout 300 mutants out of 1000 were chosen. In the second round, only three mutants were selected. As shown in Figure 4B, if the initial state was ‘OFF' with green color, the fraction of green cells in the population was near 100% before UV stimulus, whereas less than 10% of cells remained in the green ‘OFF' state after UV stimulus (Figure 4B). This result indicates that the switch from ‘OFF' to ‘ON' is quite complete. Unfortunately, the switch from ‘ON' to ‘OFF' was not as efficient: only about one-third of the population switched to the ‘OFF' state after UV triggering (Figure 4C). Nonetheless, the switch is still significant compared with that of the population not exposed to UV irradiation (Figure 4B and C). These results show that the fine-tuned GSLC can generate different output signals under the same input on the basis of the internal state of its memory, and register the output signal into its memory as the new internal state.
To show that decoupled circuits cannot achieve the sequential logic function, we also constructed three control circuits. The bistable switch module and the NOR gate module were decoupled by removing either or both of the interconnecting parts. In the first control circuit, LacI was removed; without LacI, LexA becomes the only effective input for the NOR gate. As a consequence, upon UV stimulus, promoter PNOR always generates a high output signal, and the ‘ON' state (high CI and low CI434) is latched in the memory with the help of CIind−. Correspondingly, the color of the cells will change to red. In the other two control circuits, CIind− or both LacI and CIind− were removed. Owing to the lack of the feedback part CIind−, when the output of the promoter PNOR is ‘ON', no output signal can be registered into the memory. In this case, the memory module will spontaneously enter into the low CI/high CI434 state after UV stimulus. All experimental results are consistent with the above expectation.
Finally, to show the property of the push-on push-off switch of the circuit, we sequentially stimulated a homogeneous population of cells with the same dose of UV signal multiple times. The first UV stimulus caused the fraction of green cells in the population to decrease from 99.3% to 8.4%, so that more than 90% of the population switched from the ‘OFF' to the ‘ON' state. The second UV stimulus resulted in the fraction of green cells increasing from 8.4% to 34.5%. Therefore, only 26.1% of the population switched back to the ‘OFF' state. These results are comparable to the results of switching efficiency measurement shown in Figure 4B and C. With repeated exposure to UV irradiation, the population increasingly appeared like a mixture of the two states, the ratio of which gradually reached a steady state. The push-on–push-off function of the circuit was thus lost at the population level.
In summary, we successfully assembled a bistable switch module and a combinatorial NOR gate module into a functional sequential logic circuit. We combined rational design with directed evolution to generate the desired system behavior. In this work, we showed that simultaneous mutation of multiple RBS targets, followed by directed evolution, is a powerful tool to search the in vivo parameter space to generate functional circuits from multiple rationally designed synthetic device modules. We anticipate that this approach will lend itself well to the next step in synthetic biology, combining multiple circuits, each composed of several device modules, to create useful synthetic systems that perform sophisticated computation.
Design and synthesis of basic functional circuits are the fundamental tasks of synthetic biologists. Before it is possible to engineer higher-order genetic networks that can perform complex functions, a toolkit of basic devices must be developed. Among those devices, sequential logic circuits are expected to be the foundation of the genetic information-processing systems. In this study, we report the design and construction of a genetic sequential logic circuit in Escherichia coli. It can generate different outputs in response to the same input signal on the basis of its internal state, and ‘memorize' the output. The circuit is composed of two parts: (1) a bistable switch memory module and (2) a double-repressed promoter NOR gate module. The two modules were individually rationally designed, and they were coupled together by fine-tuning the interconnecting parts through directed evolution. After fine-tuning, the circuit could be repeatedly, alternatively triggered by the same input signal; it functions as a push-on push-off switch.
doi:10.1038/msb.2010.2
PMCID: PMC2858441  PMID: 20212522
bistable switch; coupling modules; genetic sequential logic circuit; NOR gate; push-on push-off switch
4.  Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen 
A synthetic genetic system is designed and characterized that allows Escherichia coli to sense and eradicate Pseudomonas aeruginosa, providing a novel antimicrobial strategy that could potentially be applied to fighting infectious pathogens.
We have engineered and demonstrated a novel genetic circuit that enables Escherichia coli to produce and release pyocin upon quorum sensing detection of Pseudomonas aeruginosa, which in turn kills P. aeruginosa.The quorum sensing device, which comprises an LasR transcription factor constitutively expressed by a pTetR promoter and a downstream pLuxR inducible promoter, has a switch point of 1.2 × 10E-7 M 3OC12HSL and is able to sense 3OC12HSL natively produced by P. aeruginosa.The E7 lysis device when coupled downstream of the quorum sensing device enhances pyocin release eight-fold.The engineered E. coli, which carries the sensing, lysing, and killing devices, effectively inhibits the growth of planktonic and biofilm P. aeruginosa by 99 and 90%, respectively.
In this study, we have made progress toward developing a novel antimicrobial strategy, based on an engineered microbial system, using the synthetic biology framework. Our final system was designed to (i) detect AHLs produced by P. aeruginosa; (ii) produce pyocin S5 upon the detection; and (iii) lyse the E. coli cells by E7 lysis protein so that the produced pyocin S5 is released from the cells, leading to the killing of P. aeruginosa.
Figure 1 shows a schematic of our sensing and killing genetic system. The sensing device was designed based on the Type I quorum sensing mechanism of P. aeruginosa. The tetR promoter, which is constitutively on, produces a transcriptional factor, LasR, that binds to AHL 3OC12HSL. The luxR promoter, to which LasR-3OC12HSL activator complex reportedly binds, was adopted as the inducible promoter in our sensing device (Gray et al, 1994). Next, the formation of the LasR-3OC12HSL complex, which binds to the luxR promoter, activates the killing and lysing devices, leading to the production of pyocin S5 and lysis E7 proteins within the E. coli chassis. Upon reaching a threshold concentration, the lysis E7 protein perforates membrane of the E. coli host and releases the accumulated pyocin S5. Pyocin S5, which is a soluble protein, then diffuses toward the target pathogen and damages its cellular integrity, thereby killing it.
To evaluate and characterize the sensing device, the gene encoding the green fluorescent protein (GFP) was fused to the sensing device and the GFP expression was monitored at a range of concentrations of 3OC12HSL. From the measured GFP synthesis rates, we observed a basal expression level of 0.216 RFU per OD per minute without induction, followed by a sharp increase in GFP production rate as the concentration of 3OC12HSL was increased beyond 1.0E-7 M. A transfer function that describes the static relationship between the input (3OC12HSL) and output (GFP production rate) of the sensing device was determined by fitting an empirical mathematical model (Hill equation) to the experimental data where the input 3OC12HSL concentration is <1.0E-6 M. The resulting best fit model demonstrated that the static performance of the sensing device follows a Hill equation below the input concentration of 1.0E-6 M 3OC12HSL. The model showed that the sensing device saturated at a maximum output of 1.96 RFU per OD per minute at input concentration >3.3E-7 M but <1.0E-6 M 3OC12HSL, and the switch point for the sensing device was 1.2E-7 M 3OC12HSL, the input concentration at which output is at half-maximal. Since this switch point concentration is smaller than the concentration of 3OC12HSL present (1.0E-6 to 1.0E-4 M) within proximity to the site of P. aeruginosa infection as earlier reported in the literature (Pearson et al, 1995; Charlton et al, 2000), the sensing device would be sensitive enough to detect the amount of 3OC12HSL natively produced by P. aeruginosa.
