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Vol 2, No 3 (March 2018) /
Microphysiological Systems: automated fabrication via extrusion bioprinting
Margaret Elizabeth Prendergast1, Gabriel Montoya1, Taciana Pereira1, Jakub Lewicki1,2, Ricky Solorzano1, Anthony Atala3
1Allevi Inc., Philadelphia, PA, USA; 2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 3Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA
Contributions: (I) Conception and design: ME Prendergast, G Montoya; (II) Administrative support: R Solorzano, A Atala; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: None; (V) Data analysis and interpretation: None; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
Correspondence to: Margaret Elizabeth Prendergast. Allevi Inc., Philadelphia, PA, USA. Email: margaret@allevi3d.com.
Abstract: Microphysiological systems (MPS) offer great potential for improving pre-clinical testing for pharmaceutical treatments and novel therapies. These advanced in vitro models are designed to recapitulate the basic functions of living tissues or organs through dynamic culture and biomimetic microarchitecture. Increasing advancements in MPS design require advanced fabrication methods, such as extrusion bioprinting. Extrusion bioprinting presents the ability to automate fabrication of these systems in a simplified, one-step process, while providing the ability to fabricate complex, reproducible designs that incorporate dynamic culture, 3D microtissues and integrated sensors for analysis into a single system. This work reviews the main components that constitute these systems, current state-of-the-art MPS fabricated via extrusion bioprinting, and future considerations for the development of MPS.
Keywords: Extrusion bioprinting; biofabrication; Microphysiological systems (MPS); 3D culture
Received: 17 November 2017; Accepted: 31 January 2018; Published: 22 March 2018.
In 1965, Gordon Moore predicted computing would increase in power and decrease in cost at an exponential rate. While this law has continued to uphold in computing, advances in drug development have followed a troubling alternative trend, referred to as EROOM's law (1). Since 1950, the number of novel treatments approved per billion US dollars spent on R & D has halved approximately every 9 years (1). A large factor in EROOM's law is the high failure rate of potential therapies in clinical trials, highlighting the ineffectiveness of current in vitro testing methods. Many current in vitro models use static 2D cultures that bear little resemblance to native tissue environments. In 2015, less than 12% of drugs entering clinical trials resulted in an approved medicine, costing millions of dollars and exposing patients to harmful side effects (2). MPS models have provided great promise for improving the rate of innovation in drug development and off-setting EROOM's law through more physiologically relevant in vitro models (3). These systems are designed to recapitulate the functions of living tissues or organs through relevant microarchitecture and dynamic stimulation of cultures (4).
The fabrication of these devices involves two main parts: microfabrication of the tissue cultures and design of the microfluidics, sensors and other dynamic systems for the delivery of nutrients, chemicals, biological factors and mechanical or electrophysiological signals in a controlled microenvironment. Several techniques are available for this fabrication process, including photolithography, soft lithography, dry etching, sputter coating, chemical vapor deposition and stereolithography (4). While each of these fabrication methods provides individual advantages, many involve non-biocompatible hazardous reagents and require laborious multi-step processes (4).
Recently, extrusion biofabrication methods have been applied for the fabrication of MPS. Bioprinting technologies allow scientists to pattern cell types and create more complex tissue structures, creating more physiologically relevant models useful in translational research (5). This biocompatible process allows the potential for a single-step, automated fabrication of MPS, creating a more efficient fabrication process. Additionally, bioprinting allows for the integration of 3D tissue designs within MPS devices, as opposed to 2D cultures often used with other methods. This review article details design components and considerations for these devices, recent advances in MPS fabricated through extrusion biofabrication and future considerations for this technology.
3D biofabrication technologies
Advantages of 3D culture, such as cell adhesive sites in three dimensions and tunable mechanical properties, became apparent with the development of spheroid cultures in the 1970s (6). As research on 3D cultures progressed, technologies were developed for increased control over the 3D microenvironment. The development of the first 3D bioprinter in the 1990s allowed scientists to design more intricate 3D microarchitectures (7,8). Since the 1990s, biofabrication technologies have become increasingly complex. Today there are a variety of biofabrication methods available, which can be split into two general groups: jetting-based and extrusion-based bioprinting (9).
Jetting-based bioprinting involves non-contact techniques to fabricate 2D or 3D structures using picolitre bioink droplets layered onto a substrate (9). Common jetting-based methods include piezoelectric, thermal, pneumatic and laser-assisted technology (9). Piezoelectric actuators apply a piezo-crystal pulse to eject a small droplet of a bioink (7,9). Thermal methods create bioink droplets by locally increasing temperature within the bioink compartment to generate a bubble (9). Pneumatic pressure and valves generate droplets through opening and closing of microvalves (9). Focused laser energy methods generate vaporization through laser systems to produce small droplets (9). Jetting-based technologies offer advantages such as high resolution ranging from 20–100 µm, but are limited to low viscosity bioinks and have a long processing time, limiting the size of structures that can be created with these methods (7,9).
Extrusion-based bioprinting dispenses continuous filaments of bioinks through micro-nozzles to build 2D or 3D structures (9). These methods dispense bioinks through pneumatic pressure or syringe pumps (9). The speed and volume of material dispensed can be adjusted by controlling the pressure level or displacement of the piston or pump (7,9). While these methods offer lower resolution compared to jet-based technologies, they allow for the fabrication of constructs with clinically relevant sizes in a realistic time-period and provide versatility in material compatibility. Because of these advantages, extrusion-based bioprinting is often regarded as the most promising and is the most common biofabrication method used to develop 3D microphysiological systems (MPS) (4,7,10,11). A more detailed review on various biofabrication technologies can be found in Seol et al. (9).
MPS design components and considerations
The components of MPS can be broken into four key elements: a microfluidic chip or culture chamber, live microtissues, components for dynamic culture and sensors for results readout (12). A visualization of these systems is demonstrated in Figure 1.
Figure 1 Components of MPS. The components of MPS can be broken into four key elements: (A) the housing, or culture chamber; (B) the live microtissue, the primary component of any MPS; (C) controlled flow and dynamic culture components for applied mechanical, shear and electrical forces as well as (D) integrated sensors for real-time analysis of constructs. MPS, Microphysiological systems.
Microfluidic housing/culture chamber
The microfluidic chip contains the housing, or culture chamber, for the live microtissues and often connects this chamber to a dynamic flow system for perfusion of nutrients, bioactive molecules and drug compounds to the live microtissues. Ideally, a culture chamber provides a sterile environment that can be fabricated or molded into custom geometries and can be easily analyzed noninvasively. For this portion of the device, many microfluidics have utilized silicone-based inks such as poly(dimethylsiloxane) (PDMS), which are highly compatible with a variety of fabrication methods and provide optical clarity, low cost and high reproducibility (13). However, PDMS comes with some significant disadvantages for MPS including deformation, evaporation, absorption, leaching and hydrophobic recovery (13-15). The most prominent disadvantage of this material for use in MPS is the absorption of proteins, drugs and hydrophobic molecules, complicating the pharmacokinetic and pharmacodynamic models of drugs. A variety of alternatives to PDMS have been developed, with some of the most promising including acrylonitrile butadiene styrene (ABS), poly(lactic acid) (PLA) and polycaprolactone (PCL). These materials, which overcome the disadvantages of deformation, absorption, leaching and hydrophobic recovery with PDMS, can be used with transparent glass or polystyrene substrates to fabricate chambers for MPS. Additionally, because of their printability, these materials can be used in extrusion biofabrication platforms for one step biofabrication of both the culture chamber and the live microtissues. A summary of these materials, along with advantages and disadvantages, is presented in Table 1. Many recent publications have attempted to move away from the use of PDMS towards these alternative materials.
Table 1 Materials for microfluidic housing
Live microtissues
The primary component of any MPS is the live microtissue. Live microtissues can vary in complexity, from simple 2D cultures along the walls or grooves of the microfluidic chip to intricate, multicellular 3D microarchitectures. Extrusion biofabrication offers the capability to create both simple, uniform cell layers and more complex 3D architectures. A variety of tissue types have been modeled as MPS through extrusion bioprinting, some of which are detailed in Section IV below and in Table 2.
Table 2 MPS fabricated via extrusion bioprinting
3D Microtissues
Biofabrication of MPS allows for spatial control of cell-laden materials to mimic multi-cellular microphysiological architectures. A variety of materials can be used for this purpose. Briefly, common materials used in 3D microtissues for cell deposition include naturally-derived and synthetic hydrogels. While some MPS utilize scaffold-free cell slurries, matrix bioinks allow for enhanced resolution and control over cell deposition. Additionally, use of matrix bioinks allows for control over substrate mechanical properties and biological factors. This material and cell selection are not the primary focus of this review, although more can be learned from our previous publication on this topic (8). Some examples of matrix bioinks used in MPS can be found in Table 2.
Channel fabrication in live 3D microtissues
Vasculature is a major component of many tissues and is critical in modeling physiologically relevant in vitro models. Vasculature is particularly important for drug screening technologies, as many compounds are injected directly into the blood stream. Many MPS incorporate some type of channel to mimic vascular flow, ranging from simple straight channels to more complex branched features.
Extrusion biofabrication offers a variety of methods for incorporation of microchannels to apply flow and recapitulate vasculature structures within microtissues (4,11,22,24). Often, sacrificial bioinks are used to fabricate complex channels within a microtissue. Some common materials used for this purpose are pluronic F127, agarose, gelatin and carbohydrate glass (8). Bioinks such as pluronic, agarose and gelatin offer a thermal reversible gelation that allow the materials to be printed at room temperature then washed away either through heating or cooling. Bioinks such as carbohydrate glass offer increased mechanical stiffness compared to other softer sacrificial materials that allow for easier construction of more complex designs (8).
Temporary support baths utilizing the sacrificial material gelatin have also demonstrated great promise for the fabrication of microtissues with complex channels (8). The FRESH method, further described in a previous publication, provides temporary support of soft, fragile bioinks during fabrication. This method has not yet been used for the fabrication of MPS, but has great potential for the automation of highly complex vasculature (8,25).
Dynamic culture: flow, mechanical and electrical stimulation
MPS may also contain extra components to stimulate flow or incorporate additional mechanical or electrical stimulation of tissues during culture.
Stimulating flow: pumps for MPS
To create better in vitro models of dynamic in vivo cellular systems, pumps are often used to expose cells to flowing media, as compared to static in vitro cultures. Pumps can also act as an automated way to change cell media, and can be either "closed" (recirculating liquid) or "open" (refillable main liquid reservoir) systems. The application should determine the type of pump used, as different types offer various advantages and disadvantages. Some characteristics to consider are flow rate, flow type (pulsatile or constant), duration of flow, mechanical effects, and electrical requirements. The most common pumps used in MPS are gravity-driven flow, peristaltic pumps, syringe pumps and electroosmotic pumps (Table 3, Figure 2). For a more comprehensive review on pumps, refer to reference (26).
Table 3 Pumps for MPS culture
Figure 2 Pumps for applied flow [adapted from (26)]. To create better in vitro models of dynamic in vivo cellular systems, pumps are often used to expose cells to flowing media. The most common pumps used in MPS are syringe pumps (A); electroosmotic pumps (B); peristaltic pumps (C); and gravity-driven flow (D). Gravity-driven flow systems can be set up with reservoirs oriented vertically (top) or horizontally (bottom). ∆h, change in height; Vapp, voltage applied; arrows indicate direction of flow. MPS, Microphysiological systems.
Gravity-driven flow
Gravity-driven flow is a passive flow technique that is simple to implement, as it requires no mechanical parts or power. It is comprised of only two liquid reservoirs with a height difference between them, creating a flow of liquid from the higher reservoir to the lower one (Figure 2). In a closed system, the flow rate will decrease as the height difference between the reservoirs decreases. However, this effect can be minimized by orienting the reservoirs horizontally (Figure 2). In an open system, refilling the higher reservoir will also negate this problem and prolong the flow. While gravity-driven pumps are only able to produce a constant flow rate, the simplicity of these pumps can be beneficial in complex applications where tubing and multiple pumps would otherwise be needed.
Peristaltic pumps are a type of active, positive-displacement pump that results in sinusoidal, pulsatile flow (24,26). With these systems, liquid is pushed through a tube that is wrapped around rotors that are turned by a rotating shaft (Figure 2). Peristaltic pumps are very versatile as they are bi-directional, have large ranges of flow rates, and can be used as either closed or open systems. However, peristaltic pumps require power and have mechanical components that can wear with time. Additionally, these systems can have a large footprint, which brought about the push for smaller pumps such as the peristaltic micropump (28). Wagner et al. utilized a peristaltic micropump in a multi-organ-chip system that was integrated directly onto the MPS (32). This pump, no more than a few millimeters in width, provided pulsatile flow at a circulation rate of 40 µL/min to two culture compartments (32). Continued development and miniaturization of peristaltic pumps provides great promise for the advancement of MPS.
Another type of active, positive-displacement pump is the syringe pump (22,29,30). These pumps utilize a motor-driven piston to create either pulsatile or constant flow stemming from a syringe full of liquid (Figure 2). Since all liquid must originate from the syringe, it is a closed system. This can become an obstacle in longer term projects where the piston syringe would need to be refilled. However, these pumps offer significant control over flow, making them very popular and widely available.
Electroosmotic pumps
As opposed to many other pumps, electroosmotic pumps are not mechanical, meaning that they have no moving parts. Instead, these pumps use electricity for active flow generation in microchannels in an open system. A surface-ionizable material, such as silica, becomes negatively charged due to deprotonation (31). This charge attracts cations, which form a "positively charged solution layer" close to the channel's surface. When an external electric field is applied, the cations move, pulling the solution with them. The opposite can be done with a positively charged surface. Electroosmotic flow (EOF) is simple but requires caution, as electric fields above 1 kV/cm can cause cell lysis (27).
