The gastrointestinal tract is a unique organ system that provides an epithelial barrier between our underlying immune system and luminal pathogens. Disruption of gastrointestinal homeostasis, as a result of impaired barrier function, is associated with numerous pathologies including inflammatory bowel disease and colorectal cancer. In parallel to the clinical development of endoscopy technologies to monitor and diagnose these pathologies in humans, advanced mouse colonoscopy techniques are being developed. When these technologies are coupled with model systems of human disease, which are essential to our understanding of the pathophysiology of gastrointestinal diseases, the requirement for euthanasia of multiple cohorts of mice is eliminated. Here we highlight the suitability of white light endoscopy to monitor the progression of colitis in mice. We further outline the experimental power of combined standard endoscopy with confocal microendoscopy, which permits visualization of fluorescent markers in a single animal in real-time. Together, these technologies will enhance our understanding of the interplay between components of the gastrointestinal microenvironment and their role in disease. •Monitoring of mucosal damage using white light endoscopy•Monitoring of the epithelial barrier using confocal endomicroscopy•Monitoring of the vasculature architecture using confocal endomicroscopy Crosstalk between intestinal epithelial cells (IEC) and the immune system plays a critical role in maintaining barrier function by detecting and responding to environmental stimuli including pathogens, dietary metabolites, food antigens and the commensal bacteria that comprise the microbiota. Breakdown of these networks leads to intestinal immunopathology, including infection, coeliac disease, and inflammatory bowel disease (IBD).IBD describes a group of relapsing–remitting inflammatory disorders, including Crohn's disease (CD) and ulcerative colitis (UC). As a result of chronic bowel inflammation, IBD patients have an increased risk of developing colorectal cancer (CRC) depending on genetic and environmental factors as well as the severity and duration of the disease. CRC is one of the leading causes of cancer-related deaths worldwide; however, if colon polyps are detected early using techniques such as colonoscopy, the removal of these pre-cancerous lesions can dramatically reduce the incidence of advanced CRC . While sRecent innovative techniques combining traditional white light endoscopy with fluorescence endoscopy have enhanced our understanding of the pathogenesis of IBD and increased our ability to detect dysplasia and precancerous lesions of the gastrointestinal tract . These nStandard white light endoscopy is also increasingly used as an experimental tool with genetic or chemically induced mouse models of IBD and CRC to grade inflammation and monitor tumor development in an individual animal over time. These mouse models mimic different features of the pathophysiology of intestinal diseases, including loss of epithelial barrier integrity, chronic inflammation and epithelial hyperplasia. Here we describe new confocal laser endomicroscopy methods suitable for use in mice, which take advantage of the combined used of standard white light endoscopy and confocal microendoscopy. These novel methods permit the generation of high-magnification cross-sectional images of the colonic mucosa for immediate visualization of immune, endothelial or epithelial cell behavior without the need for a biopsy or euthanasia and tissue collection.22.12.1.12.1.1.1Endovision Tricam and 3-Chip camera head 2.1.1.2Xenon 175 light source with anti-fog pump (cat # 20131501)2.1.1.3Hopkins straight forward telescope, diameter 1.9 mm (cat # 64301A)2.1.1.4Examination and protection sheath (cat # 61209C)2.1.1.5Fiber optic light cable and air hose (cat # 495NL)2.1.1.6Computer and standard video softwareKarl Storz Coloview miniendoscopic system2.1.22.1.2.1Confocal processor (cat# CDB-0001)2.1.2.2Proflex microprobe, Mini-Z, diameter 0.94 mmCellVizio Dual Band confocal endoscopy system2.1.32.1.3.1Induction box2.1.3.2Nose coneIsoflurane machine2.22.2.1Dextran sulfate sodium 2.2.2FIuorescein isothiocyanate–dextran 2.32.3.1All procedures were approved and conducted in accordance with the Walter and Eliza Hall Institute Animal Ethics Committee.2.3.2To minimize variation in gut microflora, all animals within an experimental cohort should be bred in the same room and housed on the same rack in a specific pathogen-free barrier facility.2.42.4.1Baseline weights should be determined for individual mice on Day 0.2.4.2Prepare 2% (w/v) DSS in sterile mouse drinking water, allowing for approximately 5 mL of water per mouse/day of the experiment.2.4.3Provide DSS water to the mice for 5 consecutive days.2.4.4On Day 5 return the mice to normal drinking water for a further three days.2.4.5Throughout the 8 day cycle, monitor mice for weight-loss, stool consistency, the presence of blood in the stools, lethargy, ruffled fur, hunching and reduced movement.2.4.6Animals that experience > 15% transient weight-loss over three days should be ethically euthanized.2.52.5.1The Coloview white light endoscopy equipment and the CellVizio confocal endoscopy equipment should be assembled according to the manufacturer's guidelines.2.5.2Clean and sterilize the Karl Storz endoscopy sheath using 70% ethanol and an anti-bacterial agent.2.5.3Anesthetize live mice in an induction chamber with 3% isoflurane and 100% oxygen at a flow rate of 0.4 L/min.2.5.4Secure the head of the mouse in a nose cone, with the mouse positioned ventral side up and the hind legs comfortably stretched out behind.2.5.5Maintain the isoflurane at 0.5–2% for the endoscopy procedure.2.5.6Confirm the rate of airflow by placing the tip of the endoscopy sheath in a beaker of water. One air bubble at a time should emerge from the sheath.2.5.7Insert the endoscope sheath up to 3 cm into the rectum.2.5.8Record endoscopy videos using standard media software, such as iMovies.2.5.9Record clinical disease scores for the Murine Endoscopic Index of Colitis Severity (MEICS). Scoring parameters are provided in 2.62.6.1Initialize the CellVizio 488 laser scanning unit, turn on the confocal processor and launch the CellVizio Dual and ImageCell software. The laser requires approximately 20 min to initialize.2.6.2Insert a microprobe of choice into the laser scanning unit. Use the Quantikit 488 to calibrate the probe as per the manufacturer instructions, and adjust the laser power to approximately 30%.2.6.3Insert the fiber optic probe down the working channel of the Karl Storz endoscopy sheath.2.6.4Initiate video recording from the Karl Storz Coloview system, and insert the endoscopy sheath up to 3 cm into the rectum of the mouse. Place the sheath against the colonic mucosa for fluorescent imaging.2.6.5Initiate the 488 laser using the foot pedal and use the ImageCell software to record fluorescent videos in real-time. Simultaneous recording using the white light endoscopy unit is also possible.2.6.6Re-calibrate the system following the manufacturer protocols prior to imaging each mouse.2.6.8Place the mouse back in its cage and monitor for recovery from anesthetic.2.72.7.1Prior to fluorescent imaging, inject 100 μL FITC–dextran  intravenously.2.7.2Monitor the time after injection, image the colon as described in 2.7.3CellVizio Vessel Detection software can be used to quantify vessel segmentation, length, area and diameter.2.82.8.1Clean the Karl Storz endoscopy sheath, Karl Storz light probe and CellVizio fiber optic probes thoroughly in accordance with manufacturer suggestions.2.8.2Disassemble and store the equipment as per the manufacturer suggestions.3The increased use of mouse colonoscopy over the past decade has allowed us to monitor clinical disease progression in an individual mouse over time. The recent development of new imaging technologies, including miniaturized confocal laser endomicroscopy, has enabled high resolution in vivo tracking of fluorescently labeled proteins throughout the course of disease. These advancements will allow for an enhanced understanding of the pathophysiology of IBD and CRC.4A clear workbench, ideally within a specific pathogen free animal facility, is required for the set-up of both of the endoscopy units . This wiIn the absence of an available white light endoscopy system, changes in mucosal biology can be tracked through histopathology C, and seIn contrast to the white light imaging achieved with the Karl Storz system, the CellVizio confocal microendoscopy system allows for more advanced imaging in real-time of fluorescent markers in live mice. The CellVizio laser scanning unit (class IIIB laser) generates excitation at both 488 nm and 660 nm, and couples the laser beam into a unique fiber optic probe chosen based on the application, with probe specs from 0.35 to 4.2 mm. Each fiber within the probe is a point detector, equivalent to a pinhole used in standard confocal imaging. In general, 9–50 frames per second are imaged up to a 1.4 μm lateral resolution, 10–70 μm optical section and 225–600 μm field of view, with a z-axis signal integration of 200 μm surface depth. This minimally invasive procedure can be applied, for example, to imaging of the brain, cornea, lung, heart, kidney, liver, pancreas, spleen and gastrointestinal tract . Given tCdx2-Cre , and the scanning frame rate of 9 to 50 frames per second. However, CellVizio has developed software that permits stitching of images together to create a larger static image of a region of interest. Our results were generated using the Mini-Z series probe, which has a working distance of 50 μm and a lateral resolution of 3.5 μm to permit high resolution imaging of the epithelial cells and vasculature. However, a number of probes are available with a range of 1.4–3.5 μm lateral resolution, which would permit the visualization of individual immune cells. We have demonstrated the power of taking advantage of the 488 laser, and YFP or FITC–dextran monitoring; however, the CellVizio confocal systems are available with dual laser capabilities, which permit simultaneous monitoring of two parameters through use of both 488 and 660 nm excitation wavelengths.6ex vivo or in vitro.The ability to perform high-resolution confocal imaging of the colon of live mice in situ over a defined time course will have a significant impact on biomedical research. By using the techniques described, our understanding of IBD and CRC onset and progression will be transformed through the generation of a new understanding of cell communication, trafficking and microenvironment relationships, in a manner not possible Supplementary material.Confocal laser endoscopy tracking of fluorescently tagged epithelial cells.Video recording of YFP positive epithelial cells and crypt structures in the distal colon from a mouse expressing YFP under the control of a constitutive CDX promoter. Scale bar = 20 μm.Confocal laser endoscopy tracking of fluorescently labeled vasculature.Video recording of FITC dextran moving through the vasculature within the distal colon from a mouse intravenously injected approximately 10 min prior to imaging. Scale bar = 20 μm.The following are the supplementary data related to this article.The authors have none to declare.