In line with the objective of the E7 lysis device in mediating the export of pyocin, we studied the efficiency of the lysis device in the final system by measuring the amount of the released protein. While distinct bands that corresponded to pyocin S5 were observed on the SDS–PAGE of the final system, no bands were seen in lanes without the lysis device. We further validated the results by estimating the protein concentrations in the supernatant with Bradford assay and showed that the amount of pyocin released by our final system was eight times higher than the system without the lysis device.
To verify that our engineered E. coli can inhibit P. aeruginosa in a mixed culture, we monitored the growth of P. aeruginosa co-cultured with the engineered E. coli in the ratio 1:4 by CFU count. The result shows that our engineered E. coli with the final system effectively inhibited the growth of P. aeruginosa by 99% while continuous growths were apparent in P. aeruginosa co-cultured with incomplete E. coli systems missing either the pyocin S5 or E7 lysis devices.
To examine the potential application of our engineered system against a pseudo disease state of Pseudomonas, a static biofilm inhibition assay was performed. Figure 6A shows that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%. This observation is in stark contrast to the pyocin-resistant control strain PAO1 and pyocin-sensitive clinical isolate ln7 subjected to treatment with E. coli having the systems missing either the pyocin S5 or E7 lysis devices. To visualize the extent of biofilm inhibition, biofilm cells with green fluorescence were grown in the presence of engineered E. coli on glass slide substrate and examined with confocal laser scanning microscopy. Figure 6B shows that the morphology of Pseudomonas biofilm treated with the engineered E. coli appeared sparse, while elaborated honey-combed structures were apparent in the control experiments. Collectively, our results suggest that our engineered E. coli carrying the final system, which contains the sensing, killing, and lysing devices, can effectively inhibit the growth of P. aeruginosa in both planktonic and sessile states.
In summary, we engineered a novel biological system, which comprises sensing, killing, and lysing devices, that enables E. coli to sense and eradicate pathogenic P. aeruginosa strains by exploiting the synthetic biology framework. More importantly, our study presents the possibility of engineering potentially beneficial microbiota into therapeutic bioagents to arrest Pseudomonas infection. Given the stalled development of new antibiotics and the increasing emergence of multidrug-resistant pathogens, this study provides the foundational basis for a novel synthetic biology-driven antimicrobial strategy that could be extended to include other pathogens such as Vibrio cholera and Helicobacter pylori.
Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.
doi:10.1038/msb.2011.55
PMCID: PMC3202794  PMID: 21847113
genetic circuits; Pseudomonas aeruginosa; pyocin; quorum sensing; synthetic biology
5.  Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results  
Study type: Basic science
Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3
Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3 b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)—Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time
Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics.
Methods: Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5).
Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: Bone
Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc space
Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a–c H&E (overall tissue staining for light micrsocopy) d–f Alcian blue (proteoglycans) g–i Picrosirius red (collagen I and II)
Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body bone
MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NP
7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP)
Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months.
Conclusions: This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d–f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.
doi:10.1055/s-0028-1100918
PMCID: PMC3623095  PMID: 23637671
6.  Genetic programs constructed from layered logic gates in single cells 
Nature  2012;491(7423):249-253.
Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits2–6, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits1,7. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.
doi:10.1038/nature11516
PMCID: PMC3904217  PMID: 23041931
7.  Cross-synaptic synchrony and transmission of signal and noise across the mouse retina 
eLife  2014;3:e03892.
Cross-synaptic synchrony—correlations in transmitter release across output synapses of a single neuron—is a key determinant of how signal and noise traverse neural circuits. The anatomical connectivity between rod bipolar and A17 amacrine cells in the mammalian retina, specifically that neighboring A17s often receive input from many of the same rod bipolar cells, provides a rare technical opportunity to measure cross-synaptic synchrony under physiological conditions. This approach reveals that synchronization of rod bipolar cell synapses is near perfect in the dark and decreases with increasing light level. Strong synaptic synchronization in the dark minimizes intrinsic synaptic noise and allows rod bipolar cells to faithfully transmit upstream signal and noise to downstream neurons. Desynchronization in steady light lowers the sensitivity of the rod bipolar output to upstream voltage fluctuations. This work reveals how cross-synaptic synchrony shapes retinal responses to physiological light inputs and, more generally, signaling in complex neural networks.
DOI: http://dx.doi.org/10.7554/eLife.03892.001
eLife digest
The human eye is capable of detecting a single photon of starlight. This level of sensitivity is made possible by the high sensitivity of photoreceptors called rods. There are around 120 million rods in the retina, and they support vision in levels of light that are too low to activate the photoreceptors called cones that allow us to see in color. This is why we cannot see colors in the dark.
Signals are relayed through the retina via a circuit made up of multiple types of neurons. The activation of rods leads to activation of cells known as ‘rod bipolar cells’ which, in turn, activate amacrine cells and ganglion cells, with the latter sending signals via the optic nerve to the brain. All of these neurons communicate with one another at junctions called synapses. Activation of a rod bipolar cell, for example, triggers the release of molecules called neurotransmitters: these molecules bind to and activate receptors on the amacrine cells, enabling the signal to be transmitted.
For the brain to detect that a single photon has struck a rod, the eye must transmit information along this chain of neurons in a way that is highly reliable while adding very little noise to the signal. Grimes et al. have now revealed a key step in how this is achieved.
Electrical recordings from the mouse retina revealed that, in the dark, small fluctuations in the activity of rod bipolar cells lead to the near-deterministic release of neurotransmitters. This reduces the impact of random fluctuations in neurotransmitter release produced at individual synapses and ensures that the signals from rod bipolar cells (and thus from rods) are transmitted faithfully through the circuit with minimal added noise. As light levels increase, this tight synchrony of transmitter release breaks down, reducing the sensitivity to individual photons.
Given that many other brain regions share the features that enable retinal cells to coordinate the release of neurotransmitters, this mechanism might be used throughout the brain to increase the signal-to-noise ratio for the transmission of information through neural circuits.
DOI: http://dx.doi.org/10.7554/eLife.03892.002
doi:10.7554/eLife.03892
PMCID: PMC4174577  PMID: 25180102
synaptic transmission; signal processing; synchronization; mouse
8.  Exercises in Molecular Computing 
Accounts of Chemical Research  2014;47(6):1845-1852.
Conspectus
The successes of electronic digital logic have transformed every aspect of human life over the last half-century. The word “computer” now signifies a ubiquitous electronic device, rather than a human occupation. Yet evidently humans, large assemblies of molecules, can compute, and it has been a thrilling challenge to develop smaller, simpler, synthetic assemblies of molecules that can do useful computation. When we say that molecules compute, what we usually mean is that such molecules respond to certain inputs, for example, the presence or absence of other molecules, in a precisely defined but potentially complex fashion. The simplest way for a chemist to think about computing molecules is as sensors that can integrate the presence or absence of multiple analytes into a change in a single reporting property. Here we review several forms of molecular computing developed in our laboratories.