Electrical stimulation and integrated sensors
A variety of biocompatible conductive materials have been used to provide electrical stimulation to microtissues (33,34). Electroactive biomaterials have the ability to deliver electrical, electrochemical and electromechanical stimulation to cells and can also be used to create integrated sensors in MPS (35). Bioprinting provides the opportunity to incorporate these materials directly into microtissues and MPS. Some common conductive materials utilized in bioprinting include gold, graphene, carbon and silver-based inks as well as electrically conductive polymers such as polymer polypyrrole (PPy).
Gold nanoparticles are commonly incorporated into biomaterials for a variety of applications and offer an array of benefits, including high surface energy, high conductivity, and biocompatibility (36). Gold-based materials have been utilized for the fabrication of biosensors and have been incorporated into live microtissues for functionalization and electrical stimulation. A gold nanorod-incorporated (GNR) gelatin methacrylate (GelMA)-based ink was used to print functional cardiac tissue constructs. These GNR constructs demonstrated increased cell adhesion and organization as well as promotion of synchronized contraction of cardiac cells compared to non-GNR constructs (37).
Due to its unique structure, graphene provides a variety of advantages including high fracture strength, electrical and thermal conductivity, fast mobility of charge carriers and high biocompatibility (38). This material was incorporated into a GelMA-based ink to fabricate graphene nanoplatelets for nerve tissue regeneration (39). The conductive substrate provides cues to developing cells to reinforce electrical connections and promotes formation of a neural network (39).
Carbon-based inks are another popular material for biofabrication due to their electrical conductivity and mechanical properties. Several carbon-based bioinks have been fabricated over the past year, but a particularly promising one from Adams et al. demonstrates a biocompatible carbon-nanotube ink that can fabricate 3D functionalized structures (40). This bioprinted MPS demonstrated high cell attachment and differentiated actin cytoskeletal structures when electrical impulse was applied compared to unstimulated scaffolds (40).
Recently, electroconductive materials have also been used in MPS to incorporate sensors for automated, quantifiable results readout. Extrusion biofabrication presents the opportunity to fabricate these sensors directly into the live microtissues, as described in the cardiac MPS from Lewis et al. shown in Section IV and Figure 3 below (15). This system utilized a silver particle-filled, polyamide (Ag:PA) ink to fabricate electrical leads and contact pads, as well as a TPU-CB-TPU (TPU, thermoplastic polyurethane; CB, carbon black nanoparticles) ink within the structures (15). These sensors allowed for noninvasive analysis of the MPS (15).
Figure 3 Cardiac microphysiological device principle and microscale 3D-printing procedure [adapted from (15)]. (A) Principle sketch of the device. Contraction of an anisotropic engineered cardiac tissue [1] deflects a cantilever substrate [2], thereby stretching a soft strain gauge embedded in the cantilever. This generates a resistance change proportional to the contractile stress of the tissue [3]; (B) the fully printed final device. Insert 1: confocal microscopy image of immuno-stained laminar NRVM cardiac tissue on the cantilever surface. Blue: DAPI nuclei stain, White: α-actinin, scale bar 10 µm. Insert 2: still images of a cantilever deflecting upon tissue contraction. Insert 3: example resistance signal; (C-I) automated printing of the device on a 2 inch × 3 inch glass slide substrate in 7 sequential steps. For each step, a corresponding still image from the printing procedure is displayed.
Electroactive biomaterials have also been used for mechanical stimulation in MPS. Svennersten et al. utilized a PPy, which exhibits good biocompatibility, chemical stability and high conductivity, to fabricate a microactuator system on a chip that delivered mechanical stimulation to renal epithelial cells (35,41). This stimulation resulted in increased internal calcium levels, a documented response to mechanical stimulation (35).
Mechanical stimulation
Many tissues are exposed to continuous mechanical stimulation in vivo. To properly mimic this environment, many MPS aim to include mechanical stimulation (42,43). In addition to use of electroactive biomaterials, this stimulation can also be achieved through application of uniaxial, cyclical, or biaxial strain.
Huh et al. successfully applied uniaxial strain in a lung on a chip model through generation of displacement along one axis (43,44). This model incorporated 3D microarchitecture and dynamic mechanical activity to recapitulate the function of the alveolar-capillary interface (44). In a different study, cyclical stain was used to promote differentiation of murine embryonic stem cells into cardiomyocytes, demonstrating upregulation of α-MHC in mechanically stimulated embryoid bodies (43). While sometimes more complex than uniaxial or cyclical strain, biaxial strain has high potential for high-throughput devices and can be incorporated in a variety of ways. Biaxial strain was applied with a stretching device and applied vacuum chamber on a lung-on-a-chip device (43). Alternatively, another model designed wells with a deformable bottom to apply biaxial strain to cardiac progenitor cells, demonstrating a successful activation of β-catenin (43).
Mechanically active MPS have the potential to reproduce dynamic physical forces that are critical for organ function and disease development. To effectively recapitulate in vivo environments, careful consideration should be made in designing these forces. A more comprehensive review of these forces and methods to apply them can be found in reference (45). Biofabrication platforms have the potential to simplify the fabrication of these incorporated components through the automation of chamber designs used to apply these forces.
MPS fabricated via extrusion bioprinting
Utilizing the components described above, many MPS of varying tissues have been fabricated through extrusion biofabrication. Some of these models are summarized in Table 2.
The blood-brain barrier (BBB) plays an integral role in regulating transport phenomena of small- and macro-molecules as well as maintaining homeostasis of the brain microenvironment (16). Dysfunction of the BBB is associated with a variety of neurological disorders, including neurodegenerative disease and ischemia, and as such, there is a high interest in developing more effective in vitro models of this system (16). Kim et al. developed a model by 3D co-printing a polymeric frame with dissolvable poly(vinyl alcohol) to create a microvasculature structure to encase cell-laden collagen. This model was used to develop a time-dependent evolution of the barrier function for up to 3 weeks (16).
Cardiovascular disease prevails as the leading cause of death worldwide (46). Additionally, a large number of treatments for other diseases present cardiotoxic side effects, leading to withdrawal of therapies from the market (46). While a biomimetic cardiac model is clearly necessary for the advancement of medicine, the complex geometry and mechanisms of the heart is especially difficult to recapitulate and requires advanced fabrication methods.
Vasculature in cardiac models is necessary for the culture of thick cardiac tissue, but its complexity is not able to be replicated through traditional fabrication methods. Zhang et al. explored a novel strategy for fabricating endothelialized myocardial tissues via the use of extrusion bioprinting, microfluidics and stem cells (17). When combined with a microfluidic perfusion bioreactor, the bioprinted model remained viable for up to 15 days with perfusion, compared to significant cell death by day 7 without perfusion (17). This model was also tested with hiPSC-cardiomyocytes and with further development has a potential for use in personalized drug screening (17).
While 3D models provide enhanced physiological relevance, the increased complexity often causes challenges when analyzing tissue constructs. The Lewis and Parker group worked to integrate embedded sensors into a 3D bioprinted cardiac MPS for easier analysis of contractile forces of cells (15). Using a custom extrusion biofabrication platform, the group was able to sequentially print multiple functional materials for a one-step fabrication of a comprehensive chip to test contractile force and drug response of cardiac tissues (15). The constructs were fabricated with an array of PDMS wells as the microfluidic housing with a sandwich structure of TPU-CB-TPU printed inside each well. The electrical conductivity of the embedded CB structures is affected by strain and was used to measure the contractile forces of cells cultured on the constructs (15).
While 20% of failures in Phase III clinical trials are caused from renal toxicity, only 2% of drugs tested in pre-clinical phases fail from this issue (11). Therefore, it is imperative to create more physiologically relevant 3D models of the kidney tissue to improve pre-clinical testing accuracy of therapeutics and drugs (11). The proximal tubule, responsible for 65–80% of nutrient absorption and transport from the renal filtrate to the blood, is the most frequently damaged site in the kidney from prescription drugs (11). Prior to the study by Homan et al., models of the proximal tubule consisted of either 3D static cultures limited to 1 mm in size or perfusion models of 2D culture (11). Through extrusion bioprinting, this group was able to fabricate complex 3D perfusable architecture that was cultured for over 2 months. The model was tested with a nephrotoxin, cyclosporine A, and demonstrated disruption of the epithelial barrier in a dose dependent manner (11). This model significantly enhanced epithelial morphology and functional properties relative to the same cell types grown on 2D controls or without perfusion. The automated, customizable, one-step fabrication method demonstrates the power of extrusion biofabrication for modeling MPS (11).
Drug-induced-liver-injury (DILI) is the most common reason for regulatory actions post drug approval, yet almost 50% of drugs that cause substantial DILI produce little or no toxicity in animal or preclinical models (47,48). In vitro liver models have increased in complexity over the past few decades, from simple organoids of hepatocytes to 3D patterned cells in microfluidic chambers (49). Comparisons of various models have demonstrated the superiority of 3D cultures as well as fluid flow over 2D, static cultures (48-50). Extrusion biofabrication presents the capability to create advanced 3D architectures and culture chambers in one step, and a variety of liver-on-chip models have been fabricated through this method (51-53).
Bhise et al. utilized extrusion biofabrication to control deposition, placement and thickness of spheroid-laden GelMA solutions to fabricate a 3D hepatic construct (10). Through bioprinting technology, variables such as spheroid concentration, hydrogel composition and geometry of printed constructs could be easily changed and controlled (10). The biofabrication process was designed to interact with a pre-fabricated bioreactor and culture chamber, which was used to provide a controlled flow rate of media to constructs at 200 µL/h (10) Cultured spheroids maintained viability for up to 30 days of culture and demonstrated similar response to APAP treatment as animal models, demonstrating potential for its use as a model for drug toxicity analysis (10).
Lee et al. not only created geometrically complex multicellular constructs, but also utilized extrusion bioprinting for a one-step fabrication process of the construct and culture chamber (20). The group tested various geometries, including a 2D printed scaffold-free model, a single-cell 3D model and a multi-cellular 3D model, all encased in a 3D printed polycaprolactone or PDMS chamber (20). Results demonstrated lower protein absorption in PCL chamber constructs compared to platforms fabricated with PDMS, and liver function was significantly improved in 3D models compared to 2D ones (20) .
Lung-on-a-chip
One of the most common methods of entry for drugs, pathogens and other agents into the human body are the lungs. Modeling this process via MPS is important for studying disease pathways and to analyze the transport of aerosol based drugs (49). Most systems aim to model the epithelial-endothelial layers that create a semi-permeable barrier that separate air and fluid channels (49).
The automation of fabricating simple cell layers through biofabrication was demonstrated by Horváth et al. by printing cell-laden Matrigel layers of human alveolar epithelial type II cell line A549 and hybrid human cell line EA hy926 onto transwell culture plate inserts (23). Compared to constructs fabricated via pipetting, the cultures fabricated via extrusion bioprinting demonstrated improved uniformity and tightness of mono- and co-cultures (23). This automated fabrication process, combined with pneumatic channels and cyclic wall stretching used in Huh et al., described in Section III above, could create a truly superior model for analysis (44). Additionally, the chambers utilized in Huh et al. could be potentially bioprinted as well, creating a simplified, one-step fabrication process (44).
Nervous-system-on-a-chip
Enhanced models of the nervous system are necessary to better understand the complex neurological phenomena and to develop effective treatment for neurological disorders. Johnson et al. developed a bioprinted nervous system model through fabricated PCL microchannels with compartmentalized grease and silicone chambers and cell suspensions of rat embryonic hippocampal neurons, rat embryonic sensory neurons, rat embryonic Schwann cells and porcine kidney epithelial cells (21). These components migrated throughout the channels to form an interconnected nervous system that was used to investigate the preferential transmission of pseudorabies virus (PRV) from the cell body of PNS, CNS or the terminal site (21).
Vessels-on-chips
In vitro models focused on vascular-related diseases are integral for developing improved treatments of these diseases. Thrombosis in particular is a leading cause for morbidity and mortality from cardiovascular diseases (22). Through the commercially available Allevi (previously BioBots) platform, Zhang et al. fabricated a fibroblast laden GelMA hydrogel with endothelialized channels and induced thrombi through the infusion of blood into the model (22). Migration of cells in response to the clot and the pathology of fibrosis were analyzed with this model. To form the vascular channels, Zhang et al. utilized pluronic F127, a sacrificial material that can be washed away after fabrication (22). This model is illustrated in Figure 4 (22).
Figure 4 Thrombosis-on-a-chip [adapted from (22)]. (A) Schematics and (B) photographs showing the sacrificial bioprinting method to fabricate thrombosis-on-a-chip. (A) i,ii) bioprinting of a pluronic template; iii) dried template is placed on a PDMS support; iv) the mold is filled with GelMA followed by crosslinking; v) dissolution of the sacrificial channels and frame to produce; vi) the final construct with hollow channels; (B) photographs showing the experimental depiction of the corresponding steps of the sacrificial bioprinting process illustrated in (A); (C) endothelialization of the hollow microchannels inside the GelMA construct for (i) a linear and (ii) bifurcating microchannels; (iii) CD31 (green) and nuclei (blue) staining of the confluent layer of HUVECs. PDMS, poly(dimethylsiloxane); GelMA, gelatin methacrylate; UV, ultraviolet light; HUVECs, human umbilical vein endothelial cells.
Cancer-on-chips
In addition to healthy tissue models, there is also a large need for disease models for in vitro testing. Tumor MPS have the potential to offer more physiologically relevant models through tumor heterogeneity, vasculature incorporation and biomimetic spheroid structures (18). Bioprinting has demonstrated improvement over previous fabrication methods for cell deposition into microfluidic devices, and offers great potential for advanced cancer in vitro models (18).
Multi-tissue platforms: towards "Body-on-a-chip" devices