In this work, we report the potential use of novel carbon nanofibers (CNFs), dispersed during fabrication of glass fiber composites to monitor damage propagation under static loading. The use of CNFs enables a transformation of the typically non-conductive glass fiber composites into new fiber composites with appreciable electrical conductivity. The percolation limit of CNFs/epoxy nanocomposites was first quantified. The electromechanical responses of glass fiber composites fabricated using CNFs/epoxy nanocomposite were examined under static tension loads. The experimental observations showed a nonlinear change of electrical conductivity of glass fiber composites incorporating CNFs versus the stress level under static load. Microstructural investigations proved the ability of CNFs to alter the polymer matrix and to produce a new polymer nanocomposite with a connected nanofiber network with improved electrical properties and different mechanical properties compared with the neat epoxy. It is concluded that incorporating CNFs during fabrication of glass fiber composites can provide an innovative means of self-sensing that will allow damage propagation to be monitored in glass fiber composites. In recent years, textile fabric composites have attracted widespread attention owing to their superior properties, making them attractive materials for many applications like smart fabrics and the aerospace, marine, and automobile industries ,2. GlassIn this sense, nanomaterials play an important and significant role in controlling the intrinsic properties of glass fiber composites. Carbonaceous nanomaterials such as CNTs and CNFs are the most common and the most promising additives used to fabricate multifunctional glass textile nanocomposites with enhanced capabilities. For instance, CNFs are attractive candidates in conductivity-related applications because of their relatively high electrical conductivity . A percoTo date, the ability of CNFs in monitoring damage propagation in glass textile-reinforced polymer composites has not yet been reported. In this study, the ability of CNFs to improve electrical conductivity and alter the mechanical properties of epoxy nanocomposites is investigated. The conductive CNFs/epoxy nanocomposite is then used to monitor damage propagation in glass textile-reinforced polymer composites under static loading.® 37-127 epoxy system supplied by U.S. Composites, Inc.  The epoxy resin is low viscosity, 100% reactive diluted liquid based on Bisphenol-A containing glycidyl ether. The hardener was Aliphatic Amine EPOTUF® 37-614. The resin to hardener mixing ratio was 2:1. The bidirectional S-Glass fiber fabric was supplied by ACP Composites, Inc. .CNFs were supplied by Nanostructured & Amorphous Materials Inc. They had diameter of 80–200 nm and a length of 0.5-20 µm and thus an aspect ratio ranging between 6.3 and 100. The epoxy used in fabrication was EPOTUFThe performance of the CNFs-polymer nanocomposite is affected by the homogeneity of nanofibers dispersion into the polymer matrix. Several dispersion methods were recommended in the literature to obtain homogeneously dispersed CNFs in the polymer matrix and to avoid agglomeration. CNFs with different contents  were first hand-stirred into the epoxy resin, and then sonicated in a path sonicator for 1 h at 40 °C and frequency of 40 kHz. The resin-CNFs mixture was further dispersed using a high shear mixer at speed 11,000 rpm at a temperature of 90 °C for 1 h. The resin-CNFs mixture was then mechanically stirred at temperature of 90 °C for 2 h and a speed of 800 rpm. The resin-CNFs mixture was degassed to remove the bubbles for 30 min at 50 °C and then left to cool for 1 h at room temperature. After cooling, the epoxy hardener was hand-stirred into the resin-CNFs mixture for 5 min and left overnight. CNFs/epoxy nanocomposite was then cured for 2.5 days at 110 °C to ensure full curing. −1 wave number.To prepare glass fiber reinforced (GFRP) composites, after adding the epoxy hardener to the resin-CNFs mixture, that mixture was then used to fabricate the GFRP using the hand layup technique. Six layers of bidirectional plain weave glass fiber textile fabrics were laid in 0° direction, and then vacuum pressure was applied for 24 h. The glass fiber composite plates were then cured for 2.5 days at 110 °C to insure complete curing. 2 wt % CNFs were used to fabricate glass fiber composites. The CNFs content used for producing the glass fiber composites was based on the electrical percolation observations of epoxy-CNFs nanocomposites discussed below. The electrical conductivity of CNFs/epoxy polymer nanocomposites was determined according to ASTM D257 . MeasureA is the cross sectional area, L is the length, and R is the measured electrical resistance.Silver paint was used to ensure good contact between the specimens and the electrode. The electrical conductivity (σ) was calculated using Equation (1).To identify the significance of CNF on the elastic properties of CNFs/epoxy nanocomposites, three specimens of 20 mm × 10 mm × 3 mm were tested. Dynamic mechanic analysis (DMA) was performed on a Triton Instruments, operating in the tension mode at an oscillation frequency of 1 Hz. Data were collected from room temperature to 150 °C at a scanning rate of 10 °C/min. For static tension properties of CNFs/epoxy nanocomposites, three specimens of 20 mm × 10 mm × 2 mm were tested using Triton Instruments, operating in the static tension mode with preload force 0.01 N, load rate 0.2 N/min and to maximum force 2 N.® Bionex servo hydraulic machine. A displacement control protocol was used in the static tension tests according to the ASTM standards methods D3039/D3039M [Three GFRP composite coupons of 19 mm × 150 mm were tested under off-axis  static monotonically increasing tension stress. The reason for choosing off-axis loading was to simulate realistic loading conditions where stresses are generated in any direction to the glass fiber composite. It is also well established that behavior of the fiber composites in the off-axis direction is governed by the polymer matrix rather than the fibers . Therefo9/D3039M  with a lED(t) was calculated using Equation (2).ED(t) is the electrical damage measured at time t, σ(t0) is the initial electrical conductivity of the composite prior to load application at time 0t, and σ(t) is the electrical conductivity of the composite at time t. Moreover, a metric of damage based on change of modulus of elasticity, representing mechanical damage and denoted MD(t) was calculated using Equation (3):E(0t) is the initial tangent modulus of elasticity of the glass fiber composite coupon at 0t and E(t) is the tangent modulus of elasticity of the glass fiber composite coupon at time t. A minimum tangent modulus of zero was assumed to account for the descending stress-strain.Damage in glass fiber composite coupons was estimated in terms of the change of the electrical conductivity during loading. The electrical conductivity was measured and a metric of damage based on electrical conductivity change denoted E) versus the CNFs concentrations are shown in The mechanical properties of CNFs/epoxy nanocomposites can be observed from a static tension test, shown in cE, mE and fE are the moduli of composite, matrix and filler respectively and fV is the filler/fiber volume fraction.To better understand the significance of fillers/fibers on the mechanical properties of polymer composites, a number of models exist in the literature ,24,25. WcE except at CNFs contents above 1.0 wt % CNFs. At low CNFs contents, the significance of the CNFs on the epoxy matrix is negligible, and thus CNFs work as fibers that reinforce the epoxy matrix and improve its mechanical properties as a composite. This behavior follows classical composite theory and thus good agreement with the model can be observed. Conversely, at 2.0 wt % and 2.5 wt % CNFs , CNFs start to induce a chemical effect on the epoxy matrix, hindering the reaction between the resin and the hardener and affecting epoxy crosslinking. This adversely affects the mechanical properties of CNFs/epoxy nanocomposite. The effect of CNFs on epoxy crosslinking is not considered in Christensen’s model [’s model  and all −1), epoxide ring (~830 cm−1), N–H of primary amines (1590–1640 cm−1), O–H groups (3200–3600 cm−1), C–N (1040–1120 cm−1) and ether (~1250 cm−1). To further confirm the above hypothesis of interaction between the CNFs and epoxy and the chemical significance of high CNFs on epoxy, FTIR analysis was performed. FTIR is utilized to observe chemical changes of the epoxy matrix after incorporating CNFs. The FTIR spectra of the epoxy matrix with and without CNFs is presented in −1) at this wt % of CNFs compared with other wt %. It is obvious from the FTIR spectra in −1. This is attributed to the fact that at such low CNFs concentrations, there is a minor effect from CNFs on epoxy curing. Incorporating CNFs concentrations of 1.5, 2.0 and 2.5 wt % into the epoxy matrix appeared to shift the O–H peak to 3370 cm−1, 3320 cm−1 and 3310 cm−1, respectively. This may be attributed to the effect of CNFs on the curing behavior of the epoxy matrix [−1 is attributed to the combined effect of the differently associated hydroxyl groups, i.e., hydrogen bonding between hydroxyl and hydroxyl/carbonyl groups of different strength and hydrogen bonding of water molecules. In addition, a matrix having O–H groups could undergo two modes of hydrogen bonding; inter- and intramolecular hydrogen bonds between O–H groups [Comparing the FTIR spectrum of 2.0 wt % CNFs/epoxy with other spectra of CNFs/epoxy polymer nanocomposites and the neat epoxy, it can be observed that incorporating 2.0 wt % CNFs in the epoxy matrix caused an increase in the epoxide ring and primary N–H band intensity. In addition, the band of the hydroxyl group is significantly shifted to a lower intensity and to a lower wave number value (3410–3320 cmy matrix . It is wH groups . Consequ−1 toward lower wave numbers (3320 cm−1) can be attributed to redistribution in the arrangement of hydroxyl group association due to the different geometry caused by lowered cross-linked matrix.Overall, the O–H band intensity decreases and is shifted to a lower wave number. This observed progressive shift of the (υ O–H) band from about 3410 cmE is the storage modulus at (Tg + 50), T is the absolute temperature of (Tg + 50) K and R is the gas constant. The molecular weight between crosslinks was given using Equation (6).cM is molecular weight between crosslinks and ρ is the density of the polymer composite. The degree of crosslinking linkX of the polymer composites is suggested as an inverse for the molecular weight between crosslinks in a unit volume as in Equation (7):To further confirm the above hypothesis, DMA testing was conducted on neat epoxy and CNFs/epoxy nanocomposites. According to the rubber elasticity theory modified by Nielsen , the crolinkX is presented in The calculated degree of crosslinking −7 S/m. When CNFs were added to epoxy resin, the electrical conductivity increased. The percolation phenomenon was observed when, at low CNFs content, the conductivity of the CNFs/epoxy nanocomposites was very close to the conductivity of the epoxy matrix, because no conductive CNFs network was formed. As more CNFs were added, the fibers moved close together, and at a certain concentration (percolation threshold), a continuous conductive network of carbon nanofibers was formed and the conductivity increased by several orders of magnitude.The epoxy matrix has inherently poor electrical conductivity. The most effective method to overcome this limitation is to add conducting fillers. 