When we began our work, combinatorial approaches to using DNA for computing were used to search for solutions to constraint satisfaction problems. We chose to work instead on logic circuits, building bottom-up from units based on catalytic nucleic acids, focusing on DNA secondary structures in the design of individual circuit elements, and reserving the combinatorial opportunities of DNA for the representation of multiple signals propagating in a large circuit. Such circuit design directly corresponds to the intuition about sensors transforming the detection of analytes into reporting properties. While this approach was unusual at the time, it has been adopted since by other groups working on biomolecular computing with different nucleic acid chemistries.
We created logic gates by modularly combining deoxyribozymes (DNA-based enzymes cleaving or combining other oligonucleotides), in the role of reporting elements, with stem–loops as input detection elements. For instance, a deoxyribozyme that normally exhibits an oligonucleotide substrate recognition region is modified such that a stem–loop closes onto the substrate recognition region, making it unavailable for the substrate and thus rendering the deoxyribozyme inactive. But a conformational change can then be induced by an input oligonucleotide, complementary to the loop, to open the stem, allow the substrate to bind, and allow its cleavage to proceed, which is eventually reported via fluorescence. In this Account, several designs of this form are reviewed, along with their application in the construction of large circuits that exhibited complex logical and temporal relationships between the inputs and the outputs.
Intelligent (in the sense of being capable of nontrivial information processing) theranostic (therapy + diagnostic) applications have always been the ultimate motivation for developing computing (i.e., decision-making) circuits, and we review our experiments with logic-gate elements bound to cell surfaces that evaluate the proximal presence of multiple markers on lymphocytes.
doi:10.1021/ar5000538
PMCID: PMC4063495  PMID: 24873234
9.  Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism 
A comprehensive genome-scale metabolic network of Chlamydomonas reinhardtii, including a detailed account of light-driven metabolism, is reconstructed and validated. The model provides a new resource for research of C. reinhardtii metabolism and in algal biotechnology.
The genome-scale metabolic network of Chlamydomonas reinhardtii (iRC1080) was reconstructed, accounting for >32% of the estimated metabolic genes encoded in the genome, and including extensive details of lipid metabolic pathways.This is the first metabolic network to explicitly account for stoichiometry and wavelengths of metabolic photon usage, providing a new resource for research of C. reinhardtii metabolism and developments in algal biotechnology.Metabolic functional annotation and the largest transcript verification of a metabolic network to date was performed, at least partially verifying >90% of the transcripts accounted for in iRC1080. Analysis of the network supports hypotheses concerning the evolution of latent lipid pathways in C. reinhardtii, including very long-chain polyunsaturated fatty acid and ceramide synthesis pathways.A novel approach for modeling light-driven metabolism was developed that accounts for both light source intensity and spectral quality of emitted light. The constructs resulting from this approach, termed prism reactions, were shown to significantly improve the accuracy of model predictions, and their use was demonstrated for evaluation of light source efficiency and design.
Algae have garnered significant interest in recent years, especially for their potential application in biofuel production. The hallmark, model eukaryotic microalgae Chlamydomonas reinhardtii has been widely used to study photosynthesis, cell motility and phototaxis, cell wall biogenesis, and other fundamental cellular processes (Harris, 2001). Characterizing algal metabolism is key to engineering production strains and understanding photobiological phenomena. Based on extensive literature on C. reinhardtii metabolism, its genome sequence (Merchant et al, 2007), and gene functional annotation, we have reconstructed and experimentally validated the genome-scale metabolic network for this alga, iRC1080, the first network to account for detailed photon absorption permitting growth simulations under different light sources. iRC1080 accounts for 1080 genes, associated with 2190 reactions and 1068 unique metabolites and encompasses 83 subsystems distributed across 10 cellular compartments (Figure 1A). Its >32% coverage of estimated metabolic genes is a tremendous expansion over previous algal reconstructions (Boyle and Morgan, 2009; Manichaikul et al, 2009). The lipid metabolic pathways of iRC1080 are considerably expanded relative to existing networks, and chemical properties of all metabolites in these pathways are accounted for explicitly, providing sufficient detail to completely specify all individual molecular species: backbone molecule and stereochemical numbering of acyl-chain positions; acyl-chain length; and number, position, and cis–trans stereoisomerism of carbon–carbon double bonds. Such detail in lipid metabolism will be critical for model-driven metabolic engineering efforts.
We experimentally verified transcripts accounted for in the network under permissive growth conditions, detecting >90% of tested transcript models (Figure 1B) and providing validating evidence for the contents of iRC1080. We also analyzed the extent of transcript verification by specific metabolic subsystems. Some subsystems stood out as more poorly verified, including chloroplast and mitochondrial transport systems and sphingolipid metabolism, all of which exhibited <80% of transcripts detected, reflecting incomplete characterization of compartmental transporters and supporting a hypothesis of latent pathway evolution for ceramide synthesis in C. reinhardtii. Additional lines of evidence from the reconstruction effort similarly support this hypothesis including lack of ceramide synthetase and other annotation gaps downstream in sphingolipid metabolism. A similar hypothesis of latent pathway evolution was established for very long-chain fatty acids (VLCFAs) and their polyunsaturated analogs (VLCPUFAs) (Figure 1C), owing to the absence of this class of lipids in previous experimental measurements, lack of a candidate VLCFA elongase in the functional annotation, and additional downstream annotation gaps in arachidonic acid metabolism.
The network provides a detailed account of metabolic photon absorption by light-driven reactions, including photosystems I and II, light-dependent protochlorophyllide oxidoreductase, provitamin D3 photoconversion to vitamin D3, and rhodopsin photoisomerase; this network accounting permits the precise modeling of light-dependent metabolism. iRC1080 accounts for effective light spectral ranges through analysis of biochemical activity spectra (Figure 3A), either reaction activity or absorbance at varying light wavelengths. Defining effective spectral ranges associated with each photon-utilizing reaction enabled our network to model growth under different light sources via stoichiometric representation of the spectral composition of emitted light, termed prism reactions. Coefficients for different photon wavelengths in a prism reaction correspond to the ratios of photon flux in the defined effective spectral ranges to the total emitted photon flux from a given light source (Figure 3B). This approach distinguishes the amount of emitted photons that drive different metabolic reactions. We created prism reactions for most light sources that have been used in published studies for algal and plant growth including solar light, various light bulbs, and LEDs. We also included regulatory effects, resulting from lighting conditions insofar as published studies enabled. Light and dark conditions have been shown to affect metabolic enzyme activity in C. reinhardtii on multiple levels: transcriptional regulation, chloroplast RNA degradation, translational regulation, and thioredoxin-mediated enzyme regulation. Through application of our light model and prism reactions, we were able to closely recapitulate experimental growth measurements under solar, incandescent, and red LED lights. Through unbiased sampling, we were able to establish the tremendous statistical significance of the accuracy of growth predictions achievable through implementation of prism reactions. Finally, application of the photosynthetic model was demonstrated prospectively to evaluate light utilization efficiency under different light sources. The results suggest that, of the existing light sources, red LEDs provide the greatest efficiency, about three times as efficient as sunlight. Extending this analysis, the model was applied to design a maximally efficient LED spectrum for algal growth. The result was a 677-nm peak LED spectrum with a total incident photon flux of 360 μE/m2/s, suggesting that for the simple objective of maximizing growth efficiency, LED technology has already reached an effective theoretical optimum.