The advancement of single MPS platforms has led to research on multi-tissue devices towards full "body-on-a-chip" or "human-on-a-chip" devices. These devices aim to create more systemic in-vitro models of metabolic pathways within the human body for more relevant pharmacokinetic and pharmacodynamic analysis.
One notable multi-tissue MPS fabricated through extrusion bioprinting is a three-tissue organ-on-a-chip model from Skardal et al. (54). To mimic inter-organ responses and create a multi-tissue biomimetic model, Skardal et al. developed a three-tissue MPS consisting of liver, heart and lung microtissues connected in a closed, circulatory perfusion system. The system demonstrated secondary toxicity in cardiac tissues likely caused from the release of inflammatory cytokines from lung microtissue after exposure to a known toxic compound bleomycin (54). Skardal et al. utilized biofabrication to construct liver structures and the PCL microchambers (54).
While few multi-tissue systems have been developed through biofabrication, there is high potential for advancement of multi-tissue systems with this method, which lends easily to the scaled complexity of these systems (55,56). A more comprehensive review of other multi-tissue MPS can be found in reference (57).
Discussion: current limitations and future considerations
Advances in biofabrication technologies, specifically in extrusion biofabrication, have led to the progression of increasingly biomimetic MPS. Extrusion biofabrication offers many advantages over other methods, including biocompatibility, the versatility to pattern a variety of materials, and the automated one-step fabrication of multi-material, complex microstructures. These advantages have led to the development of superior tissue models previously impossible to fabricate through other methods. These models incorporate 3D cell culture, advanced sensors for analysis and dynamic culture of systems through controlled flow and electrical and mechanical forces.
Yet many fabricated MPS still utilize outdated, laborious methods for fabrication of these models. This lack of adoption is likely due to the initial barrier of access to the advanced technology of extrusion biofabrication. Many models above were fabricated on expensive, complex, custom-built platforms, which most labs do not have the expertise or funding to develop. These custom-built devices, while extremely powerful, limit the ability for reproducibility or collaboration. Likewise, initial commercially available extrusion biofabrication platforms were extremely expensive and difficult to use. This landscape is changing, however, with accessible, low-cost biofabrication platforms that incorporate the advanced technologies of these other platforms. The potential of these standardized, low-cost platforms is evident through advancements such as the thrombosis model depicted in Figure 4 and developed by Zhang et al. (22). With a standardized system like the one used in this work, advancements in the field can be easily reproduced and scaled. This capability will be especially important for the translation and mass production of these systems. A standardized platform also presents promise for translation of MPS into clinical practice for personalized medicine. Personalized therapies are becoming more and more relevant, and it will be necessary to develop methods to test compatibility of such therapies with individual patients (58,59). Consideration not only in MPS design but also manufacturing method, scalability and fabrication time must be considered. Standardized extrusion biofabrication platforms present great promise for this development and translation. In addition to standardization of these systems, continued improvement in extrusion biofabrication technology, as well as matrix bioinks, is necessary to provide enhanced resolution and manufacturing capabilities for the continued advancement of MPS.
Many of the current MPS fabricated with extrusion biofabrication incorporate some but not all advantages of this method. Systems focus on uniform patterning of 2D cells, or controlled architecture of vasculature channels, but do not always incorporate sensors for analysis, multiple cell types, 3D culture environments or design for application of additional mechanical or electrical stimuli. As more research institutions gain access to this technology and the field continues to mature, we predict the development of even more biomimetic, complex MPS that incorporate all of these features. Likewise, more multi-tissue platforms will likely be developed for the prediction of multiorgan toxicities and analysis. As these advancements rapidly develop, it would be useful to create a standardized method of analysis for success or failure of each of these systems.
These methods of analysis will need to be tailored for each tissue and tissue model developed, and should be based on functions measured in the clinic. MPS should be compared not only to current in vitro models, as many often are, but also to the in vivo systems or pathology the component is attempting to model. Clear markers of improvement and success should be determined prior to readout. Computational models should be developed to compare measurements in MPS to in vivo systems. Programs such as the National Institute of Health (NIH) MPS Program run by the NIH, the Defense Advanced Research Projects Agency (DARPA) and the U.S. Food and Drug Administration (FDA) not only provide funding for MPS research but also provide a set of standards and compounds for the testing of new models (60). This set of standards involves minimal functional requirements for each system and a set of training compounds based on known organ-specific toxicities in the human population (60). These types of standards should be further developed and applied to all projects regardless of funding for effective development of new systems (60).
In addition to standards for testing new systems, it is imperative to also consider the analysis of these systems and how data for this analysis will be collected. Common methods such as staining or anti-body based assays are often destructive and time-consuming (61). MPS must move toward real-time, non-destructive read-out capabilities for active monitoring and technologies with realistic scalability. Advances in soft sensor technologies and developments of bioinks for fabrication of integrated electrode arrays, such as the ones in Lewis et al., demonstrate an ideal approach to MPS design. More advancements in bioinks and incorporation of these soft sensor technologies are needed for the advancement of MPS.
Current methods of in vitro testing are highly ineffective and costly. MPS provide great promise to improve the effectiveness and decrease the cost of this testing. For truly advanced in vitro models, however, advanced fabrication methods must be implemented to recapitulate complex in vivo environments. Extrusion biofabrication platforms allow for the complexity, versatility and reproducibility necessary to fabricate truly physiologically relevant models. These systems have the potential to save billions of dollars and minimize exposure of harmful treatments to both animals and humans. Through automated fabrication via extrusion biofabrication, advancements in MPS have the potential to advance the rate of discovery of therapies to improve human health, hopefully creating a pace of advancement closer to Moore's law.
Conflicts of Interest: Margaret Prendergast, Ricardo Solorzano and Taciana Pereira work for Allevi, Inc. The other authors have no conflicts of interest to declare.
Scannell JW, Blanckley A, Boldon H, et al. Diagnosing the decline in pharmaceutical R&D efficiency. Nat Rev Drug Discov 2012;11:191-200. [Crossref] [PubMed]
Pharmaceutical Research and Manufacturers of America. 2016 biopharmaceutical research industry profile. Washington, DC: PhRMA, 2016.
Fabre KM, Livingston C, Tagle DA. Organs-on-chips (microphysiological systems): tools to expedite efficacy and toxicity testing in human tissue. Exp Biol Med (Maywood) 2014;239:1073-7. [Crossref] [PubMed]
Chandra P, Kengla C, Jin S. MIcrofabrication and 3D-Bioprinting of Organ-on-a-Chip. In: Murphy SV, Atala A, editor. Regenerative Medicine Technology: On-a-Chip Applications for Disease Modeling, Drug Discovery and Personalized Medicine. CRC Press: Taylor & Francis Group, 2016.
Ravi M, Paramesh V, Kaviya SR, et al. 3D cell culture systems: advantages and applications. J Cell Physiol 2015;230:16-26. [Crossref] [PubMed]
Benien P, Swami A. 3D tumor models: history, advances and future perspectives. Future Oncol 2014;10:1311-27. [Crossref] [PubMed]
Malda J, Visser J, Melchels FP, et al. 25th anniversary article: Engineering hydrogels for biofabrication. Adv Mater 2013;25:5011-28. [Crossref] [PubMed]
Prendergast ME, Solorzano RD, Cabrera D. Bioinks for biofabrication: current state and future perspectives. Journal of 3D Printing in Medicine 2016. [Epub ahead of print].
Seol YJ, Kang HW, Lee SJ, et al. Bioprinting technology and its applications. Eur J Cardiothorac Surg 2014;46:342-8. [Crossref] [PubMed]
Bhise NS, Manoharan V, Massa S, et al. A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication 2016;8:014101. [Crossref] [PubMed]
Homan KA, Kolesky DB, Skylar-Scott MA, et al. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips. Sci Rep 2016;6:34845. [Crossref] [PubMed]
Yang Q, Lian Q, Xu F. Perspective: Fabrication of integrated organ-on-a-chip via bioprinting. Biomicrofluidics 2017;11:031301. [Crossref] [PubMed]
Berthier E, Young EW, Beebe D. Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip 2012;12:1224-37. [Crossref] [PubMed]
Esch EW, Bahinski A, Huh D. Organs-on-chips at the frontiers of drug discovery. Nat Rev Drug Discov 2015;14:248-60. [Crossref] [PubMed]
Lind JU, Busbee TA, Valentine AD, et al. Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing. Nat Mater 2017;16:303-8. [Crossref] [PubMed]
Kim JA, Kim HN, Im SK, et al. Collagen-based brain microvasculature model in vitro using three-dimensional printed template. Biomicrofluidics 2015;9:024115. [Crossref] [PubMed]
Knowlton S, Joshi A, Yenilmez B, et al. Advancing cancer research using bioprinting for tumor-on-a-chip platforms. International Journal of Bioprinting 2016;2:3-8. [Crossref]
Kizawa H, Nagao E, Shimamura M, et al. Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery. Biochem Biophys Rep 2017;10:186-91. [Crossref] [PubMed]
Lee H, Cho DW. One-step fabrication of an organ-on-a-chip with spatial heterogeneity using a 3D bioprinting technology. Lab Chip 2016;16:2618-25. [Crossref] [PubMed]
Johnson BN, Lancaster KZ, Hogue IB, et al. 3D printed nervous system on a chip. Lab Chip 2016;16:1393-400. [Crossref] [PubMed]
Zhang YS, Davoudi F, Walch P, et al. Bioprinted thrombosis-on-a-chip. Lab Chip 2016;16:4097-105. [Crossref] [PubMed]
Horváth L, Umehara Y, Jud C, et al. Engineering an in vitro air-blood barrier by 3D bioprinting. Sci Rep 2015;5:7974. [Crossref] [PubMed]
Kolesky DB, Homan KA, Skylar-Scott MA, et al. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci U S A 2016;113:3179-84. [Crossref] [PubMed]
Hinton TJ, Jallerat Q, Palchesko RN, et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv 2015;1:e1500758. [Crossref] [PubMed]
Byun CK, Abi-Samra K, Cho YK, et al. Pumps for microfluidic cell culture. Electrophoresis 2014;35:245-57. [Crossref] [PubMed]
Laser DJ, Santiago JG. A review of micropumps. Journal of Micromechanis and Microengineering 2004;14:R35. [Crossref]
Materne EM, Maschmeyer I, Lorenz AK, et al. The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture. J Vis Exp 2015.e52526. [PubMed]
Park JY, Morgan M, Sachs AN, et al. Single cell trapping in larger microwells capable of supporting cell spreading and proliferation. Microfluid Nanofluidics 2010;8:263-8. [Crossref] [PubMed]
Li Z, Mak SY, Sauret A, et al. Syringe-pump-induced fluctuation in all-aqueous microfluidic system implications for flow rate accuracy. Lab Chip 2014;14:744-9. [Crossref] [PubMed]
Wang W, Gu C, Lynch KB, et al. High-pressure open-channel on-chip electroosmotic pump for nanoflow high performance liquid chromatography. Anal Chem 2014;86:1958-64. [Crossref] [PubMed]
Wagner I, Materne EM, Brincker S, et al. A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture. Lab Chip 2013;13:3538-47. [Crossref] [PubMed]
Truby RL, Lewis JA. Printing soft matter in three dimensions. Nature 2016;540:371-8. [Crossref] [PubMed]
Xu Y, Wu X, Guo X, et al. The Boom in 3D-Printed Sensor Technology. Sensors (Basel) 2017;17:E1166. [Crossref] [PubMed]
Balint R, Cassidy NJ, Cartmell SH. Conductive polymers: towards a smart biomaterial for tissue engineering. Acta Biomater 2014;10:2341-53. [Crossref] [PubMed]
Kumar S, Ahlawat W, Kumar R, et al. Graphene, carbon nanotubes, zinc oxide and gold as elite nanomaterials for fabrication of biosensors for healthcare. Biosens Bioelectron 2015;70:498-503. [Crossref] [PubMed]
Zhu K, Shin SR, van Kempen T, et al. Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs. Advanced Functional Materials 2017;27:1605352. [Crossref]
Gardin C, Piattelli A, Zavan B. Graphene in Regenerative Medicine: Focus on Stem Cells and Neuronal Differentiation. Trends Biotechnol 2016;34:435-7. [Crossref] [PubMed]
Wei Zhu, Harris BT, Zhang LG. Gelatin methacrylamide hydrogel with graphene nanoplatelets for neural cell-laden 3D bioprinting. Conf Proc IEEE Eng Med Biol Soc 2016;2016:4185-8.
Adams SD, Ashok A, Kanwar RK, et al. Integrated 3D printed scaffolds and electrical stimulation for enhancing primary human cardiomyocyte cultures. Bioprinting 2017;6:18-24. [Crossref]
Svennersten K, Berggren M, Richter-Dahlfors A, et al. Mechanical stimulation of epithelial cells using polypyrrole microactuators. Lab Chip 2011;11:3287-93. [Crossref] [PubMed]
Huh D, Hamilton GA, Ingber DE. From 3D cell culture to organs-on-chips. Trends Cell Biol 2011;21:745-54. [Crossref] [PubMed]
Uzel SG, Pavesi A, Kamm RD. Microfabrication and microfluidics for muscle tissue models. Prog Biophys Mol Biol 2014;115:279-93. [Crossref] [PubMed]
Huh DD. A human breathing lung-on-a-chip. Ann Am Thorac Soc 2015;12:S42-4. [Crossref] [PubMed]
Davis CA, Zambrano S, Anumolu P, et al. Device-based in vitro techniques for mechanical stimulation of vascular cells: a review. J Biomech Eng 2015;137:040801. [Crossref] [PubMed]
Kurokawa YK, George SC. Tissue engineering the cardiac microenvironment: Multicellular microphysiological systems for drug screening. Adv Drug Deliv Rev 2016;96:225-33. [Crossref] [PubMed]
Xu D, Peltz G. Can Humanized Mice Predict Drug "Behavior" in Humans? Annu Rev Pharmacol Toxicol 2016;56:323-38. [Crossref] [PubMed]
Soldatow VY, Lecluyse EL, Griffith LG, et al. In vitro models for liver toxicity testing. Toxicol Res (Camb) 2013;2:23-39. [Crossref] [PubMed]
Skardal A, Shupe T, Atala A. Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling. Drug Discov Today 2016;21:1399-411. [Crossref] [PubMed]
Choi K, Pfund WP, Andersen ME, et al. Development of 3D dynamic flow model of human liver and its application to prediction of metabolic clearance of 7-ethoxycoumarin. Tissue Eng Part C Methods 2014;20:641-51. [Crossref] [PubMed]
Snyder JE, Hamid Q, Wang C, et al. Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip. Biofabrication 2011;3:034112. [Crossref] [PubMed]
Faulkner-Jones A, Fyfe C, Cornelissen DJ, et al. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D. Biofabrication 2015;7:044102. [Crossref] [PubMed]
Skardal A, Murphy SV, Devarasetty M, et al. Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Sci Rep 2017;7:8837. [Crossref] [PubMed]
Xiao S, Coppeta JR, Rogers HB, et al. A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle. Nat Commun 2017;8:14584. [Crossref] [PubMed]
Yi HG, Lee H, Cho DW. 3D Printing of Organs-On-Chips. Bioengineering (Basel) 2017;4:E10. [Crossref] [PubMed]
Lee SH, Sung JH. Organ-on-a-Chip Technology for Reproducing Multiorgan Physiology. Adv Healthc Mater 2018.7. [PubMed]
Hirsch T, Rothoeft T, Teig N, et al. Regeneration of the entire human epidermis using transgenic stem cells. Nature 2017;551:327-32. [Crossref] [PubMed]
National Cancer Institute. CAR T-Cell Therapy Approved for Some Children and Young Adults with Leukemia. Cancer Currents Blog 2017. Available online: https://www.cancer.gov/news-events/cancer-currents-blog/2017/tisagenlecleucel-fda-childhood-leukemia
Sutherland ML, Fabre KM, Tagle DA. The National Institutes of Health Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline. Stem Cell Res Ther 2013;4 Suppl 1:I1. [Crossref] [PubMed]
Capulli AK, Tian K, Mehandru N, et al. Approaching the in vitro clinical trial: engineering organs on chips. Lab Chip 2014;14:3181-6. [Crossref] [PubMed]
Cite this article as: Prendergast ME, Montoya G, Pereira T, Lewicki J, Solorzano R, Atala A. Microphysiological Systems: automated fabrication via extrusion bioprinting. Microphysiol Syst 2018;2:2.