3 S/m. The illustration in The CNFs/epoxy percolated at 1.5 wt % which was determined by the conventional method, i.e., the peak position of dlogσ/dC  and the The stress-strain curves of GFRP composite coupons with and without 2.0 wt % CNFs under static tension are presented in The source of nonlinearity is in the off-axis direction; the polymer matrix dominates the tension behavior rather than the fibers . It was The mechanical and electrical properties of epoxy incorporating CNFs were examined. The results of the electrical measurements show that CNFs can significantly improve the electrical conductivity of epoxy. It was also observed that a significant improvement in the electrical conductivity of CNFs/epoxy nanocomposite is achieved with a percolation threshold equal to 1.5 wt %. The electromechanical behavior of GFRP incorporating 2.0 wt % CNFs was then examined under monotonically increased static loading. Electromechanical measurements of GFRP coupons under off-axis static tension tests showed that electrical damage based on the change in electrical conductivity of GFRP can be correlated well with growing damage in the GFRP coupons under static tension loads. The measurements also showed that damage propagation monitoring in CNFs/GFRP using electrical conductivity has a lower sensitivity than mechanical damage propagation. This might be attributed to the pronounced effect of microcracking on mechanical behavior compared with its influence on electrical conductivity. Nevertheless, it is evident that using CNFs during fabrication of GFRP composites allows damage initiation and damage propagation in GFRP to be monitored with acceptable repeatability and resolution.


LMT exhibited efficient adsorption of cobalt ions (Co(II)), and the adsorbed Co(II) was readily desorbed by nitric acid (HNO3). All parameters affecting the adsorption and/or desorption of Co(II), including initial Co(II) concentration, pH value, temperature, HNO3 concentration, and time, were optimized. The kinetic data analysis showed that the adsorption followed the pseudo-second-order kinetic model and fit well into the Langmuir isotherm equation. Notably, the nanocomposite can be used four times without significantly losing adsorbent capability. The Energy-Dispersive X-ray (EDX) and FTIR spectra analysis also revealed that the adsorption mechanism may be mainly a chemical adsorption dominated process.A lignocellulose/montmorillonite (LMT) nanocomposite was prepared as a reusable adsorbent for cobalt(II) ions, and characterized by nitrogen (N High levels of cobalt may cause paralysis, diarrhea, low blood pressure, lung irritation, and bone defects in humans [Industrial wastewater contains various toxic heavy metals that pose significant health risks if they enter the human food chain ,2. Cobaln humans . Therefon humans ,5,6,7; un humans ,9.Lignocellulose (LC), a natural renewable polymer, exists widely in plants and is primarily composed of cellulose, hemicellulose, and lignin ,11. LC hOn the other hand, montmorillonite (MT) is a type of silicate mineral with a nanolamellar structure, and has a very limited adsorption capacity of heavy metal ions due to low affinity, swelling, and dispersion in water ,18,19,202+), and cadmium (Cd2+). Polymer/clay nanocomposites have become a promising alternative for the expansion of industrial and economic activities and the satisfaction of increasingly stringent environmental conditions [2 adsorption/desorption isotherm, X-ray Diffraction (XRD), Scanning Electron Microscope (SEM), Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR), and performed a detailed study on its adsorption and desorption of Co(II). To focus on ecosystem stability and public health, this study was completed on the adsorption of 59Co(II). To our knowledge, no studies have been completed on the adsorption capacity of Co(II) using a LMT nanocomposite. Hence, a reusable LMT nanocomposite was synthesized in this study. All possible parameters affecting adsorption and/or desorption of Co(II), such as initial Co(II) concentration, pH value, temperature, HNO3 concentration, and time, were optimized. Then, the LMT nanocomposite absorption procedure was kinetically analyzed, and the reusability of LMT nanocomposite was systematically tested. The prepared LMT nanocomposite showed potential for the adsorption for Co(II) for removal from aqueous solutions.Bunhu and Tichagwa  preparednditions ,26,27,283)2·6H2O) was purchased from Shanghai Jinshan Chemical Co., Shanghai, China. All other chemicals and reagents used in the work were of analytical grade and used without further purification. All solutions were prepared by using deionized water.LC (SAM-100) was purchased from Beijing Huaduo Biotech Ltd., Beijing, China. MT (cation exchange capacity (CEC) = 100 meq/100 g) was purchased from Zhejiang Feng Hong Clay Chemical Co., Huzhou, China, was washed by deionized water and dried overnight at 70 °C, milled, and sieved to a 200-mesh particles size. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O, and diluted to desired concentrations (from 0.0015 to 0.0060 mol/L). The pH value of each Co(II) solution was adjusted by using either hydrochloric acid  or sodium hydroxide  solutions. All solutions were freshly made for each experiment. The distribution coefficients of 59Co(II) and the ratio of the concentration of 59Co(II) adsorbed to that in solution was 3.7 × 102 Bq/L.Aqueous stock Co(II) solution (1 mol/L) was prepared by using Co(NOThe LMT nanocomposite was prepared as follows: a certain amount of LC was added into aqueous NaOH solution (ω = 0.20) with 1:30 ratio of weight of LC (g) to volume of NaOH solution. The mixture was stirred at room temperature until a uniform LC suspension was formed. Then, the LC-NaOH mixture was added into a suspension of MT (1.0 g in 30 mL of distilled deionized water), and stirred at room temperature for 0.5 h. The resulting mixture was heated at 60 °C and stirred for 6 h and centrifuged, and supernatant was separated, and pH value was adjusted to 7.0 by using a pH meter . Finally, the supernatant was completely dried under vacuum at 105 °C for 5 h. All samples were ground and sieved to a 200-mesh particle size, and stored in an airtight plastic container until testing.tq,1 (mg/g) is the capacity of adsorption at time t (min); C0 and tC,1 (mol/L) are the Co(II) initial and final concentrations at time t (min), respectively; V1 (mL) is the volume of Co(II) solution; and m1 (g) is the mass of adsorbent. For calculating tq,1, we assumed no loss of Co(II) ions occurred in all experimental procedures.LMT nanocomposite (0.1000 g) was accurately weighed and added into 50 mL of Co(II) solution. The suspension was stirred at a uniform speed of 120 rpm in a thermostatic shaker . During the experimental procedure, the pH value of each Co(II) solution was adjusted to a constant value. When the adsorption equilibrium was reached, the mixtures were centrifuged at 6000 rpm for 5 min. Then, the initial and final concentrations of Co(II) were determined using a double beam ultraviolet (UV)-visible spectrophotometer  . The adstq,2 (mg/g) is the desorption amount at time t (min); tC,2 (mol/L) is the concentration of Co(II) in HNO3 solution at time t (min); V2 (mL) is the volume of HNO3 solution; and m2 (g) is final mass of the adsorbent after releasing Co(II).The Co(II)-loaded LMT nanocomposite (0.1000 g) was accurately weighted, transferred into 0.5 M different 50 mL desorption eluents, and put into an ultrasonic cleaning machine . When the desorption equilibrium was reached, the suspension was centrifuged, and the concentration of the desorbed Co(II) was determined using the same method as in the adsorption experiment. The effects of different desorption eluents and concentration, desorption temperatures, and ultrasonic desorption times were studied. Similarly, desorption experiments were repeated three times, and reproducibility was within ±3%. The desorption capacity of the Co(II)-loaded LMT nanocomposite was calculated according to the following equation ,32:(2)qt2) precipitation, based on the solubility product constant (Ksp) for Co(OH)2 being 5.92 × 10−15 (pH = 9.36). The solvent was heated for evaporation. Then, Co(OH)2 precipitation was concentrated, separated, precipitated, and loaded into an closed container, diluted, and used for further experiments.To investigate the reusability of the LMT nanocomposite, repeated adsorption–desorption experiments were performed. The LMT nanocomposite, after the first batch reaction, was washed with 15 mL distilled deionized water twice to remove the remaining acid, and dried in a vacuum at 70 °C for the next adsorption of Co(II). The regenerated the LMT nanocomposite was used in four consecutive cycles of adsorption–desorption experiments. Meanwhile, the pH of the waste solution containing Co(II) ions was adjusted to a value greater than 10.0 by using 0.1 M NaOH solution, and was completely converted into cobalt(II) hydroxide (Co(OH)SBET) and total pore volume (Vtot) were determined according to the manufacturer’s software. The micropore surface area (Smic), mesopore surface area (Smeso), and the mesopore volume (Vmeso) were evaluated using the t-plot method. The density functional theory pore size distribution of the LMT was obtained using the Autosorb software package with medium regularization. FTIR spectra were recorded in KBr pellets by FTIR spectrophotometer . X-ray Diffraction (XRD) analysis of the powdered samples was completed using an X-ray power diffractometer with a copper (Cu) anode , running at 40 kV and 30 mA, scanning from 4° to 18° at 3°/min. Morphological changes and surface analysis of the samples were recorded by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) . TEM image analysis of the samples was completed with a TEM  at 200 kV.The Brunauer–Emmett–Teller (BET) surface area of the LMT was determined from nitrogen adsorption–desorption isotherms measured at −196 °C using a surface