In summary, the C. reinhardtii metabolic network iRC1080 that we have reconstructed offers insight into the basic biology of this species and may be employed prospectively for genetic engineering design and light source design relevant to algal biotechnology. iRC1080 was used to analyze lipid metabolism and generate novel hypotheses about the evolution of latent pathways. The predictive capacity of metabolic models developed from iRC1080 was demonstrated in simulating mutant phenotypes and in evaluation of light source efficiency. Our network provides a broad knowledgebase of the biochemistry and genomics underlying global metabolism of a photoautotroph, and our modeling approach for light-driven metabolism exemplifies how integration of largely unvisited data types, such as physicochemical environmental parameters, can expand the diversity of applications of metabolic networks.
Metabolic network reconstruction encompasses existing knowledge about an organism's metabolism and genome annotation, providing a platform for omics data analysis and phenotype prediction. The model alga Chlamydomonas reinhardtii is employed to study diverse biological processes from photosynthesis to phototaxis. Recent heightened interest in this species results from an international movement to develop algal biofuels. Integrating biological and optical data, we reconstructed a genome-scale metabolic network for this alga and devised a novel light-modeling approach that enables quantitative growth prediction for a given light source, resolving wavelength and photon flux. We experimentally verified transcripts accounted for in the network and physiologically validated model function through simulation and generation of new experimental growth data, providing high confidence in network contents and predictive applications. The network offers insight into algal metabolism and potential for genetic engineering and efficient light source design, a pioneering resource for studying light-driven metabolism and quantitative systems biology.
doi:10.1038/msb.2011.52
PMCID: PMC3202792  PMID: 21811229
Chlamydomonas reinhardtii; lipid metabolism; metabolic engineering; photobioreactor
10.  Controlling fertilization and cAMP signaling in sperm by optogenetics 
eLife  null;4:e05161.
Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.
DOI: http://dx.doi.org/10.7554/eLife.05161.001
eLife digest
Tiny hair-like structures called cilia on the outside of cells play many important roles, including detecting physical and chemical signals from the environment. Special cilia—called flagella—help cells to move around and perhaps the most well-known of these are sperm flagella, which propel sperm in their quest to fertilize the egg. A chemical messenger called cAMP is essential for the movement of sperm flagella.
When a sperm cell enters the female reproductive tract, an enzyme called SACY is activated. Within seconds, SACY produces cAMP and, thereby, causes the flagella to beat faster so that the sperm cell speeds toward the egg. cAMP also controls sperm maturation, which is needed to penetrate the egg. However, the precise details of the role of cAMP in sperm cells are not clear.
Here, Jansen et al. have investigated this role using a cutting-edge technique—called optogenetics—that was originally developed to study brain cells in living organisms. Jansen et al. genetically engineered a mouse so that exposing sperm to blue light activates a light-sensitive enzyme called bPAC that increases cAMP levels in sperm.
In these mice, the activation of bPAC by light accelerated the beating of the flagella so the sperm moved faster, in a way that was similar to the effects that are normally observed after the activation of the SACY enzyme. In mice lacking among other things the SACY enzyme—whose sperm cells are unable to move or fertilize an egg—activating the light-sensitive bPAC enzyme restored sperm motility and enabled the sperm to fertilize an egg.
These results show that optogenetics may be a useful tool for studying how flagella and other types of cilia work.
DOI: http://dx.doi.org/10.7554/eLife.05161.002
doi:10.7554/eLife.05161
PMCID: PMC4298566  PMID: 25601414
cyclic nucleotide signaling; sperm; capacitation; cAMP; calcium; optogenetics; mouse
11.  Robustness of Circadian Clocks to Daylight Fluctuations: Hints from the Picoeucaryote Ostreococcus tauri 
PLoS Computational Biology  2010;6(11):e1000990.
The development of systemic approaches in biology has put emphasis on identifying genetic modules whose behavior can be modeled accurately so as to gain insight into their structure and function. However, most gene circuits in a cell are under control of external signals and thus, quantitative agreement between experimental data and a mathematical model is difficult. Circadian biology has been one notable exception: quantitative models of the internal clock that orchestrates biological processes over the 24-hour diurnal cycle have been constructed for a few organisms, from cyanobacteria to plants and mammals. In most cases, a complex architecture with interlocked feedback loops has been evidenced. Here we present the first modeling results for the circadian clock of the green unicellular alga Ostreococcus tauri. Two plant-like clock genes have been shown to play a central role in the Ostreococcus clock. We find that their expression time profiles can be accurately reproduced by a minimal model of a two-gene transcriptional feedback loop. Remarkably, best adjustment of data recorded under light/dark alternation is obtained when assuming that the oscillator is not coupled to the diurnal cycle. This suggests that coupling to light is confined to specific time intervals and has no dynamical effect when the oscillator is entrained by the diurnal cycle. This intringuing property may reflect a strategy to minimize the impact of fluctuations in daylight intensity on the core circadian oscillator, a type of perturbation that has been rarely considered when assessing the robustness of circadian clocks.
Author Summary
Circadian clocks keep time of day in many living organisms, allowing them to anticipate environmental changes induced by day/night alternation. They consist of networks of genes and proteins interacting so as to generate biochemical oscillations with a period close to 24 hours. Circadian clocks synchronize to the day/night cycle through the year principally by sensing ambient light. Depending on the weather, the perceived light intensity can display large fluctuations within the day and from day to day, potentially inducing unwanted resetting of the clock. Furthermore, marine organisms such as microalgae are subjected to dramatic changes in light intensities in the water column due to streams and wind. We showed, using mathematical modelling, that the green unicellular marine alga Ostreococcus tauri has evolved a simple but effective strategy to shield the circadian clock from daylight fluctuations by localizing coupling to the light during specific time intervals. In our model, as in experiments, coupling is invisible when the clock is in phase with the day/night cycle but resets the clock when it is out of phase. Such a clock architecture is immune to strong daylight fluctuations.
doi:10.1371/journal.pcbi.1000990
PMCID: PMC2978692  PMID: 21085637
12.  Construction and Enhancement of a Minimal Genetic AND Logic Gate▿ †  
The ability of genetic networks to integrate multiple inputs in the generation of cellular responses is critical for the adaptation of cellular phenotype to distinct environments and of great interest in the construction of complex artificial circuits. To develop artificial genetic circuits that can integrate intercellular signaling molecules and commonly used inducing agents, we have constructed an artificial genetic AND gate based on the PluxI quorum-sensing promoter and the lac repressor. The hybrid promoter exhibited reduced basal and induced expression levels but increased expression capacity, generating clear logical responses that could be described using a simple mathematical model. The model also predicted that the AND gate's logic could be improved by altering the properties of the LuxR transcriptional activator and, in particular, by increasing its rate of transcriptional activation. Following these predictions, we were able to improve the AND gate's logic by ∼1.5-fold using a LuxR mutant library generated by directed evolution, providing the first example of the use of mutant transcriptional activators to improve the logic of a complex regulatory circuit. In addition, detailed characterizations of the AND gate's responses shed light on how LuxR, LacI, and RNA polymerase interact to activate gene expression.
doi:10.1128/AEM.01684-08
PMCID: PMC2632134  PMID: 19060164
13.  Modular Design of Artificial Tissue Homeostasis: Robust Control through Synthetic Cellular Heterogeneity 
PLoS Computational Biology  2012;8(7):e1002579.
Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of β-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation) are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for ‘synthetic cellular heterogeneity’ that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism), demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a ‘phenotypic sensitivity analysis’ method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in isolation, and its impact on overall system performance once integrated.
Author Summary
Over the last decade several relatively small synthetic gene networks have been successfully implemented and characterized, including oscillators, toggle switches, and intercellular communication systems. However, the ability to engineer large-scale synthetic gene networks for controlling multicellular systems with predictable and robust behavior remains a challenge. Here we present a novel combination of computational methods to aid the iterative design and optimization of such synthetic biological systems. We apply these methods to the design and analysis of an artificial tissue homeostasis system that exhibits coordinated control of cellular proliferation, differentiation, and cell-death. Achieving artificial tissue homeostasis would be therapeutically relevant for diseases such as Type I diabetes, for instance by transplanting genetically engineered stem cells that stably maintain populations of insulin-producing beta-cells despite normal cell death and autoimmune attacks. To manage complexity in the design process, we employ principles of logic abstraction and modularity and investigate their limits in biological networks. In this work, we find factors often associated with robustness (e.g., multicellular synchronization and noise attenuation) to be actually detrimental, and overcome these problems by engineering genetic modules that generate beneficial population heterogeneity. A combination of computational methods elucidates how these modules function to enhance robust control, and provides guidance for experimental implementation.
doi:10.1371/journal.pcbi.1002579
PMCID: PMC3400602  PMID: 22829755
14.  Detecting and Removing Inconsistencies between Experimental Data and Signaling Network Topologies Using Integer Linear Programming on Interaction Graphs 
PLoS Computational Biology  2013;9(9):e1003204.
Cross-referencing experimental data with our current knowledge of signaling network topologies is one central goal of mathematical modeling of cellular signal transduction networks. We present a new methodology for data-driven interrogation and training of signaling networks. While most published methods for signaling network inference operate on Bayesian, Boolean, or ODE models, our approach uses integer linear programming (ILP) on interaction graphs to encode constraints on the qualitative behavior of the nodes. These constraints are posed by the network topology and their formulation as ILP allows us to predict the possible qualitative changes (up, down, no effect) of the activation levels of the nodes for a given stimulus. We provide four basic operations to detect and remove inconsistencies between measurements and predicted behavior: (i) find a topology-consistent explanation for responses of signaling nodes measured in a stimulus-response experiment (if none exists, find the closest explanation); (ii) determine a minimal set of nodes that need to be corrected to make an inconsistent scenario consistent; (iii) determine the optimal subgraph of the given network topology which can best reflect measurements from a set of experimental scenarios; (iv) find possibly missing edges that would improve the consistency of the graph with respect to a set of experimental scenarios the most. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGFR/ErbB signaling against a library of high-throughput phosphoproteomic data measured in primary hepatocytes. Our methods detect interactions that are likely to be inactive in hepatocytes and provide suggestions for new interactions that, if included, would significantly improve the goodness of fit. Our framework is highly flexible and the underlying model requires only easily accessible biological knowledge. All related algorithms were implemented in a freely available toolbox SigNetTrainer making it an appealing approach for various applications.
Author Summary
Cellular signal transduction is orchestrated by communication networks of signaling proteins commonly depicted on signaling pathway maps. However, each cell type may have distinct variants of signaling pathways, and wiring diagrams are often altered in disease states. The identification of truly active signaling topologies based on experimental data is therefore one key challenge in systems biology of cellular signaling. We present a new framework for training signaling networks based on interaction graphs (IG). In contrast to complex modeling formalisms, IG capture merely the known positive and negative edges between the components. This basic information, however, already sets hard constraints on the possible qualitative behaviors of the nodes when perturbing the network. Our approach uses Integer Linear Programming to encode these constraints and to predict the possible changes (down, neutral, up) of the activation levels of the involved players for a given experiment. Based on this formulation we developed several algorithms for detecting and removing inconsistencies between measurements and network topology. Demonstrated by EGFR/ErbB signaling in hepatocytes, our approach delivers direct conclusions on edges that are likely inactive or missing relative to canonical pathway maps. Such information drives the further elucidation of signaling network topologies under normal and pathological phenotypes.
doi:10.1371/journal.pcbi.1003204
PMCID: PMC3764019  PMID: 24039561
15.  Synthetic in vitro transcriptional oscillators 
A fundamental goal of synthetic biology is to understand design principles through engineering biochemical systems.Three in vitro synthetic transcriptional oscillators were constructed and analyzed: a two-node-negative feedback oscillator, an amplified negative-feedback oscillator, and a three-node ring oscillator.The in vitro oscillators are governed by similar design principles as previous theoretical studies and synthetic oscillators in vivo.Because of unintended reactions that arise even without the complexity of living cells, several challenges remain for predictive and robust oscillator performance.
Fundamental goals for synthetic biology are to understand the principles of biological circuitry from an engineering perspective and to establish engineering methods for creating biochemical circuitry to control molecular processes—both in vitro and in vivo (Benner and Sismour, 2005; Adrianantoandro et al, 2006). Here, we make use of a previously proposed class of in vitro biochemical systems, transcriptional circuits, that can be modularly wired into arbitrarily complex networks by changing the regulatory and coding sequence domains of DNA templates (Kim et al, 2006; Subsoontorn et al 2011). Using design motifs for inhibitory and excitatory regulations, three different oscillator designs were constructed and characterized: a two-switch negative-feedback oscillator, loosely analogous to the p53–Mdm2-feedback loop (Bar-Or et al, 2000); the same oscillator augmented with a positive-feedback loop, loosely analogous to a synthetic relaxation oscillator (Atkinson et al, 2003); and a three-switch ring oscillator analogous to the repressilator (Elowitz and Leibler, 2000).
DNA and RNA hybridization reactions (Figure 1B) can be assembled to create either an inhibitable switch (Figure 1A, right and bottom) with a threshold set by the total concentration of its DNA activator strand (Figure 1C, bottom), or an activatable switch (Figure 1A, left and top) with a threshold set by its DNA inhibitor strand concentration (Figure 1C, top). This threshold mechanism is analogous to biological threshold mechanisms such as ‘inhibitor ultrasensitivity' (Ferrell, 1996) and ‘molecular titration' (Buchler and Louis, 2008). Using these design motifs, we constructed a two-switch negative-feedback oscillator (Figure 1A, inset): RNA activator rA1 activates the production of RNA inhibitor rI2 by modulating switch Sw21, while RNA inhibitor rI2, in turn, inhibits the production of RNA activator rA1 by modulating switch Sw12. A total of seven DNA strands are used, in addition to the two enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H. The fact that such a negative-feedback loop can lead to temporal oscillations can be seen from a mathematical model of transcriptional networks. Experimental results showed qualitative agreement with predicted oscillator behavior from simple model simulations.
The fully optimized system revealed five complete oscillation cycles with a nearly 50% amplitude swing (Figure 3A) until, after ∼20 h, the production rate could no longer be sustained in the batch reaction. Gel measurements verified oscillations in RNA concentrations and switch states (Figure 3B and C). However to our surprise, rather than oscillations with constant amplitude and constant mean, the RNA inhibitor concentration builds up after each cycle. An extended mathematical model that incorporated an interference reaction from ‘waste' product (Figure 3B and C) could qualitatively capture this behavior.