If you want to get to Tbilisi for weekend, make sure to choose last Sunday of October. This is when the festival Tbilisoba takes place, during which locals commonly feast and thank for summer harvest.
One of the most characteristic elements of architecture of the capital Georgia is the Bridge of Peace, that is adjusted to pedestrians. It perfectly fits into the surroundings, despite its futuristic style.
On the summit of the mountain Sololaki, in the western part of the city, there is a twenty-metres high monument of Mother Georgia (Kartlis deda). For small fee you can get there by cableway to admire the beauty of the whole Tbilisi.
What's worth seeing in Tbilisi?
Tbilisi is called the Pearl of Caucasus – there is nothing short of exaggeration in that statement. The city, which is situated on the joint of Europe and Asia, allows one to feel a genuine oriental vibe, that is composed of lovely views, unique monuments, and above all hospitable guests.
What to pay attention to while walking around the capital of Georgia? To balconies, definitely. Those, which you will see in that spot are real masterpieces – hand-made sculptures, wooden balustrades, the older, the more beautiful and more creative. They are mostly located on second floors of buildings.
Make sure to go to the oldest district of the city – Abanotubani. It is known mainly for baths. The name Tbilisi reportedly comes from sulphur springs, whose are beneficially used by citizens, as tbili means in local language something hot. Baths are topped with copulas, that after dark, when the steam is lingering above them, make huge impression!
In the last few years Tbilisi has been changing, especially when it comes to infrastructure. New restaurants, bars and clubs are founded, old buildings and roads are modernized. Where then should you go when you feel hungry? Exquisite Georgian cuisine you can try in Barbarestan at D. Aghmashenebeli 132. You can also except very pleasant vibe and great, regional cuisine in Old City Wall, whose address is Baratashvili 1. For Asian dishes step by Umami Asian Fusion at 1 Rose Revolution Square.
When upon visiting the town, you would like also to see, how traditional trading spot looks like, go to the Dry Bridge bazaar. Its name refers to a nearby bridge. You can buy there various things, but above all it is worth to step by to hear burble of locals that deal with all kind of business there.