area analyzer . The pore structure of the LMT was assessed from the isotherms according to conventional procedures. The surface area  ions, the surface area and total pore volume of Co(II)-loaded LMT were dropped, at 112.3 m2/g and 1.117 cm3/g, respectively. Especially, the most obvious decrease is the BET specific surface area reduced from 73.4% to 41.1% and pore volume reduced from 69.4% to 30.6% for the mesopores. These results imply that the contribution of the mesopores was dominant and that the porous structure of LMT was well developed. Based on the data in The adsorptive capacity of the LMT nanocomposite is related to its specific surface and pore volume. The textural parameters of MT, LMT and Co(II)-loaded LMT in −1 (−1 (2O). The adsorption band of C–H stretching at 2909 cm−1 (−1 (−1 (2O) in MT. In addition, the band at 1437 cm−1, due to C–H bending on methyl and methylene  was an effective way to prove the intercalation of MT and LC. XRD spectra of MT and LMT nanocomposite are shown in  with LC b, the moThe morphologies of purified MT and LMT are shown in TEM images of purified MT and LMT are shown in The initial Co(II) concentration is an important driving force for overcoming mass transfer resistance of Co(II) between the aqueous and solid phases. The effects of different initial Co(II) concentrations on the adsorption capacity of LC, MT, and LMT nanocomposite are shown in Ksp) for Co(OH)2 of 5.92 × 10−15, and the adsorption pH value should be less than 9.36. The relationship between pH value and adsorption capacity are shown in −) increased, and the coordination and chelation ability of Co(II) with LMT gradually increased. However, when the pH was higher than 5.6, Co(II) could react with a basic pH regulator, which resulted in facile hydroxide complexation or precipitation, and therefore caused a reduction in adsorption capacity [The pH value is another important factor affecting adsorption of Co(II) on adsorbents, including LMT nanocomposite, LC, and MT. The maximum pH value of the Co(II) solution must avoid the formation of hydrolyzed species. The precipitation was calculated using the concentration of Co(II) and the solubility product constant (capacity . We deteThe relationship between adsorption capacity and adsorption temperature is shown in The effects of adsorption time on the LMT nanocomposite adsorption capacity of Co(II) are shown in tq increased significantly with increasing time t in minutes at the early stage and slowly dropped afterward. The maximum adsorption capacity of 93.02 mg/g was found at 60 min. We concluded that as the majority of the LMT nanocomposite adsorption sites (or pockets) remain, the adsorption rate is faster.The adsorption rate of LMT nanocomposite of Co(II) was investigated by assuming pseudo-first-order and pseudo-second-order models. eq (mg/g) is the amount of adsorption at equilibrium, tq (mg/g) is the adsorption amount at time t (min), k1 (min−1) is the rate constant of the pseudo-first-order adsorption kinetic equation, and k2 (g/(mg/min)) is the rate constant of the pseudo-second-order adsorption kinetic equation.The adsorption kinetic curve of the LMT nanocomposite was modeled by fitting the pseudo-first-order adsorption kinetic data into Equation (3) and fitting the pseudo-second-order adsorption kinetic data into Equation (4) : (3)log onto the LMT nanocomposite [The results of adsorption kinetics are tabulated in omposite .eq and adsorption equilibrium concentration eC. In An adsorption isotherm describes the equilibrium of the sorption of a material on the surface of an adsorbent. In this study, two adsorption isotherms, the Langmuir and Freundlich adsorption isotherm models, were used to interpret experimental data. b (L/mg) is the Langmuir constant relating to the adsorption capacity; n and fk are Freundlich constants; eC (mol/L) is the concentration of Co(II) at equilibrium; qmax (mg/g) is the monolayer saturation adsorption; and eq (mg/g) is the adsorption capacity at equilibrium.Isothermal adsorption curves were fitted and plotted by employing the Langmuir and Freundlich equations, shown in Equations (5) and (6), respectively : (5)CeqeRL) based on the following equation [LK (L/mg) is the Langmuir adsorption constant and C0 is the optimal concentration of Co(II) ions. The LR value indicates the nature of the isotherm is unfavorable (LR > 1), linear (LR = 1), favorable (0 < LR < 1), or irreversible (LR = 0).The essential characteristics of the Langmuir isotherm can be represented according to a dimensionless equilibrium parameter (equation : (7)RL=1R2 value and fitting curve, the adsorption of Co(II) ions by the LMT nanocomposite falls into the Langmuir isothermal adsorption model, indicating that the mechanism involved is monolayer adsorption [The result of the isotherm equation and the fitting curves are shown in sorption .eC/eq versus eC  value calculated from the Langmuir model was 93.43 mg/g, which was almost the same as found in the experimental data, 93.02 mg/g. This result may be due to the homogeneous distribution of the activated sites on the surface of the LMT. Furthermore, the value of LR for the Langmuir isotherm was between 0 and 1, indicating a favorable process. Obviously, the Langmuir model more accurately described the adsorption of Co(II) onto LMT. The Langmuir model corresponds to a dominant electrostatic attraction, ion exchange, and coordination mechanism. This means that the adsorption process involved physical adsorption and monolayer coverage chemical complexation at the interface and the outer heterogeneous surface of the LMT nanocomposite.Comparisons of the isotherm models for the adsorption of Co(II) onto LMT were performed by comparing each linear plot of ersus Ce a, and lnsus lnCe b. The ca3, HCl, H2SO4, H3PO4, NaOH and Na2CO3, separately, as the desorbing eluents to investigate their effects on desorption  ions from LMT nanocomposite. The desorption capacity of NaOH was slightly higher than Na2CO3, but still showed lower desorption capacity when compared with the acid eluents. Among the four acidic desorption eluents, HNO3 was found to be the most appropriate desorption eluent for the regeneration of Co(II)-loaded LMT. This result was expected because the surface of LMT was protonated by H+ ions under acidic conditions, and electrostatic interactions occurred between H+ and the activated sites of the surface of LMT, thereby allowing the desorption of positively charged Co(II). Furthermore, ion-exchange, electrostatic attraction and coordination mechanisms were determined, and HNO3 could be an effective desorption eluent for the regeneration of Co(II)-loaded LMT in this work.An important characteristic of the LMT nanocomposite is the possibility of its regeneration for further use. Repeated adsorption/desorption cycles experiments were conducted in the present work using HNOsorption . Figure 3 concentrations on the desorption capacity of Co(II)-loaded LMT are shown in 3 concentration.The effects of different HNO+ appeared to be the driving force for desorption of Co(II), to a limit, by positive ion exchange and increased the concentration gradients of Co(II) and H+, further facilitating the desorption of loaded-Co(II). However, high concentrations of H+ beyond this limit would not increase the desorption capacity, similar to a report in the literature [3 concentration of 0.5 mol/L, suggesting that the adsorption of Co(II) onto LMT partially occurred via electrostatic attraction and ion exchange, substantiating the results for pH values with respect to adsorption.The concentrated Hterature . The rel+ and Co(II) may compete for the activated sites, leading to an increase in desorption efficiency. In addition, a higher desorption temperature may impair the adsorption efficiency of the active sites and lead to the decomposition of LMT with damage to the 3D structures, having a detrimental effect on the desorption process. This further supports the results for adsorption temperature [The effects of different desorption temperatures on the desorption capacity of Co(II)-loaded LMT nanocomposite are shown in perature ,43. TherThe effects of different desorption times on the Co(II)-loaded LMT nanocomposite are shown in Recovering and reusing an adsorbent would decrease the processing cost, and is an important parameter for practical applications . The reu−1 (−1 (−1 (−1 (−1 (−1 (−1 (−1 (Fourier Transform Infrared Spectroscopy (FTIR) spectra of pure LMT, Co(II)-loaded LMT, and recovered LMT are shown in −1 a are att−1  images of LMT, Co(II)-loaded LMT, and recovered LMT are shown in The Energy-Dispersive X-ray (EDX) is an analytical technique used for elemental analysis. EDX analysis of LMT was performed to confirm the existence of Co(II) on Co(II)-loaded LMT. EDX spectra of pure LMT a, Co(II)The values of adsorption capacity by the other adsorbents from the literature are given in 2 adsorption/desorption isotherm, FTIR, XRD, SEM, and TEM. The results demonstrated that the slender and scattered LC was dispersed into the MT nanoplatelets, and an intercalated-exfoliated nanostructure was formed in the LMT, displaying a higher surface area of 245.1 m2/g and total pore volume of 1.692 cm3/g, greatly improving the nano-effect. LMT can be effectively applied for the adsorption of Co(II) ions from aqueous solutions. The maximum adsorption capacity, with 0.10 g of LMT for Co(II), reached 93.02 mg/g under optimal conditions of an initial Co(II) concentration of 0.0040 mol/L, a pH of 5.6, an adsorption temperature of 75 °C, and an adsorption time of 60 min. The adsorption kinetics and isotherms were well-fitted to both the pseudo-second-order adsorption kinetics equation, with an R2 value of 0.9992, and the Langmuir isothermal adsorption model, with an R2 of 0.9987. These results indicate that the adsorption equilibrium was mainly dominated by monolayer chemical adsorption in the experimental range.A reusable LMT nanocomposite was prepared using a chemical intercalation reaction. The LMT nanocomposite was characterized by N3 as a desorption agent in the ultrasonic oscillation treatment. The optimum conditions of desorption were as follows: the concentration of HNO3 was 0.5 mol/L, the desorption temperature was 60 °C, and the time for ultrasonic desorption was 55 min. Under the optimum conditions, the maximum desorption capacity was determined as 87.34 mg/g.The effects on the desorption capacity of the Co(II)-loaded-LMT were observed by using HNOThe adsorption/desorption experiments demonstrated that the adsorption, desorption capacity, and desorption efficiency of LMT remained at a relatively high level after four rounds of adsorption/desorption recycling. This study showed that the LMT nanocomposite is an excellent and renewable potential adsorbent for the removal of Co(II) ions from aqueous solutions.