Using a new autoregulatory switch Sw11, we added a positive-feedback loop to the two-node oscillator to make an amplified negative feedback oscillator (Design II, Figure 1D). Further, we replaced the excitatory connection of Sw21 by a chain of two inhibitory connections, Sw23 and Sw31, to construct a three-switch ring oscillator (Design III, Figure 1D). All three oscillator designs could be tuned to reach the oscillatory regime in parameter space.
Reassuringly, our in vitro oscillators exhibit several design principles previously observed in vivo. (1) Introducing delay in a simple negative-feedback loop can help achieve stable oscillation (Novák and Tyson, 2008; Stricker et al, 2008). (2) The addition of a positive-feedback self-loop to a negative-feedback oscillator provides access to rich dynamics and improved tunability (Tsai et al, 2008). (3) Oscillations in biochemical ring oscillators (such as the repressilator) are sensitive to parameter asymmetry among individual components (Tuttle et al, 2005). (4) The saturation of degradation machinery and the management of waste products could play an important role.
However, several significant difficulties remain for predictive and robust oscillator performances: limited lifetime of closed batch reactions, interference from waste products, and asymmetry of switch components make quantitative modeling and predictio difficult. As a complementary approach to top-down view of systems biology, cell-free in vitro systems offer a valuable training ground to create and explore increasingly interesting and powerful information-based chemical systems (Simpson, 2006). In vitro oscillators could be used to orchestrate other chemical processes such as DNA nanomachines (Dittmer and Simmel, 2004) and to provide embedded controllers within prototype artificial cells (Noireaux and Libchaber, 2004; Griffiths and Tawfik, 2006).
The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells.
doi:10.1038/msb.2010.119
PMCID: PMC3063688  PMID: 21283141
cell free; in vitro; oscillation; synthetic biology; transcriptional circuits
16.  Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion 
Biochemistry  2012;51(8):1638-1647.
Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca2+-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves and quantitates the rapidly diffusing monomer and slowly diffusing heterodimer subpopulations. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing average diffusion coefficient of the fluorescent PH-CaM population, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ∼102-fold greater in the membrane system than in solution, apparently due both to faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.
doi:10.1021/bi201743a
PMCID: PMC3318961  PMID: 22263647
peripheral membrane protein; pleckstrin homology domain; calmodulin; 2-dimensional diffusion; reversible oligomerization; calcium-regulated dimerization
17.  Active regulation of receptor ratios controls integration of quorum-sensing signals in Vibrio harveyi 
Single-cell quantification of the input–output relation of the quorum-sensing circuit reveals how Vibrio harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity.
We identify the role of multiple feedback loops in the quorum-sensing circuit of the model bacterium, Vibrio harveyi. Single-cell microscopy and genetic analysis demonstrate that a novel feedback loop regulates receptor ratios to control the integration of multiple signals.Quantitative investigation of cells with all feedback loops present as well as mutants with specific feedback loops disrupted reveals that the multiple feedback loops expand the input dynamic range and compress the output dynamic range of signal transmission, and also control the noise level of the output.Our experimental observations can be interpreted in terms of a simple model of the quorum-sensing network. Plotting output after reparameterizing the input variables directly reveals how feedback controls receptors ratios.
Organisms detect multiple environmental cues simultaneously and use the information to coordinate their behaviors. Correctly integrating signals generally requires complex signal transduction pathways (Pawson and Scott, 2010). In addition to accurately integrating signals, regulatory circuits must ensure signal transmission fidelity. Information can be lost or corrupted by internal or external perturbations, so circuits must be designed to function robustly in the presence of such fluctuations. For example, the circadian clock in Neurospora (Virshup and Forger, 2009) and the chemotaxis network in Escherichia coli (Oleksiuk et al, 2011) accurately compensate for temperature variation. However, while signal integration and signal transmission have been addressed separately, little is known about mechanisms cells use to solve both tasks simultaneously. In this study, we report how the model bacterium Vibrio harveyi simultaneously integrates and faithfully transmits multiple chemical signals.
In a process called quorum sensing, bacteria communicate by synthesizing, releasing, and detecting signal molecules called autoinducers (AIs). To study the integration of such signals, we studied a strain of V. harveyi that integrates two AI signals into its quorum-sensing circuit: AI-1, an intra-species signal, and AI-2, a ‘universal' inter-species signal. Each signal is detected by a cognate receptor AI-1 by LuxN, and AI-2 by LuxPQ (Figure 4A). The information encoded in the two AIs is transduced through a shared signaling pathway into the master quorum-sensing regulator LuxR. In this study, the AIs serve as inputs and LuxR serves as the output of the quorum-sensing circuit. Interestingly, there are five distinct feedback loops in the V. harveyi quorum-sensing circuit (Figure 4A). How does the circuit use shared components to distinguish between the two AI inputs and what role does each feedback loop have in signal integration and transmission?
Using single-cell microscopy, we assayed the activity of the quorum-sensing circuit with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input–output relation both in cells with all feedback loops present (Figure 4A) as well as in mutants with specific feedback loops disrupted (Figure 4E, I, M, and Q). We compared the mean LuxR level (Figure 4B, F, J, N, and R) and noise level (Figure 4C, G, K, O, and S) for the input–output relation of five strains. We discovered that the LuxN feedback loop regulates receptor ratios (LuxN to LuxPQ) to control the integration of two signals. We also found that the multiple feedback loops expand the input dynamic range and compress the output dynamic range of signal transmission, and also control the noise in the output.
In summary, we used single-cell microscopy to quantify the integration of quorum-sensing signals in V. harveyi. Multiple feedback loops in the quorum-sensing circuit actively regulate receptor ratios to control signal integration, sculpt the input–output dynamic range, and regulate the noise level. This system presents a paradigm for how complex circuitry allows cells to appropriately detect and respond to multiple signals in a dynamically changing environment.
Quorum sensing is a chemical signaling mechanism used by bacteria to communicate and orchestrate group behaviors. Multiple feedback loops exist in the quorum-sensing circuit of the model bacterium Vibrio harveyi. Using fluorescence microscopy of individual cells, we assayed the activity of the quorum-sensing circuit, with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input–output relation both in cells with all feedback loops present as well as in mutants with specific feedback loops disrupted. We found that one of the feedback loops regulates receptor ratios to control the integration of multiple signals. Together, the feedback loops affect the input–output dynamic range of signal transmission and the noise in the output. We conclude that V. harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity.
doi:10.1038/msb.2011.30
PMCID: PMC3130561  PMID: 21613980
feedback loops; quorum sensing; signal integration; single-cell fluorescence microscopy
18.  Solving a Hamiltonian Path Problem with a bacterial computer 
Background
The Hamiltonian Path Problem asks whether there is a route in a directed graph from a beginning node to an ending node, visiting each node exactly once. The Hamiltonian Path Problem is NP complete, achieving surprising computational complexity with modest increases in size. This challenge has inspired researchers to broaden the definition of a computer. DNA computers have been developed that solve NP complete problems. Bacterial computers can be programmed by constructing genetic circuits to execute an algorithm that is responsive to the environment and whose result can be observed. Each bacterium can examine a solution to a mathematical problem and billions of them can explore billions of possible solutions. Bacterial computers can be automated, made responsive to selection, and reproduce themselves so that more processing capacity is applied to problems over time.