text:
#include <GLTools.h>            // OpenGL toolkit
#include <GLShaderManager.h>    // Shader Manager Class

#ifdef __APPLE__
#include <glut/glut.h>          // OS X version of GLUT
#else
#define FREEGLUT_STATIC
#include <GL/glut.h>            // Windows FreeGlut equivalent
#endif

GLBatch	triangleBatch;
GLShaderManager	shaderManager;

///////////////////////////////////////////////////////////////////////////////
// Window has changed size, or has just been created. In either case, we need
// to use the window dimensions to set the viewport and the projection matrix.
void ChangeSize(int w, int h)
    {
	glViewport(0, 0, w, h);
    }

///////////////////////////////////////////////////////////////////////////////
// This function does any needed initialization on the rendering context.
// This is the first opportunity to do any OpenGL related tasks.
void SetupRC()
	{
	// Blue background
	glClearColor(0.0f, 0.0f, 1.0f, 1.0f );

	shaderManager.InitializeStockShaders();

	// Load up a triangle
	GLfloat vVerts[] = { -0.5f, 0.0f, 0.0f,
		                  0.5f, 0.0f, 0.0f,
						  0.0f, 0.5f, 0.0f };

	triangleBatch.Begin(GL_TRIANGLES, 3);
	triangleBatch.CopyVertexData3f(vVerts);
	triangleBatch.End();
	}

///////////////////////////////////////////////////////////////////////////////
// Called to draw scene
void RenderScene(void)
	{
	// Clear the window with current clearing color
	glClear(GL_COLOR_BUFFER_BIT | GL_DEPTH_BUFFER_BIT | GL_STENCIL_BUFFER_BIT);

	GLfloat vRed[] = { 1.0f, 0.0f, 0.0f, 1.0f };
	shaderManager.UseStockShader(GLT_SHADER_IDENTITY, vRed);
	triangleBatch.Draw();

	// Perform the buffer swap to display back buffer
	glutSwapBuffers();
	}

///////////////////////////////////////////////////////////////////////////////
// Main entry point for GLUT based programs
int main(int argc, char* argv[])
	{
	gltSetWorkingDirectory(argv[0]);

	glutInit(&argc, argv);
	glutInitDisplayMode(GLUT_DOUBLE | GLUT_RGBA | GLUT_DEPTH | GLUT_STENCIL);
	glutInitWindowSize(800, 600);
	glutCreateWindow("Triangle");
    glutReshapeFunc(ChangeSize);
    glutDisplayFunc(RenderScene);

	GLenum err = glewInit();
	if (GLEW_OK != err) {
		fprintf(stderr, "GLEW Error: %s\n", glewGetErrorString(err));
		return 1;
		}

	SetupRC();

	glutMainLoop();
	return 0;
	}

redpajama_set_name: RedPajamaGithub


bitsquare is a decentralized open-source exchange, desktop application that allows you to buy and sell bitcoins in exchange for national currencies, or alternative crypto currencies, without the need to entrust funds to a third party or middleman, meaning that the transactions occur directly between the buyer and seller. This approach makes trading Bitcoin much safer as the exchange is not vulnerable to hacks or thefts and since the software is completely peer-to-peer it is also impervious to DOS attacks.
Using Bitsquare is not only safer than using a centralized exchange, it is also much more private. Centralized exchanges require users to verify their identity, while Bitsquare will only require you to reveal your identity to the person you're selling Bitcoin to, which is anonymous and untraceable like you since the Bisquare network operates using Tor hidden services, and all the private data sent over the wire is end-to-end encrypted.
However, not everything in Bitsquare is superior, as advanced trading features are not available and since everything is decentralized, order books are hosted by the user. This means that both parties must be online for the exchange to begin. Furthermore, both the buyer and seller have to signal certain actions within the trade for it to be successful, meaning that not only the computer must be turned on, but the trader must also be online.