The neuromuscular reflex arc is the system that integrates the proprioceptive information for muscle length and activity (sensory afferent), to modify motoneuron output to achieve graded muscle contraction (actuation efferent). The sensory portion of the arc is composed of proprioceptive sensory neurons and the muscle spindle, which is embedded in the muscle tissue and composed of intrafusal fibers. The gamma motoneurons (γ-MNs) that innervate these fibers regulate the intrafusal fiber’s stretch so that they retain proper tension and sensitivity during muscle contraction or relaxation. This mechanism is in place to maintain the sensitivity of proprioception during dynamic muscle activity and to prevent muscular damage. In this study, a co-culture system was developed for innervation of intrafusal fibers by human γ-MNs and demonstrated by morphological and immunocytochemical analysis, then validated by functional electrophysiological evaluation. This human-based fusimotor model and its incorporation into the reflex arc allows for a more accurate recapitulation of neuromuscular function for applications in disease investigations, drug discovery, prosthetic design and neuropathic pain investigations.Investigation of neuromuscular deficits and diseases such as SMA, as well as for next generation prosthetics, utilizing  While animal models have been the standard for disease and drug evaluation, many of the effects seen in these models are incongruent with the effects seen in humans. Furthermore, various organizations have restricted the use of animal models for ethical considerations. To avoid these difficulties, in vitro systems that integrate human cell culture with BioMEMs devices are being investigated for their ability to recapitulate functional human organ systems8. The high content analysis capabilities, reproducibility, ethical considerations, and biological flexibility with relevant cell types have increased the demand for realistic functional in vitro systems. The neuromuscular reflex arc is a highly complex biological circuit, where the actuation segment has been successfully evaluated using in vitro systems14. We have previously shown functional neuromuscular junction formation in vitro for both animal and human cells on a non-biological substrate14. We have also demonstrated the fundamental sensory portion of the arc by animal and human sensory neuron innervation of intrafusal fibers15. Further elaboration of this system, by incorporating additional sensory components, allows for a more accurate recapitulation of in vivo functionality and serves as a better representative platform for investigating prosthetic design, neuromuscular diseases and understanding the mechanism of action for relevant drugs and their targets17.There has been a recent emphasis on the development of 18. The regulatory control of tension and contraction is mediated by innervating α-motoneurons. Embedded within the extrafusal fiber tissue are muscle spindles, the sensory organ for afferent (sensory) signals to regulate muscle function18. The spindles are composed of intrafusal fibers encapsulated in collagen and innervated by both sensory and gamma-motoneurons (γ-MNs)20. The primary role of intrafusal fibers is the detection of the magnitude and speed of stretch or flexion of the muscle and the position of the limbs, or proprioception19. Upon a change in muscle tension, the muscle spindles send signals through afferent sensory neurons which are relayed to neurons within the spinal cord. Motoneurons receiving afferent information can then signal to intrafusal or extrafusal fibers to relax or contract in response to sensory input18. With the afferent sensory feedback, the reflex arc acts as an automated closed loop so that voluntary movement can be achieved accurately and properly.The stretch reflex arc is the physiological system that regulates skeletal muscle movement and tension. This arc can be broken down into two primary components: the efferent and afferent domains. The efferent domain regulates mechanical actuation, muscle tension and relaxation through extrafusal fibers18. The Type Ia sensory neurons innervate the intrafusal fibers with annulospiral wrappings around the equatorial region and Type II sensory neurons employ flower spray endings towards the peripheral ends of the fiber. γ-MNs innervate the peripheral ends of the intrafusal fibers via flower-spray endings as well. The peripheral ends of the intrafusal fibers slowly relax or contract under the control of γ-MNs. These γ-MNs modulate the tension, sensitivity, and length of the intrafusal fibers so muscle spindles can maintain constant sensitivity during dynamic muscle action and prevent overextension, which can lead to undue stress on the muscle as well as tendon and joint damage19. Unlike α motoneurons, γ-MNs do not directly induce muscle tissue contraction or relaxation but modulate the sensitivity of the muscle spindle instead18, which then modifies the activity of α-motoneurons (α-MN) and subsequent muscle contraction. Human-based in vitro models for the innervation of extrafusal fibers by α-MNs and intrafusal fibers by proprioceptive sensory neurons have been established, but an in vitro model for the innervation of intrafusal fibers by γ-MNs has yet to be developed, despite being crucial for proper function of the neuromuscular system. Development and incorporation of this γ-motoneuron - intrafusal fiber system into the reflex arc would provide a better platform for the study of neuromuscular development, prosthetic design, relevant diseases and the evaluation of potential drug candidates.Several types of neurons innervate both the extrafusal and intrafusal fibers of the muscle tissue. The extrafusal fibers are primarily innervated by α motoneurons while intrafusal fibers are innervated by both sensory neurons and γ-MNsin vitro studies demonstrating this particular cellular interaction, especially with human cells. Although γ-MNs have a specific function in the neuromuscular system, their developmental similarity to α-MNs has made it difficult to identify them for study. γ-MNs originate in the ventral spinal cord along with three subtypes of α-MNs19. Recently, molecular markers have become available for identifying γ-MNs in co-culture with α-MNs from murine tissue26. However, functional data of postnatal in vitro γ-MNs has yet to be observed and no human systems have been studied.Investigations surrounding γ-MN interactions have been sparse due to the nature of the cell types involved, and the lack of 27. These data imply that animal models may be insufficient to recapitulate the human form of the disease and more accurate, reproducible, and modular models are needed to fully understand the mechanisms of disease onset and progression. There is also a distinct possibility that this circuit can be involved in neuropathic pain in some cases28 and a model system that could investigate this possibility should significantly advance our understandings in this field. In this study, we aimed to develop a de novo defined human-based functional in vitro fusimotor system in which the innervation of intrafusal fibers by γ-MNs was evaluated by both immunocytochemical and electrophysiological approaches.Utilization of human reflex arc test platforms with the inclusion of γ-MNs could allow for more appropriate analysis of neuromuscular diseases, particularly in relation to proprioceptive function. This is specifically applicable for Spinal Muscular Atrophy (SMA) research. The mechanism for SMA progression in murine models indicated a difference in motoneuron subtype (α versus γ) survival rates compared to analysis of human cells37. Figure et al.15 and Guo et al.34. Cellular proliferation and differentiation of intrafusal fibers and neurons were temporally monitored via phase contrast microscopy and assessed via morphological analysis as shown in Fig. in vivo38. Phase contrast images demonstrated both chain and bag morphologies of intrafusal fibers. Direct physical contacts observed morphologically between intrafusal fibers and neurons indicated possible intrafusal fiber innervation by MNs  on trimethoxysilylpropyldiethylenetriamine (DETA) coated glass coverslips in serum-free medium. This surface modification has been used as a means of providing a surface amenable for the proliferation and maturation of an array of cell types for long term experimentationMNs Fig. .Figure 139. Upon the binding of neuregulin to ERB2, the receptor becomes phosphorylated (pErbB2)40, leading to an increase in the transcription factor EGR3, which initiates the expression of genes for intrafusal differentiation41. Immunocytochemistry indicated positive identification of both bag and chain intrafusal fibers -based neuromuscular interaction42. In order to detect the possible innervation of intrafusal fibers by γ-MNs in the co-culture, intrafusal fibers were visualized by fluorescently-labeled bungarotoxin (BTX), a toxin that specifically binds to ACh receptors  was found to be the most robust. Other markers were evaluated, including Wnt7a (marker for γ -MNs) and NeuN (marker for α-MNs), but did not have as robust and repeatable of a signal as ERRγ  as the control. There was found to be a 5.3 fold increase in ERRγ expression in differentiated human motoneurons compared to the control ). Glutamate is an excitatory neurotransmitter that has been previously used to stimulate neurons in co-cultures with muscle without directly initiating myotube contractionure Fig. . Theoreture Fig. . To confved Fig. . This deved Fig. . The fewin vitro fusimotor system in which functional innervation of intrafusal fibers by γ-MNs was demonstrated by morphological, immunocytochemical and electrophysiological analysis. Neither the culture of human γ-MNs nor their innervation by intrafusal fibers has previously been shown in vitro. The recapitulation of the spindle fusimotor circuit in vitro with human cells provides a defined model to investigate the physiology of this circuit, which is otherwise extremely difficult to observe for human systems. The use of MNs and muscle derived from iPSCs also offers the possibility for their incorporation into human-on-a-chip systems designed for the investigation of patient specific neuromuscular diseases and