Results
We programmed bacteria with a genetic circuit that enables them to evaluate all possible paths in a directed graph in order to find a Hamiltonian path. We encoded a three node directed graph as DNA segments that were autonomously shuffled randomly inside bacteria by a Hin/hixC recombination system we previously adapted from Salmonella typhimurium for use in Escherichia coli. We represented nodes in the graph as linked halves of two different genes encoding red or green fluorescent proteins. Bacterial populations displayed phenotypes that reflected random ordering of edges in the graph. Individual bacterial clones that found a Hamiltonian path reported their success by fluorescing both red and green, resulting in yellow colonies. We used DNA sequencing to verify that the yellow phenotype resulted from genotypes that represented Hamiltonian path solutions, demonstrating that our bacterial computer functioned as expected.
Conclusion
We successfully designed, constructed, and tested a bacterial computer capable of finding a Hamiltonian path in a three node directed graph. This proof-of-concept experiment demonstrates that bacterial computing is a new way to address NP-complete problems using the inherent advantages of genetic systems. The results of our experiments also validate synthetic biology as a valuable approach to biological engineering. We designed and constructed basic parts, devices, and systems using synthetic biology principles of standardization and abstraction.
doi:10.1186/1754-1611-3-11
PMCID: PMC2723075  PMID: 19630940
19.  Metacontrast masking and the cortical representation of surface color: dynamical aspects of edge integration and contrast gain control 
Advances in Cognitive Psychology  2008;3(1-2):327-347.
This paper reviews recent theoretical and experimental work supporting the idea that brightness is computed in a series of neural stages involving edge integration and contrast gain control. It is proposed here that metacontrast and paracontrast masking occur as byproducts of the dynamical properties of these neural mechanisms. The brightness computation model assumes, more specifically, that early visual neurons in the retina, and cortical areas V1 and V2, encode local edge signals whose magnitudes are proportional to the logarithms of the luminance ratios at luminance edges within the retinal image. These local edge signals give rise to secondary neural lightness and darkness spatial induction signals, which are summed at a later stage of cortical processing to produce a neural representation of surface color, or achromatic color, in the case of the chromatically neutral stimuli considered here. Prior to the spatial summation of these edge-based induction signals, the weights assigned to local edge contrast are adjusted by cortical gain mechanisms involving both lateral interactions between neural edge detectors and top-down attentional control. We have previously constructed and computer-simulated a neural model of achromatic color perception based on these principles and have shown that our model gives a good quantitative account of the results of several brightness matching experiments. Adding to this model the realistic dynamical assumptions that 1) the neurons that encode local contrast exhibit transient firing rate enhancement at the onset of an edge, and 2) that the effects of contrast gain control take time to spread between edges, results in a dynamic model of brightness computation that predicts the existence Broca-Sulzer transient brightness enhancement of the target, Type B metacontrast masking, and a form of paracontrast masking in which the target brightness is enhanced when the mask precedes the target in time.
doi:10.2478/v10053-008-0034-z
PMCID: PMC2864963  PMID: 20517518
edge integration; brightness; lightness; achromatic color; brightness induction; masking; metacontrast; paracontrast; type B masking
20.  Noise Reduction by Diffusional Dissipation in a Minimal Quorum Sensing Motif 
PLoS Computational Biology  2008;4(8):e1000167.
Cellular interactions are subject to random fluctuations (noise) in quantities of interacting molecules. Noise presents a major challenge for the robust function of natural and engineered cellular networks. Past studies have analyzed how noise is regulated at the intracellular level. Cell–cell communication, however, may provide a complementary strategy to achieve robust gene expression by enabling the coupling of a cell with its environment and other cells. To gain insight into this issue, we have examined noise regulation by quorum sensing (QS), a mechanism by which many bacteria communicate through production and sensing of small diffusible signals. Using a stochastic model, we analyze a minimal QS motif in Gram-negative bacteria. Our analysis shows that diffusion of the QS signal, together with fast turnover of its transcriptional regulator, attenuates low-frequency components of extrinsic noise. We term this unique mechanism “diffusional dissipation” to emphasize the importance of fast signal turnover (or dissipation) by diffusion. We further show that this noise attenuation is a property of a more generic regulatory motif, of which QS is an implementation. Our results suggest that, in a QS system, an unstable transcriptional regulator may be favored for regulating expression of costly proteins that generate public goods.
Author Summary
Quorum sensing (QS) is a mechanism by which many bacteria regulate gene expression via the synthesis and detection of small, diffusible signals. Since its discovery, QS has been shown to control diverse physiological functions in numerous types of bacteria. It provides an elegant strategy for bacteria to sense their density and to achieve coordinated population behavior. By stochastic modeling, we show that QS can effectively reduce variability (“noise”) in the expression of its target genes. Surprisingly, the noise reduction does not significantly depend on the number of bacteria but rather results from the coupling of a bacterium and its environment through signal diffusion. Diffusion enables fast signal turnover, which, together with fast intracellular turnover of the cognate receptor of the signal, leads to noise reduction. Our work suggests a unique role of QS in achieving robust gene regulation, which is distinct from noise-regulation mechanisms that act at the intracellular level. As such, it offers novel insights into evolution of QS as well as its application in construction of synthetic gene circuits.
doi:10.1371/journal.pcbi.1000167
PMCID: PMC2507755  PMID: 18769706
21.  A cortical edge-integration model of object-based lightness computation that explains effects of spatial context and individual differences 
Previous work has demonstrated that perceived surface reflectance (lightness) can be modeled in simple contexts in a quantitatively exact way by assuming that the visual system first extracts information about local, directed steps in log luminance, then spatially integrates these steps along paths through the image to compute lightness (Rudd and Zemach, 2004, 2005, 2007). This method of computing lightness is called edge integration. Recent evidence (Rudd, 2013) suggests that human vision employs a default strategy to integrate luminance steps only along paths from a common background region to the targets whose lightness is computed. This implies a role for gestalt grouping in edge-based lightness computation. Rudd (2010) further showed the perceptual weights applied to edges in lightness computation can be influenced by the observer's interpretation of luminance steps as resulting from either spatial variation in surface reflectance or illumination. This implies a role for top-down factors in any edge-based model of lightness (Rudd and Zemach, 2005). Here, I show how the separate influences of grouping and attention on lightness can be modeled in tandem by a cortical mechanism that first employs top-down signals to spatially select regions of interest for lightness computation. An object-based network computation, involving neurons that code for border-ownership, then automatically sets the neural gains applied to edge signals surviving the earlier spatial selection stage. Only the borders that survive both processing stages are spatially integrated to compute lightness. The model assumptions are consistent with those of the cortical lightness model presented earlier by Rudd (2010, 2013), and with neurophysiological data indicating extraction of local edge information in V1, network computations to establish figure-ground relations and border ownership in V2, and edge integration to encode lightness and darkness signals in V4.
doi:10.3389/fnhum.2014.00640
PMCID: PMC4141467  PMID: 25202253
lightness; brightness; achromatic color; cortical ventral stream; neural computation
22.  Natural variation reveals that intracellular distribution of ELF3 protein is associated with function in the circadian clock 
eLife  2014;3:e02206.