Hargreaves Jones provides independent, impartial, specialist building cost advice on the full scope of construction throughout the life cycle of various projects to the property industry. We work with you to tailor the service to fulfil your criteria and deliver value.
We work in partnership with clients, consultants and contractors in both the private and public sector, including framework programmes. Our proactive approach ensures the best value for our clients, whether we are providing, high-level strategic cost and feasibility advice, procurement and contract advice, contract administration and cost management through the lifecycle, or advising on finance and taxation issues.


The Domain "bogazicikorodernegi.org" belongs to The Company "Masis Aram GOZBEK" . The owner of the bogazicikorodernegi.org and Masis Aram GOZBEK with legal rights for the same is "Masis Aram GOZBEK" .	You can reach directly by dialing 901111111111 & to discuss with Masis Aram GOZBEK. As per domain record provided by whois servers, registered address for bogazicikorodernegi.org , Masis Aram GOZBEK,masisgozbek@gmail.com is "Mahmut Sevket PaSa Cad. UGur Apt. No. 9/2 , ISTANBUL , Yesilkoy , Turkey ".
Some extra details for the bogazicikorodernegi.org and it's Owner are provided below.
Registrar Name	 : Nics Telekomünikasyon Ticaret Ltd. Şti.


text:
Q: Adding CheckBoxes to a Tab header in TabControl (WinForms) I would like to add checkboxes to the place (don't know the formal name) where the text representing the tab appears. I am attaching a sample image of how I want it to look like:

Does anyone know how I might be able to achieve this? Feel free to use any .NET language that you are comfortable with - I can easily translate to what I need.

A: If I had to do this now, I would code

*

*A class "myTab inherits System.Windows.FormsTabControl".

*Add a "Protected Overrides Sub WndProc" to capture the WM_PAINT message, in order to perform a custom paint.

But I would sure try things like that, before:
WinForms TabControl - Add New Tab Button (+)
or
http://www.codeproject.com/Articles/91387/Painting-Your-Own-Tabs-Second-Edition

A: I am thinking a HACKEY way to do this is to use the escape character for "checked" checkbox and "unchecked" checkbox in the tab HEADER string. (Don't know if this will work yet). When the user's mouse is clicked on the "unchecked" checkbox character then change the character to a "checked" checkbox or vice versa. Just simulating the checkbox checked/unchecked visual. There must be a cleaner way to do this.

redpajama_set_name: RedPajamaStackExchange


Call (954) 271-2775 Today to Redeem!
At Watson's Painting & Waterproofing Company, we know choosing a painting contractor is a tough job. So we try to make it easier. Simply pick up the phone and call! We'll be happy to answer any and all of your questions, and if you are ready to move forward, we can provide a free quote.
Watson's Painting & Waterproofing Company has been painting Deerfield Beach, FL and surrounding towns since 1996. With each new painting project, whether house painting or commercial painting, we strive to provide professional, timely, service at a fair price.
For home owners looking for house painting in Deerfield Beach, FL, whether interior or exterior, Watson's Painting & Waterproofing Company's trained painters have years of experience in doing the job right. Have a problem with paint bubbles or premature cracking? We can fix it. Paint bubbles form when moisture gets behind the paint, often originating from the inside of your home. We can fix that by adding tiny spacers to separate exterior siding and allow the moisture to escape. Inside your home, we can paint with latex, oil, or specialty paints. We generally recommend latex paint for interior painting as it affordable, easy to clean-up, and contains fewer hazardous ingredients. However, for a hard tough durable finish, oil paint may be the right choice, though some brands of latex paint are now as durable as oil.
Naturally, staining is not much different than painting, and Watson's Painting & Waterproofing Company's painters are also expert stainer. Whether you are looking to have your trim and moldings stained a natural wood color in your Deerfield Beach home, or perhaps have furniture or cabinets stained, or even have your Deerfield Beach deck power washed and waterproofed with a premium UV resistant mahogany or cedar stain, Watson's Painting & Waterproofing Company can do it.
Watson's Painting & Waterproofing Company has many years of experience brightening lives by painting condo interiors. Moving into a new apartment in Deerfield Beach? Watson's Painting & Waterproofing Company will be happy to add a fresh new layer of paint on the walls and ceiling and make your new apartment look new. No need to spend the next few years living with old faded paint or with someone else's color choice. Paint it your way, with your colors!
Want to increases sales and motivate employees? Paint your Deerfield Beach store or office. Painted walls and ceilings slowly dull over time going from the new bright happy look to drab lifeless uninspiring hue. Watson's Painting & Waterproofing Company works with Deerfield Beach business owners and office managers to send in painters after hours or on weekends so as not to interfere with customers or employees. We've been doing it since 1996.
For larger institutions near Deerfield Beach, Watson's Painting & Waterproofing Company has enough skilled painters in Deerfield Beach to paint your facility, regardless of whether its interior painting, exterior painting, or specialty painting, such as painting machines or painting factory floors.
Watson's Painting & Waterproofing Company's painters will paint your Deerfield Beach house and make it look new again. We use proven durable paints than will protect your home's exterior against UV and harsh heat or cold. And we can paint walls any color you'd like, leaving your life brighter and you feeling rejuvenated!
Leave the work to Watson's Painting & Waterproofing Company while you unwind week nights or weekends. Our commercial painters can arrive just before you leave, and will tidy up and lock up, so that when you arrive the next morning, your store or office is ready for business. Imagine how much happier customers and employee will be.
Like so many other Watson's Painting & Waterproofing Company customers, you'll be glad you met us. Our work is guaranteed, and your satisfaction with your new paint job is our #1 goal.
Watson's Painting & Waterproofing Company, proudly painting Deerfield Beach, FL and surrounding towns since 1996.
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If after completion of the verification process discrepancies are found, the Financial Aid Office will transmit the corrected information to the Central Processor. You will receive a revised Student Aid Report (SAR) reflecting the updated information. Once your file is complete, it will proceed to the packaging phase where an official award will be sent to you. The financial aid award process begins in early March.
2015 Federal Tax Return on file IRS.
Although most FAFSA of the Web applicants will be able to take advantage of IRS Data Retrieval, the following applicants cannot use this tool.


text:
Prior To Blue Line Crash, Operator Worked Long Hours, Nursed Cold, Went To Flag-Football Practice
Filed Under:Blue Line, Bob Roberts, Crash, CTA, Escalator, NTSB
This CTA train smashed into the escalator on Monday morning at the O'Hare station. (Credit: CBS)
(CBS) – The operator who dozed off and ran her CTA Blue Line train through a bumper and up an escalator one year ago this week at an O'Hare Airport terminal describes the circumstances that preceded the accident, in newly released federal records.
Brittney Haywood's 80-page interview — done the day after the March 24, 2014, accident — reveals a woman who said she liked to work overtime but knew even before the accident that staying awake was a "challenge" between 2 a.m. and 3 a.m., the time of day when the accident occurred.
"I was tired," she said in her first comments to the investigating team from the National Transportation Safety Board. "I didn't get any sleep the day before, and I might — I have — I became sleepy and I nodded off into the terminal of O'Hare."
Haywood has not spoken publicly about the accident, which injured 30 and caused approximately $9 million in damage.
She said she had worked 55.7 hours in the week before the accident, some of it as a flagman but most of it as a train operator. She had just marked her two-month anniversary as an operator.
While she said she was able to get a full day's sleep before coming to work that Sunday night, she said she had sacrificed most of her off-time the day before for a flag football practice. She estimated that she napped afterward for 50 minutes, but said even that small amount of rest made her half an hour late for work.
She was due at 4:55 p.m. but arrived at 5:22 p.m., she said. Haywood said she would be given a "miss" for reporting late. She had received another "miss" the month before when she dozed off momentarily and overshot the station platform at Belmont. She said she was aware that four "misses" in a year meant termination.
The trip on which the accident occurred was the fourth between the O'Hare Blue Line terminal and Logan Square. Five round-trips were on her schedule. She said she reported with a cold that she had been nursing for the past week, but was not on medication, just green tea. She said she began to feel tired at the Harlem station and turned off the heat to try to stay alert.
Haywood said she remembered stopping at Rosemont and seeing signals just outside the portal of the tunnel leading to the O'Hare terminal. She said she remembered nothing after that until hitting the trackside "trip" that automatically sets the emergency brakes at the end of track. She said she was trying to move the controller handle from power to emergency braking but "was too late."
Asked for her opinion about the CTA and safety, Haywood told investigators the transit agency's main goal was to "keep the trains moving."
She was quick to add: Better "safe than sorry."
CTA fired Haywood 10 days after the accident. It almost immediately lowered the speed limit on its O'Hare platforms to 15 mph from 25 mph, moved the trip arm farther from the bumper post and re-emphasized its fatigue-awareness training program for rail operators.