deficits.We have developed a human-based in vitro and γ-MN identification and functional innervation had not been demonstrated with human cells at all. Here, a co-culture of human intrafusal fibers and human MNs was established to evaluate the interactions between these two important cell types from the sensory portion of the reflex arc. Initial morphological analysis via phase contrast microscopy indicated the two cell types were compatible in a serum-free defined system and formed close physical contacts suggesting synaptic interactions. Immunocytochemical analysis confirmed the identity of the cell types in these intercellular interactions. Intrafusal fibers stained positive for pERB2 and EGR3, while the γ-MNs were identified by immunostaining with a γ-MN specific marker ERRγ25.The functional sensory circuits, especially human based systems, have previously not been extensively investigated in vitro system.Electrophysiological analysis confirmed functional interactions between the γ-MNs and intrafusal fibers. γ-MNs were stimulated using the neurotransmitter glutamate. The majority of intrafusal fibers in co-culture demonstrated increased excitation upon the application of glutamate (28 out of 34), while only one intrafusal fiber in the monoculture responded. Furthermore, the glutamate-induced responses were terminated by the application of curare, a competitive antagonist of acetylcholine receptors. These data indicate functional innervation of human intrafusal fibers by human γ-MNs in our defined 44. Multiple mechanisms have been proposed regarding the function of glutamate receptors in muscle tissue, but none of which correspond to direct action potential generation50. Actually, a similar response was recorded from another intrafusal fiber in a muscle-only culture by adding media instead of glutamate (data not shown), indicating the addition event itself could non-specifically induce muscle excitation, although very rarely. To further confirm the muscle was not directly excited by glutamate in our system, glutamate dosage experiments (data not shown) were performed on muscle only cultures to evaluate the responsiveness of intrafusal fibers to increasing concentrations of glutamate. The lack of glutamate mediated electrical activity in these cultures, even at concentrations far higher than used in experiments reported here, demonstrates the lack of direct response to glutamate. This evidence confirms that the single cell responsive to glutamate was not the result of a glutamate induced electrophysiological mechanism in this human model.It should be pointed out that a single intrafusal fiber in monoculture conditions exhibited AP firing upon exposure to glutamate. The presence of glutamate receptors on muscle fibers is known and has been researched51. Most existing in vitro systems used to study these diseases only investigate the motor perspective of neuromuscular interactions. However, utilization and especially integration of sensory components is essential to investigate complicated neuromuscular diseases by the inclusion of afferent mechanisms. Additionally, the sensory portion of the reflex arc has been of interest to the field of pain research28. Drug discovery has been moving towards more repeatable and high-throughput organ-on-a-chip in vitro systems recently for use in preclinical compound evaluation to improve drug discovery efficiency8. These systems provide a highly controllable and repeatable platform that can be tailored to more human-specific diseases by utilizing patient-derived iPSCs. Incorporation of γ-MN and intrafusal interactions to such systems will allow for a more complete and refined model for simulating neuromuscular diseases and testing a multitude of drug response variables.Study of the entire functional reflex arc is essential to understand and further investigate many neuromuscular disorders, predominantly Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). In both reports the sparing of γ-MNs were reported in mice models as well as peripheral neuropathies53.18 mm round glass coverslips  were cleaned and functionalized by exposure to an oxygen plasma generated by a Harrick plasma cleaner (model PDC-32G) utilizing high purity oxygen gas. The surfaces were then submersed in a reaction solution of 1% v/v of trimethoxysilylpropyldiethylenetriamine  in dry toluene  and heated to just below the boiling point of toluene over a period of 30 minutes. The reaction vessel was removed from heat and allowed to cool for 30 minutes, and then rinsed in 3 serial toluene baths. Next, the surfaces were placed in dry toluene and heated to just below the boiling point of toluene over a period of 30 minutes. After the second heating step, the surfaces were cured in an oven at approximately 110 °C overnight (~15 hr). Derivatized surfaces were characterized by contact angle goniometry, with a 5 µL droplet of water, and by X-ray photoelectron spectroscopy using a Thermo Scientific ESCALAB 220i-XL instrument with aluminum Kα X-rays and a 90° take-off angleHuman intrafusal fibers were differentiated from human satellite cells provided as a gift from Dr. Herman Vandenburgh. Human skeletal muscle stem cells (hSKM SCs)/progenitors were isolated and proliferated as described in Thorrez et al.54. Briefly, the primary human skeletal muscle cells (hSKMs) were isolated by needle biopsy55 and expanded in the myoblast growth medium  plus 15% (v/v) fetal bovine serum. Biopsies were performed on adult volunteers according to procedures approved by the Institutional Clinical Review Board of the Miriam Hospital and were performed in accordance with the relevant guidelines and regulations. All samples, from the study participation and publication of images, were obtained with informed consent and de-identified before being sent to UCF. Cell preparations averaged 70% myogenic content based on desmin-positive staining56. The differentiation protocol was adapted from our protocol for extrafusal fiber differentiation by inclusion of specific factors to facilitate intrafusal differentiation15. Specifically, thawed satellite cells from liquid nitrogen were plated onto glass coverslips at a density of 100 cells per mm2. The cells were given a whole medium change of human skeletal growth medium every two days until confluent. Upon confluence, the medium was fully switched to differentiation 1 medium (DMEM (Invitrogen 11775-040), Insulin (Sigma I9278) at 10 µg/ml, BSA (Sigma A9418) at 500 µg/ml, and EGF (Invitrogen 13247-051) at 10 ng/ml)34 and fed every 2 days for 4 days. At this point, the medium was switched to differentiation 2 medium  and MNs were added to co-cultures at 50 cells per mm2. The cells were fed every two days with differentiation 2 medium and maintained for four days. After this point, the medium was given a half change with NBActive4 differentiation medium. The cells were maintained in this medium for the remainder of the culture and fed every two days until 15–30 DIV total when they were analyzed via electrophysiology or fixed for immunocytochemical analysis.Human Motoneurons were differentiated from spinal cord stem cells (SCSCs) NSI566RSC according to a previous report9. Specifically, thawed SCSC cells (0.5 E6) from liquid nitrogen were plated onto a T-25 cell culture flask coated with Poly-D-Lysine. The cells were supplemented with bFGF every day and fed every other day with N2B medium. Upon confluence, the cells were trypsinized with 0.05% Trypsin and the reaction was inhibited with the addition of 0.2% Trypsin inhibitor . The cells were replated onto a permanox dish (Diameter 60 mm) coated with Poly-D-Lysine and fibronectin. Cells were initially plated in priming medium and fed on day 2 of culture with a half change of medium. On day 4 the medium was half changed with human MN medium. The cells were maintained in the permanox dish for about 10 days. At this point cells were again trypsinized and purified via an Optiprep concentration gradient  and then made to 9.5, 7.0, 6.0, and 5.5% for fractions 1–4, bottom to top sequentially [v/v] in Neurobasal, with fractions 1 and 3 containing phenol red and fractions 2 and 4 lacking phenol red for optimizing visualization of the fractions). The cells were plated into DETA coated glass coverslips for less than 10 days before being trypsinized (as described above) and replated onto either muscle cultures or control DETA coated glass coverslips. At this point the neurons either underwent exposure to intrafusal differentiation media (co-cultures) or were maintained in human MN medium (monocultures).Cells on coverslips were fixed with 4% paraformaldehyde diluted in PBS solution for 15 minutes, then rinsed with PBS  for 5, 10, and 15 minutes and stored until staining in PBS. Cells were permeabilized with 0.1% Triton X-100 for 15 minutes then incubated for 1–2 hours at room temperature in blocking buffer (5% Donkey serum +0.5% BSA in PBS) to prevent nonspecific binding. Fixed and permeabilized cells were incubated overnight with primary antibodies (diluted in blocking buffer) at 4 °C. Antibodies used and their concentrations are listed in Table in vitro. The RNA extraction was done utilizing the Aurum™ Total RNA Mini Kit (Biorad 7326820). RT-PCR was performed using the iTaq™ Universal SYBR® Green One-Step Kit (Biorad 1725150). Reaction volumes was 25 µl total . Reactions were incubated at 50 °C for 10 minutes, 95 °C for 5 minutes, 95 °C for 10 seconds and 55 °C for 30 seconds cycled 40 times, and underwent melt curve analysis . ERRγ  expression was evaluated along with β-actin  as a positive control and template free β-actin reactions as negative controls. Samples were run in triplicate along with β-actin positive controls and template free negative controls. An MJ mini thermal cycler (Biorad) and Opticon Monitor software (Biorad) were used. Expression levels were corrected with β-actin levels.Human motoneurons and human spinal cord stem cells were harvested for RNA extraction at less than 10 days 2 0.5, MgCl2 1, EGTA 1, HEPES acid 5, HEPES base 5, Na2ATP 5. Patch pipettes were prepared  with a Sutter P97 pipette puller. Pipette resistance was 4–10 MΩ for intracellular patch clamp recordings. Experiments were performed with a Multi clamp 700B  amplifier. Signals were filtered at 2 kHz and