Natural selection of variants within the Arabidopsis thaliana circadian clock can be attributed to adaptation to varying environments. To define a basis for such variation, we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant inbred line and localized heritable variation. Extensive variation led us to identify EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal nucleotide polymorphism caused a short-period phenotype under light and severely dampened rhythm generation in darkness, and entrainment alterations resulted. We found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability to accumulate in darkness was compromised. Evidence was provided that the ELF3-Sha allele originated in Central Asia. Collectively, we showed that ELF3 protein plays a vital role in defining its light-repressor action in the circadian clock and that its functional abilities are largely dependent on its cellular localization.
DOI: http://dx.doi.org/10.7554/eLife.02206.001
eLife digest
Life on Earth tends to follow a daily rhythm: some animals are awake during the day and asleep at night, whilst others are more active at night, or during the twilight around dawn and dusk. For many living things, these cycles of activity are driven by an internal body clock that helps the organism to adapt to the daily cycle of light and dark—and similar internal clocks also exist in plants.
These internal clocks define daily—or circadian—cycles whereby multiple genes are switched ‘on’ or ‘off’ at different time points in every 24-hr period. And, because light and ambient temperatures also vary with time of the day, many organisms use these external signals as cues to reset their own internal clocks. Moreover, the hours of daylight and temperature vary around the world, and also with the seasons, so plants and animals must be able to change how these external signals influence their internal clocks so that they stay in tune with the day/night cycle. However, it is not clear how they do this.
To explore this question, Anwer et al. grew plants that were from a cross between two types of the model plant Arabidopsis thaliana from different environments: one from Germany, and the other from Tajikistan in Central Asia. These offspring were also genetically engineered so that an enzyme that could give off light was produced under the control of the internal clock. Anwer et al. found that the plants continued to glow and fade with an almost daily rhythm even after external cues, such as changes in temperature or light, had been removed.
Different offspring plants consistently glowed and faded with different rhythms such that some had, for example, a 21-hr day and others a 28-hr day. These differences were caused by many genes that differed from the original German and Tajikistan parent plants, and Anwer et al. ‘mapped’ one of these genetic differences to a single gene. Offspring that inherited a version of a gene called ELF3 from the Tajikistan parent had internal clocks that ran faster when the plant was under the light. These plants also gradually stopped glowing as brightly as the German parent when they were kept in the dark, suggesting that their internal clocks were ‘ticking more softly’. It was already known that the ELF3 gene affected the circadian clock in plants, and Anwer et al. thus concluded that the plants with Tajikistan version of this gene, called ELF3-Sha, were also less able to reset their internal clocks to synchronize in response to external cues.
Anwer et al. also showed that the normal ELF3 protein is more likely to be found in the nucleus of a plant cell than the ELF3-Sha version, which might suggest that this protein is involved in switching genes off. Further research is now needed to uncover exactly how the ELF3 protein does this to keep the plant's internal clock ‘ticking’ correctly.
DOI: http://dx.doi.org/10.7554/eLife.02206.002
doi:10.7554/eLife.02206
PMCID: PMC4071560  PMID: 24867215
circadian clock; QTL mapping/cloning; cell biology; eQTL; population analysis; Arabidopsis
23.  Hypersensitive to Red and Blue 1 and Its Modification by Protein Phosphatase 7 Are Implicated in the Control of Arabidopsis Stomatal Aperture 
PLoS Genetics  2012;8(5):e1002674.
The stomatal pores are located on the plant leaf epidermis and regulate CO2 uptake for photosynthesis and the loss of water by transpiration. Their stomatal aperture therefore affects photosynthesis, water use efficiency, and agricultural crop yields. Blue light, one of the environmental signals that regulates the plant stomatal aperture, is perceived by the blue/UV-A light-absorbing cryptochromes and phototropins. The signal transduction cascades that link the perception of light to the stomatal opening response are still largely unknown. Here, we report two new players, Hypersensitive to Red and Blue 1 (HRB1) and Protein Phosphatase 7 (PP7), and their genetic and biochemical interactions in the control of stomatal aperture. Mutations in either HRB1 or PP7 lead to the misregulation of the stomatal aperture and reduce water loss under blue light. Both HRB1 and PP7 are expressed in the guard cells in response to a light-to-dark or dark-to-light transition. HRB1 interacts with PP7 through its N-terminal ZZ-type zinc finger motif and requires a functional PP7 for its stomatal opening response. HRB1 is phosphorylated in vivo, and PP7 can dephosphorylate HRB1. HRB1 is mostly dephosphorylated in a protein complex of 193 kDa in the dark, and blue light increases complex size to 285 kDa. In the pp7 mutant, this size shift is impaired, and HRB1 is predominately phosphorylated. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation or to bring in new components for the assembly of a functional HRB1 protein complex. Guard cells control stomatal opening in response to multiple environmental or biotic stimuli. This study may furnish strategies that allow plants to enjoy the advantages of both constitutive and ABA-induced protection under water-limiting conditions.
Author Summary
Stomatal aperture is regulated by many environmental and biotic cues such as blue light, drought, elevated CO2 concentrations, high humidity, and pathogenic elicitors. Stomatal apertures vary over diurnal cycles, and stomata tend to be open during the day in response to blue light and tend to be closed at night. The blue/UV-A light-absorbing cryptochromes and phototropins are the receptors for the blue light response. We report the action of HRB1, a nuclear ZZ-type zinc finger protein, and PP7, a positive regulator of blue light signaling in the nucleus, in the signal transduction cascades downstream of blue light perception. Both hrb1 and pp7 mutants are more resistant to dehydration and show reductions in both water loss and blue light-regulated stomatal aperture. Our studies on their genetic and biochemical interactions offer novel insights on the network structure of the light signaling machinery and plant interactions with the environment. Periodic drought is one of the major environmental factors that limits biomass production and crop yield in a changing global climate. Our studies may open new possibilities to engineer plants to survive desiccation.
doi:10.1371/journal.pgen.1002674
PMCID: PMC3349726  PMID: 22589732
24.  Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology 
Nature Communications  2011;2:508-.
Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts.
Biological digital sensors require the fabrication of modular genetic logic gates. Using the Pseudomonas syringae hrp system, Wang and colleagues generate AND, NOT and NAND gates, demonstrating the ability to engineer a modular system from biological elements.
doi:10.1038/ncomms1516
PMCID: PMC3207208  PMID: 22009040
25.  Control of Daily Transcript Oscillations in Drosophila by Light and the Circadian Clock 
PLoS Genetics  2006;2(3):e39.
The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study we examine the relationship between autonomous transcript oscillations and light-driven transcript responses. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod.
Synopsis
Daily changes in sunlight dramatically affect the environment of animals like the fruit fly, a genetic model. To anticipate these changes, the fruit fly possesses a circadian molecular clock that regulates its behavioral activity and other physiology according to the time of day. The clock's mechanism is comprised of genes and their products (proteins and RNAs). One way the clock modulates physiology is by regulating other genes' RNAs. The signature of a circadian RNA is that its levels oscillate once each day. The present study uses microarrays and a novel statistical strategy to resolve how many genes are oscillating in the fly head and verify that there is only a single transcriptional clock. However, this study also finds a large number of genes that are directly regulated by the presence of light. These genes respond to light even in the absence of a functioning clock. Indeed, the light response depends on a signaling component in the retina that has an established role in vision. This represents a new mechanism by which an animal's physiology may be dynamically tuned to daily time.
doi:10.1371/journal.pgen.0020039
PMCID: PMC1413497  PMID: 16565745

Results 1-25 (1109646)