redpajama_set_name: RedPajamaCommonCrawl


text:
The Travels of Natural GasNatural Gas Safety-SMART! Home
Gas at
Your Service
Know Your Pipelines
Get Small
Want to know where natural gas comes from? Well, you'll have to use your imagination and get really small. Pretend you're a tiny natural gas molecule, hanging out in a gas trap with other natural gas molecules several miles beneath the earth's surface.
Start Underground
It's dark in there. Luckily, you don't need to breathe because there isn't any air to speak of, just a lot of methane plus some carbon dioxide, butane, ethane, propane, pentane, nitrogen, hexane, heptane, and water vapor. You and the other gas molecules have been in this one spot for a very long time—millions of years, in fact.
You're cozy and content in your little home. Then one day, out of the blue, you hear a loud roar and whoosh!—you're sucked up through a well and into a huge pipeline.
You and your little gas buddies are pumped through this pipeline to a processing plant. There, you say goodbye to the water vapor, carbon dioxide, and other molecules considered impurities. These are removed in a treatment process called "sweetening" the gas.
Next, you are pumped into a network of steel transmission pipes that range in size from 20 to 42 inches in diameter. If you could make a sound, you would probably be able to hear it echo inside these big pipes.
About every 50 to 60 miles you pass through a compressor station. Compressor stations compress (squeeze) you to push you to the next station along the line.
The compressor station works kind of like a hand around a tube of toothpaste. Squeezing the bottom of the tube causes the toothpaste to flow out the top. And the harder it's squeezed, the faster it flows. Likewise, the harder the compressor stations pressurize you, the faster you flow.
You and the other gas molecules are tightly packed together now, and you feel as if you are being pushed quickly through the pipes from behind. Compressing a gas reduces its volume, so many more of you now fit into a smaller space. Not much elbow room left, so it's lucky you don't have elbows!
A Fork in the Road
You continue traveling through the pipeline until you reach a fork—a place where the pipe splits off in different directions. Some of your friends are sent down one fork of the pipeline to distribution companies, where they are put into storage tanks for future use. The rest of you move on to a natural gas utility—a company that delivers natural gas to homes and businesses.
Once you reach the gas utility, your pressure is reduced and a chemical called mercaptan is added to you. You had no odor before, but now you can barely stand your own smell. Mercaptan contains sulfur and makes you smell like rotten eggs. It's added so people will be able to tell if natural gas is leaking from their natural gas pipes or appliances.
Under the Street
From the gas utility, you travel through small pipes called distribution mains. These pipes are between 2 and 24 inches in diameter and run below the streets. You zip along under the street, hearing the rumble of traffic overhead.
All of a sudden, the pipeline you were in gets smaller, and smaller again. Now you are in a service line that leads from the distribution main to a house. You are about to go through a gas meter! You're about to be used in someone's home! The gas meter measures the amount of gas used in the home, just as an electric meter measures electricity usage. The gas company uses this information to figure out how much to charge on people's gas bills.
Before entering the meter, you go through a gas pressure regulator where your pressurization is reduced. Then, you whiz through the meter and into gas lines—pipes that are an inch wide or less. You can relax a little now, because you're not under as much pressure. But you continue to bounce around with excitement, eager to find out which appliance you'll serve. Will it be the gas range in the kitchen? The gas clothes dryer? The water heater? The furnace? Whichever it is, you hope you'll be put to good use.


If you're linking to a wiki page, capitalization does not matter. So use what every capitalization is appropriate in your sentence.
All punctuation besides the apostrophy ('), space ( ), dash (-), and underscore (_) and period (.) are ignoreg completely. Also space, dash, apostrophy and underscore are not distinguished (ie they are interchangable as are say (a) and (A).) Periods (.) are treated as a unique character, as you might expect, except if they are at the begining of the name, in which case they are ignored completely.


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text:
Justin Bieber >
Justin Bieber Takes Paternity Test
By WENN on 18 November 2011
Follow Justin Bieber
Picture: Justin Bieber out shopping in central London London, England - 14.11.11
Pop star Justin Bieber has undergone a Dna test to put an end to allegations he fathered a child with a California woman.
Mariah Yeater, 20, hit headlines earlier this month (Nov11) after sensationally claiming the singer impregnated her in a backstage romp following a Los Angeles concert last year (10).
Yeater told reporters she had evidence proving Bieber was the father of her four-month-old son Tristyn, but she ultimately withdrew her request for a paternity test and child support last week (ends13Nov11) after the singer vehemently denied her allegations.
And in a bid to put the rumours to rest, the hitmaker voluntarily took a Dna test on Friday (18Nov11).
Speaking to U.S. TV show Extra on the eve of the singer's doctor's appointment, Bieber's manager Scooter Braun said, "(He) doesn't mind. He's like, 'I'm just going to go ahead... (with) it and be fine.'"
And Braun is still threatening to take legal action against Yeater for attempting to damage Bieber's reputation, adding, "I think it's important to hold people accountable for their actions."
Justin Bieber wants to make his wife feel 'special and valued'
Justin Bieber to perform at 2020 iHeartRadio Awards
Justin Bieber is battling Lyme disease
Justin Bieber taps Travis Scott and Post Malone for new album?
Justin Bieber releases new single Yummy
Justin Bieber to reveal exclusive footage of his wedding
Justin Bieber teases new music for Christmas Eve and New Year's Eve
Justin Bieber teases something big for 2020
Justin Bieber gets 'forever' inked on neck
Justin Bieber regrets racist remarks
Justin Bieber: Hailey Bieber makes me want to be better
Justin Bieber launches new natural deodorant with wife
Justin Bieber asks fans if they want an album before Christmas
Justin Bieber: I have no bad blood with Taylor Swift
Zoolander 2 Movie Review
With virtually the same blend of wit and idiocy as the 2001 original, this fashion-scene...
Zoolander 2 - Trailer Trailer
Derek, Hansel and some other familiar faces are back in Zoolander 2! After offering their...
Zoolander 2 - Teaser Trailer
After a disastrous return to the fashion industry following his retirement, which almost ended in...
Justin Bieber's Believe Trailer
When Justin Bieber was discovered by Scooter Braun and Usher Raymond after performing his songs...
Justin Bieber: Never Say Never Movie Review
Clearly designed with his 12-year-old female fanbase in mind, this backstage myth-making doc wants to...


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The cryptocurrencies boom forced analysts to talk about bubbles, and late "miners" to buy up video cards.
Meanwhile, many people have already earned enough money on the trend: someone did it on the appreciating prices, and someone, as in case with nVidia, profited on the growing demand for related issues.
There are a lot of debates over cryptocurrencies' potential, about possible sell-offs, and the time in which the bubble may burst.
However, while the market talks, it's worth looking around to understand the way to profit on the trend. If there is a demand for cryptocurrencies, it means the trade venues for these assets will be popular as well.
Today, there are already about 200 cryptocurrency exchanges and exchangers in the world. And it is not a limit has been reached yet. So, what if the next stage of the crypto market evolution is the boom of cryptocurrency exchanges. If so, how can we profit on that? Perhaps open your own exchange.
First, you need to resolve a legal issue. It is necessary to obtain a license. This is possible in several ways.
1. It is possible to obtain a Japanese license, but it will cost a lot. Investments may amount to at least $110K, and there is around $30K more for office expenses in Japan.
2. It is possible to register the company in jurisdictions, where there is no legal base for crypto exchange, but at least it's not banned. It all depends on what you can afford and on available resources, but even with sufficient capital you still need to find a director complying with regulators' requirements, lawyers who know all the details of licensing process, and much more.
Secondly, you need to develop the required software.
1. User Personal Account. It's a profile for client registration and verification, with deposit/withdrawal options available. To write such a program you will need to invest effort, time, and money.
2. Trading platform. This is a place through which investors and traders can open, close and manage market positions. You'll also need to think about each nuance, such as the development of gateways, connectors and bridges for platform connection. Writing the platform will consume time and money. There are major vendor lease providers of trading platforms in the world – you can choose, launch, customize and get services for the best of them.
3. Aggregator. It will allow you to connect new partners, other exchanges and even to become a market maker for some of the tools (e.g. if you create a cryptocurrency/token and want to add it to the list of trading assets at your crypto currency exchange). You must consider that your aggregator needs to process huge data volumes every second. At a rough estimate the cost of such a solution will be impressive, considering the number of expensive specialists and the time spent on development, therefore even large companies prefer not to write software, but to pick something available in the market.
All in all, the scope of work and investments required is considerable. However, there are cases when it's worth going along a streamlined path rather than searching for your own original way. Where there is demand, there is supply.
Even now at the start of this new crypto era some companies already offer the development of turnkey trading floors and exchangers, taking into account customer desires.
Such services can considerably reduce your time, efforts, and expenses.


text:
Q: Why does this not work with an imaginary exponent? Could someone please tell me why this exponentiation rule does not work when the exponent is imaginary?
The following seems to be true but is only true if $x$ is an integer.
$$e^{2\pi ix} = \left( e^{2\pi i} \right)^x = 1^x = 1$$


Always book a massage and facial during the summer. Very upscale spa. Staff is very professional. Love visiting!
Zen Spa brings more than 20 years combined experience to coastal Delaware. Our certified therapists have been trained in a variety of modalities which are incorporated into all of our services. Whether it be through maintaining balanced nutrition, living a healthy lifestyle, or integrating exercise into your life, we want to advance your journey toward achieving the body, mind, & spirit that is perfect for you.