digitized at 20 kHz with an Axon Digidata 1322 A interface. Action potentials were recorded in current-clamp mode under gap free conditions at zero holding potential. The series resistance was in the range of 5–10 MΩ and was compensated by 60% on-line. Leak currents were subtracted using a standard P/4 protocol. Before seals were established on the cells, offset potentials were nulled. Capacitance subtraction was used in all recordings. All intrafusal fibers chosen in co-culture conditions were in proximity to MNs identified via morphological analysis. To test whether the patched intrafusal fiber was innervated, glutamate was added in 30 ul doses of 200 mM to the extracellular solution in the vicinity of the patched intrafusal fiber. When excited, the MNs would excite innervated muscle fibers and the excitation was recorded. Once repetitive firing was consistent, curare was added in 30 ul doses of 500 uM to the extracellular solution.Whole-cell patch clamp recordings were performed in a recording chamber on the stage of an upright microscope . The patch-clamp recording chamber was filled with the same medium as utilized for cell culture. The intracellular solution composition was (in mM): K-gluconate 140, NaCl 4, CaClThe proportion of active intrafusal fibers that showed electrical activity in response to glutamate was compared between muscle only and co-cultured conditions by categorizing each patched cell as responsive or unresponsive and organized into a contingency table, followed by statistical analysis via Fisher’s Exact Test (α = 0.05). 57 cells were patched across multiple coverslips and platings. The standard error of the mean for each proportion was calculated for the binomial distribution. γ-MN quantification data was taken from 20 view fields from each of three separate coverslips spanning over two separate platings. For γ-motoneuron- intrafusal fiber interaction quantification, two separate co-culture coverslips from two separate platings were stained with ERRγ, BTX488, and neurofilament. Intrafusal fibers positive for BTX488 staining that had contact with neuronal processes positive for neurofilament that lead to an identifiable soma were counted. Standard deviations were calculated and expressed as percent error.Supplementary information


Cancer research, in general, is focused on targeting tumour cells to limit tumour growth. These studies, however, do not account for the specific effects of chemotherapy on tumour endothelium, in turn, affecting metastasis.We determined how endothelial deletion of Akt1 promotes prostate cancer cell invasion in vitro and metastasis to the lungs in vivo in endothelial-specific Akt1 knockdown mice.Here we show that metastatic human PC3 and DU145 prostate cancer cells invade through Akt1-deficient human lung endothelial cell (HLEC) monolayer with higher efficiency compared to control HLEC. Although the endothelial Akt1 loss in mice had no significant effect on RM1 tumour xenograft growth in vivo, it promoted metastasis to the lungs compared to the wild-type mice. Mechanistically, Akt1-deficient endothelial cells exhibited increased phosphorylation and nuclear translocation of phosphorylated β-catenin, and reduced expression of tight-junction proteins claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of β-catenin nuclear translocation using compounds ICG001 and IWR-1 restored HLEC tight-junction integrity and inhibited prostate cancer cell transendothelial migration in vitro and lung metastasis in vivo.Here we show for the first time that endothelial-specific loss of Akt1 promotes cancer metastasis in vivo involving β-catenin pathway. Hence, it is important to determine the specific role of a pathway and the effect of a drug on tumour vasculature alone so as to improve the efficacy and minimise the side effects of cancer chemotherapy.Currently, research in the development of cancer therapy more focused on the pathways promoting tumour cell growth and invasion. Studies that address the specific role of a pathway in stromal cells and how drugs affect stroma when used for cancer therapy are fewer. Among the cells in the tumour microenvironment, tumour endothelium plays a significant role not only in tumour angiogenesis, perfusion and metastasis4 including prostate cancer.5 A number of studies from our laboratory have indicated that pharmacological and genetic inhibition of Akt, particularly Akt1, inhibits prostate and bladder cancer cell function in vitro and tumour xenograft growth in vivo.8 We previously reported that, drugs such as statins and angiotensin receptor blocker candesartan, that have the ability to normalise Akt1 activity in prostate cancer by inhibiting hyperactive Akt1 in prostate cancer cells,11 and activating Akt1 from its basal state in endothelial cells, led to the inhibition of prostate cancer cell transendothelial migration in vitro.12 We have also reported that Akt1 gene knockout in mice promoted tumour vascular permeability and angiogenesis in a murine B16F10 melanoma model.13 Most recently, we demonstrated that endothelial-specific knockdown of Akt1 results in increased vascular permeability via FoxO- and β-catenin-mediated suppression of endothelial tight-junction claudin expression, mainly claudin-5.14 Since many inhibitors of Akt are in different phases of clinical trials for various types of cancers, it is important to understand the effect of Akt1 suppression in endothelial cells of tumour vasculature, and its consequences on tumour growth and metastasis.Preclinical and clinical research evidence has revealed the integral role of phosphatase and tensin homologue (PTEN)-Akt pathway in multiple cancers,13 on prostate cancer cell invasion in vitro and metastasis in vivo using murine lung colonisation model of in vivo metastasis. Our analysis revealed that Akt1 deficiency in human lung microvascular endothelial cells (HLECs) enhances the ability of human metastatic PC3 and DU145 prostate cancer cells to migrate across the endothelial monolayer in vitro, and murine RM1 prostate cancer cell metastasis to the lungs in vivo, with no changes in the growth of RM1 tumour xenografts in vivo. The akt1 loss in HLECs resulted in increased translocation of phosphorylated β-catenin from the endothelial-barrier junctions to the cytosol and the nucleus, in turn, suppressing the transcription of endothelial tight-junction proteins such as claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of β-catenin in HLEC with ICG001 and IWR-1 restored the tight-junction protein expression and inhibited DU145 cell transendothelial migration in vitro and murine RM1 cell lung metastasis in vivo. Although Akt1 is a well-known mediator of oncogenic transformation15 and prostate tumour growth,8 our current study demonstrates for the first time that endothelial-specific Akt1 loss will promote prostate cancer metastasis via nuclear translocation of β-catenin and suppression of endothelial tight-junction protein expression.In the current study, we investigated the effects of endothelial-specific knockdown of Akt1, a major endothelial isoform of AktLoxP mice with VE-Cadherin-Cre mice in the pure C57BL6 background as reported previously.14 Age-matched 8- to 12-week-old male mice were used in the study. Genotyping was performed using specific primers for A1-3Loxp: TCACAGAGATCCACCTGTGC, and A1-4113R: GCAGCGGATGATAAAGGTGT. Tamoxifen  stock solution (100 mg/ml) was prepared to dissolve in absolute ethanol and stored in an aluminum foil-covered plastic tubes at −20 °C. Right before injection, sterile corn oil was added to dilute tamoxifen to a final concentration of 10 mg/ml. Tamoxifen (1 mg/10 g dose) was administered to the mice using a 27 G needle via intraperitoneal (i.p.) injection every 24 h for 5 consecutive days. Following this, mice were maintained on a custom-made Tamoxifen diet  for the duration of the experiments.All the mouse experiments were performed with the approval of Charlie Norwood Veterans Affairs Medical Center Institutional Animal Care and Use Committee . All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals. Carbon dioxide asphyxiation followed by cervical dislocation was performed for killing. Isoflurane inhalation was used for anaesthesia. For our study, we utilised an endothelial-specific, tamoxifen-inducible Akt1 knockout mouse model (VECad-Cre-Akt1) by crossing Akt1 2 incubator at 37 °C and routinely passaged when 80–90% confluent. Stable shControl and shAkt1 (ACGCTTAACCTTTCCGCTG) HLEC cells were generated using SMART vector 2.0 lentivirus particles (109 p.f.u.) . Lentiviral particles were mixed in 1 ml Hyclone SFM4Transfx-293  and added along with 1 µl Polybrene . Three days later, transfection efficiency was tested through Turbo-GFP expression and subjected to 4 µg/ml puromycin  selection until all the cells expressed GFP. Pharmacological inhibition of Akt activity (phosphorylation) was achieved by treating the cells for 24 h with 10 µM MK-2206, an Akt inhibitor widely used in the clinical trials and GSK-3 inhibition was achieved by using 10 µM SB415286 .Primary HLECs  were maintained in EBM-2 medium with a GM-2 Bullet Kit . All cultures were maintained in a humidified 5% COImmunofluorescent staining of shControl and shAkt1 HLEC monolayers was performed using the chamber slides . HLEC were cultured to the monolayer, treated with either DMSO, 10 µM ICG001 or 10 µM IWR-1 for 24 h and washed twice with ice-cold PBS, fixed using 2% paraformaldehyde for 30 min, permeabilised with 0.1% Triton X-100 for 15 min and blocked with 2% BSA in sterile PBS for 1 h. Cell monolayers were then incubated with antibodies against claudin-5, pS33/S37/T41-β-catenin, total β-catenin, ZO-1 and ZO-2  at 4 °C overnight. Immunofluorescence was revealed using goat anti-rabbit AlexaFlour-488 secondary antibodies . Cells were mounted on a glass slide using DAPI containing mounting medium . Images were captured using a confocal microscope . The ‘blue’ nuclear staining of DAPI was pseudo-coloured to ‘green’ for a better contrast to detect co-localisation with β-catenin. Controls with no primary antibodies were analysed for any non-specific staining. For immunofluorescence staining of lung sections, lungs were fixed in 4% paraformaldehyde overnight and frozen sections were probed for Akt1 and