First of all, let's examine how token payments are functioning. Generally, they allow processing many transactions without storing customer credit card details. It means, if I run an online store with subscription options, I request credit card information from a customer and transfer this data to payment system my store is connected with. The system converts credit card data into payment profile and returns its ID to me in the form of the number or hash (let's call it 'profile ID'). Each time a customer has to pay for products he is subscribed to, I manually submit his profile ID to a payment gateway and specify the amount of money to be deducted.
Token payments are obviously insecure: numerous fraudsters can intercept the profile ID with merchant's payment gateway API credentials and write off money as much as they want.
To eliminate this problem, payment systems one by one switch to their own subscription services, when a merchant just enters client card data and defines the schedule for the system to execute payment process. For instance, the schedule can be 'deduct $3 once a week' or 'charge $100 every other day'. Thereby, a payment system serves subscription, conducts payments and monitors the proper execution of schedule logic on its own.
Besides that, transition to automatic self-organization of payment enables merchants to manage subscriptions from their accounts. It looks like this: a store owner logs in to his PayPal account and checks out correlations between schedules with payments and subscription profiles.
Moreover, operating on their own, payment systems provide merchants with additional features like offering customers trial subscriptions, when for a certain period of time clients get products at lower prices, absolutely for free or for initial subscription cost.
Summarizing all said above, payment systems abandon the use of token payments for subscription process and move to their own self-organized automatic services.
This transition is beneficial for eCommerce merchants due to the increased security of client card information, ability to manage subscriptions and the most important – making payments systems responsible for subscription execution.
In a couple of weeks we will release the 2.0 update of our Subscriptions and Recurring Payments extension that will embrace these changes and will be compatible with all PayPal services including Express Checkout and Authorize.net's ARB. It won't support token payments as it did before, but instead will provide integration with systems own payment services.
Do not worry – we will surely announce the big release of the SARP new version in our blog. Subscribe to our blog by RSS or via Email and be one of the first merchants to work with updated subscriptions payments.
Indeed a nice post. You have beautifully explained the importance and updated news on the Payment Gateway Services. Keep up the nice posting as I have subscribed to your blog.
Thanks for a great overview! There are few articles that would describe both advantages and drawbacks of payment gateways. The article was very informative for me, but it would be even more easy to compare if make as a kind of a chart, don't you think so? Thanks anyway for the post.
@Internet Payment Gateway Thanks for reading the post! Apparently, you are right - charts help digest information much easier. I appreciate your recommendation and will definitely attach charts and infographics to my future posts.


text:
View allCoulton's CatchupMedia ReleasesSpeeches
Desert Pea comes to the West
Media Releases 20 April, 2016
The Australian Government Mobile Service Centre Desert Pea will hit the road to give people in the Moree Shire, North West Slopes, and Warrumbungle Shire easy access to essential government payments and services in April and May. Minister for Human Services, Alan Tudge said the Mobile Service Centre will visit Pallamallawa, Gravesend, Coonabarabran, Binnaway, Coolah…
Big win for Western NSW owner-drivers
The Coalition Government's bill to abolish the Road Safety Remuneration Tribunal (RSRT) passed both houses of Parliament last night marking a big win for owner-drivers across Western New South Wales (NSW). Speaking on the bill in Parliament yesterday, Federal Member for Parkes, Mark Coulton expressed his support for the abolition of the RSRT following its…
New kitchen for Boggabri HACC Centre
Federal Member for Parkes, Mark Coulton last week presented the Boggabri Home and Community Care Centre with a cheque for $20,000 to support the replacement of the centre's kitchen. With the centre utilised by numerous organisations that provide crucial support services to the Boggabri community, Mr Coulton said that this project is much more than…
Hot water to flow at Warialda Sporting Complex
Gwydir Shire Council will receive Commonwealth Government funding to assist with upgrading the hot water system at the Warialda Sporting Complex. Federal Member for Parkes, Mark Coulton was in Warialda last week and presented Gwydir Shire Council with a cheque in the amount of $5,170 representing half of the estimated cost for this upgrade project.…
Road Safety Remuneration Tribunal
Speeches 18 April, 2016
Mr COULTON (Parkes—The Nationals Chief Whip): I too rise today to speak on the Road Safety Remuneration Repeal Bill 2016 and the Road Safety Remuneration Amendment (Protecting Owner Drivers) Bill 2016. I will say that I do have some credentials to speak on this: I hold here my heavy vehicle driver's licence. It is a…
COULTON'S CATCH UP 18 APRIL
Coulton's Catchup 18 April, 2016
It is very welcome news that when Federal Parliament returns this week, the Coalition Government will introduce two bills to abolish the Gillard Government's Road Safety Remuneration Tribunal (RSRT) and provide certainty to numerous small business owner-drivers across Australia. The RSRT has set a controversial minimum rates order that industry groups, farmers and truck drivers…
Encouraging stronger supply chains in Western NSW
Farmers across Western New South Wales (NSW) will soon be empowered to establish co-operatives and adopt innovative business models to boost their bargaining position in the marketplace, under the Australian Government's $13.8 million Farm Co-operatives and Collaboration Pilot Program. Federal Member for Parkes, Mark Coulton today welcomed the announcement that Southern Cross University would deliver…
$3.2 million to fix black spots across Western NSW
Road safety in New South Wales (NSW) has received a boost with a new round of funding announced under the Black Spot Programme. Minister for Infrastructure and Transport, Darren Chester and Federal Member for Parkes, Mark Coulton today announced funding of more than $3.2 million to fix nine black spots on roads in Western NSW.…
I welcome the Commonwealth-State joint-funding announcement of $2.59 million in projects to help better protect New South Wales (NSW) communities from the threat of bush fire. This funding commitment will contribute $300,000 to the NSW Rural Fire Service for aerial firefighting supply tanks in the Warrumbungle, Moree Plains, Narromine, Gwydir and Narrabri local government areas.…
Better water infrastructure for farmers in Narromine region
Media Releases 9 April, 2016
Farmers in the Narromine region are benefitting from more modern and efficient water infrastructure, with the official opening today of the Narromine Irrigation Board of Management (NIBM) Scheme. Deputy Prime Minister and Minister for Agriculture and Water Resources Barnaby Joyce and Federal Member for Parkes Mark Coulton said the Australian Government had invested more than…
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Hey there, Steve here and welcome to this week's private eye post. Last week's post was so popular (the body language of cheats), I thought it would be a good idea to a similar type of article. Today, I'll I go through the verbal signals of cheats.
All cheats lie. They may not always need to tell a direct lie, but they must account for the time they spend with the other person. All but the most accomplished of liars give off verbal clues when they aren't telling the truth. Once you know what to listen for you'll be able to decipher the "liars code." You can easily combine what you hear, with what you see from the body talk.
Disclaimer: The average person is lied to hundreds of times each day. Without training our lie spotting accuracy is about 50%. In other words, about the same as a guess. So, please consider this article as general educational information only. For help with an individual situation, please contact us or use the live chat feature on this page — your first call is free and handled in the strictest of confidence.
The easiest verbal clues to spot are the ones where you only have to think about the content of the words. The ultimate sin here, of course, is when your partner calls you by a different name when you're making love. But men are sometimes pretty stupid too — check out these dumb verbal clues.
Look at point #3 in the following post. Does your partner always seem to making these types of excuses?
Sometimes it's not the actual words, but what they actually talk about, or what they don't say. Plus add in some sentence length and verbal clues and you're really starting to dig deeper.
There's too much going on in our brains for us to be able to lie without giving off any clues. Unless your partner has been trained by security services, there will always be a code you can decipher. Take time to practice really listening to everything people say, and how they say it. If you're average, and being lied to hundreds of times each day, you're sure to get the practice you need.
Follow us on Facebook for more information on verbal clues your partner is cheating and how to get help if you think you need to.


Congratulations P-CEP Students of the Month - December 2018!
Non-profit groups - need some willing teenage help for your event?
P-CEP National Honor Society is here for you!
P-CEP Marching Band is at the Grand National Championships!
Congratulations P-CEP Students of the Month - November 2018!
Plymouth Boys Cross Country State Championship!


The journey of Lent -- from Ash Wednesday to Easter -- is traditionally one of prayer, repentance, spiritual discipline, and meditation. But coloring? Now you're invited to add the spiritual practice of "coloring the Bible" on your Lenten journey. Engage both sides of your brain as you read the scripture and color your way through each day of Lent with evocative illustrations of God's Resurrection story, from the Old Testament prophecies of Emmanuel to Jesus' victorious appearances after his crucifixion to the loyal Disciples. Dozens of drawings will draw you deeper into the story and your own place in it. This pack of five identical books is perfect for small groups, families, and womens and mens fellowships to pray and meditate together as you Color Lent!
Experience the stories of Lent in a vibrant, new way this spring as you Color Lent!
The journey of Lent -- from Ash Wednesday to Easter -- is traditionally one of prayer, repentance, spiritual discipline, and meditation. But coloring?
Now you're invited to add the spiritual practice of "coloring the Bible" on your Lenten journey. Engage both sides of your brain as you read the scripture and color your way through each day of Lent with evocative illustrations of God's Resurrection story, from the Old Testament prophecies of Emmanuel to Jesus' victorious appearances after his crucifixion to the loyal Disciples. Dozens of drawings will draw you deeper into the story and your own place in it.
Grab your crayons, colored pencils and pens, and prepare to deepen your journey to Easter this year with Coloring Lent.


Alaska is known for ever-changing weather and a laid-back attitude. We recommend packing with a "Mountain Casual" style focused on comfort and convenience. When you venture outdoors, dress in layers to be prepared for the variable weather. The temperate coastal climate here at Alyeska Resort is characterized by long summer days with temperatures in the high 60's and winter temperatures in the mid 20's. Click here for average temperatures by month.
Insulation layer such as a fleece jacket or vest, and non-cotton base layers such as long-underwear and tops. Please note: If you thinking of participating in any water sports (such as rafting or kayaking), it is important that you bring items that are not cotton. Good choices are polypropylene, nylon, and fleece.
Light ski hat, scarves & gloves. May seem silly in the summer but these items will really keep you warm if it gets unseasonably cold, or if you're feeling the effects of glacier-chilled wind, without adding a lot of weight.
Camera and/or video camera. Don't forget extra batteries, film, lens, chargers, USB cables and memory cards as these items are not as easily available for purchase in all parts of Alaska.
Lap Top - The Hotel Alyeska offers complimentary wifi and DSL connections.
Watch and/or alarm clock - many tours have early start times!
Swimsuit - The Hotel Alyeska has a wonderful saltwater pool and whirlpool with mountain views.
In case you need additional gear or left something behind, the shops at The Hotel Alyeska can provide you with a variety of options from technical apparel at the Alyeska Mountain Shop carrying brands such as The North Face or Burton to women's resort-wear and accessories at Glacier Gift & Gallery.


Now that my husband has had his surgery and life will hopefully return to some bit of normalcy, I am going full blast with business!! I have several shoots left to do this month and the first of March, so I will have some more beautiful ladies to show you all!! Working on revamping some policies and prices as well. I really do want every woman to be able to experience this ego boost so I'm working on the numbers of what I am comfortable with for my work/life balance. I'm also still wanting to find a couple more good hair/make up people to help me do what I have envisioned in my mind…not as easy as it sounds! Send some my way of you know anyone who may be interested. Thanks for stopping by!