CD31, followed by AlexaFlour secondary antibodies and mounted on DAPI. Images were taken with a fluorescent microscope . All controls gave negative results with no detectable non-specific labelling.17 Antibodies used include Akt1, Akt2, Akt3, pan-Akt, Tyr216GSK-3β, Ser33/37/Thr41β-catenin, claudin-5, ZO-1 and ZO-2  and anti-β-actin (Sigma). Densitometry was done using NIH Image J software.Cell lysates were prepared using complete lysis buffer  with protease and phosphatase inhibitor cocktails . Protein quantification was performed using Dc protein assay . Western blot analysis was performed as described previously.2 SYBR Green ROX qPCR Mastermix (Qiagen) in a real-time PCR equipment (Applied Biosystems). Sample cDNA was amplified and quantified over a large number of shorter cycles under the following conditions: an initial 10 min 95 °C period followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 15 s. To analyse the fluorescence signal, a threshold cycle (Ct) was determined, using the exponential growth phase and the baseline signal from fluorescence vs cycle number plots. The following primers were used for the messenger RNA (mRNA) analysis: claudin-5 (forward: 5′-CTGCTGGTTCGCCAACATT-3′ and reverse: 5′-TGCGACACGGGCACAG-3′), ZO-1 (forward: 5′-CAGCCGGTCACGATCTCCT-3′ and reverse: 5′-TCCGGAGACTGCCATTGC-3′), ZO-2 (forward: 5′-TTGAAGACACGGACGGTGAA-3′ and reverse: 5′-GTGATGGACGACACCAGCG-3′), Axin-2 (forward: 5′-GACGGACAGCAGTGTAGATG-3′ and reverse: 5′-GGGTTCTCGGGAAATGA-3′) and GAPDH (forward: 5′-AGCCACATCGCTCAGACAC-3′ and reverse: 5′-GCCCAATACGACCAAATCC-3′). Ct values were normalised to the level of expression of the housekeeping gene (GAPDH).Cells were grown until confluent in six-well culture plates and treated with DMSO and β-catenin inhibitors 10 µM ICG001 or 10 µM IWR-1 for 24 h. RNA was extracted using RNA isolation kit  and RNA quality was confirmed using Nanodrop 2000 spectrophotometer (Thermo Scientific). Complementary DNA (cDNA) was synthesised from 500 ng of RNA using RT2 First Strand kit (Qiagen) using a StepOne Plus thermal cycler and detection software  and quantitative real-time PCR (qRT-PCR) was performed using the RT24-well Transwell permeable plates with 8.0 µm polycarbonate membrane coated with Matrigel  were used. Briefly, cells were seeded in six-well plates and treated with either DMSO or ICG001 (10 µM) every day for a total of 72 h. Following this, cells were detached using cell dissociation buffer (sterile 20 mM EDTA in PBS [pH 7.4]) and 5000 cells were plated on the upper chamber of the transwell plates. Upper chambers were filled with serum-free medium and lower chambers were filled with DMEM/HG medium containing 20% FBS as a chemoattractant. Cells that invaded the matrigel and reached the bottom layers of the top chambers after 24 h of incubation were fixed using 3.7% paraformaldehyde then stained with 0.5% crystal violet solution. The cells were counted manually using the inverted microscope and the average was calculated from four random images.6) was injected subcutaneously in 7- to 8-week-old C57BL6 mice. Mice were killed on day 16, and tumours were dissected, weighed, and tissues were fixed in 4% PFA for immunohistochemistry analysis.Murine RM1 metastatic prostate cancer cells were grown to confluence in 75-ml flasks and were re-suspended in PBS, counted and an equal number of cells (1 × 106) suspended in normal saline were injected into each group of mice separately via the tail vein. Mice were treated with DMSO (control) or ICG001 (50 mg/kg body weight). In all the experiments, mice were evaluated for the presence of metastases 16 days after cell administration. 1.5 ml of 15% India-ink solution was injected intratracheally to stain the lungs and visualise the non-stained tumour nodules. Stained lungs were carefully resected and rinsed in Fekete’s solution , then placed in fresh Fekete’s solution overnight. The whole lungs were collected for the counting of tumour nodules. Following this, mice lungs were subjected to equal interval cross section (2 mm) analysis for RM1 cell colonisation to the lung tissues. Tumour area was measured using the NIH Image J software.Eight-week-old wild-type and VECad-Cre-Akt1 mice were divided into two groups for the administration of RM1 cells. Cells (0.5 × 10t-test (dependent t-test) and/or further confirmed with non-parametric test and Wilcoxon signed-rank test. For all other analysis, Student’s two-tailed t-test or analysis of variance test were used to determine significant differences between treatment and control values using the GraphPad Prism 4.03 software and SPSS 17.0 software.All the data are presented as mean ± SD and were calculated from multiple independent experiments performed in quadruplicates. For normalised data analysis, data was confirmed that normality assumption was satisfied and analysed using paired sample Whereas the role of Akt in oncogenic transformation and tumour progression in various cancers is well known, the specific effect of Akt1 activity modulation in endothelial cells on tumour growth and metastasis is not known. Hence, utilising shControl and shAkt1 primary HLEC, we investigated the effects of endothelial Akt1 knockdown on PC3 and DU145 cell transendothelial migration in vitro. Our study indicated that loss of Akt1 in HLEC resulted in the enhanced interaction of PC3 and DU145 cells compared to Akt intact HLEC as evidenced by their Turbo-GFP expression Fig. . Similar18 Endothelial Akt1 gene silencing was also associated with decreased phosphorylation of Akt and increased phosphorylation of β-catenin, a GSK3β substrate , and barrier protective agents like angiopoietin-1 induces activation of Akt and Src and hence they are greatly implicated in the regulation of vascular wall integrity.25 While there have been contradicting reports about the role of Akt in endothelial-barrier regulation, our recent studies have shown the integral role of Akt114 and its cross-talk with Src21 in the long-term protection of endothelial barrier in response to VEGF and angiopoietin-1. Although a number of clinical trials have been performed on the use of Akt inhibitors for cancer therapy, none has been approved so far and many studies have shown to further worsen the condition.31 As of today, the role of endothelial Akt1 in cancer metastasis and the effect of endothelial Akt1 loss on tumour growth and metastasis have not been studied.Vascular permeability is the hallmark of several diseases including cancer and organ injuries.32 Although a portion of the phosphorylated β-catenin undergoes proteolytic degradation in Akt1-deficient endothelial cells, these cells still have enough of phosphorylated β-catenin to translocate to the nucleus and function as a transcription repressor for claudins. Such a role for the phosphorylated β-catenin has been demonstrated in the normal as well as the tumour endothelial cells.33 ShAkt1 HLECs promoted interaction and invasion of PC3 and DU145 prostate cancer cells through their monolayers compared to shControl HLECs in vitro, associated with increased nuclear translocation of β-catenin. Interestingly, reduced expression of claudin-5, ZO-1 and ZO-2 in the shAkt1 HLECs was rescued upon treatment with β-catenin inhibitors ICG001 and IWR-1, which resulted in the attenuation of DU145 prostate cancer cell invasion through shAkt1, compared to a shControl HLEC monolayer in vitro. Although the mRNA levels of claudin-5 in HLECs were significantly lower in shAkt1 HLECs compared to shControl, which is reversed by treatment with β-catenin inhibitors, similar effects of Akt activity and β-catenin inhibition was not observed in the mRNA levels of ZO-1/2 suggesting that unlike claudin-5, ZO1/2 expression as result of Akt1 suppression or β-catenin inhibition is regulated via proteolytic degradation rather than transcriptional modulation. This also confirms our previous observation that Akt1-mediated endothelial-barrier modulation is predominantly through expression modulation of claudin-5 and not ZO-1/2.14 Most importantly, in a VECad-Cre-Akt1 mouse model, murine RM1 metastatic prostate cancer cells showed a significant increase in the rate of metastasis to the lungs, but not on the growth of tumour xenografts. Increased prostate cancer metastasis in these mice was significantly inhibited upon treatment with β-catenin inhibitor ICG001. Altogether, these results demonstrate the endothelial cell-specific role of Akt1 in prostate cancer metastasis.A major finding from the current study is that the lack of Akt1 in HLECs promotes prostate cancer cell transendothelial migration in vitro and metastasis to the mouse lungs in vivo involving increased nuclear localisation of β-catenin and suppression of endothelial tight-junction proteins. Phosphorylation of β-catenin in many cell types such as cancer cells results in its proteolytic degradation.34 a pro-angiogenic molecule that was initially identified as a vascular permeability factor,35 Akt activation was widely believed to promote vascular permeability and hence tumour growth and metastasis. In contrast to this dogma, our study using the systemic Akt1 knockout mice revealed increased angiogenesis in response to adeno-VEGF expression in the skin and melanoma tumour implantation.13 A major contributing factor to this phenotype was increased vascular permeability in Akt1 knockout mice because of reduced thrombospondin-1 and increased angiopoietin-2 expression, which provided a novel role of Akt1 in endothelial-barrier protection. These results were later confirmed in a different model of VE-cadherin overexpression and clustering in mice where the integral role of tight-junction proteins contributing to vascular protection by Akt1 was identified.23 Since then, a number of other researchers also reported the function of Akt1 as vascular protective in various disease models,37 demonstrating the integral role of Akt1 on endothelial-barrier and vascular protection in vivo.Since Akt phosphorylation was upregulated by VEGF,38 as well as vascular permeability blockers such as angiopoietin-1 and Robo-440 activated Akt1. Our most recent studies using the VE-Cad-CreAkt1 mice revealed that irrespective of the stimuli, Akt activation has a net endothelial-barrier protective effect14 and that the transient vascular permeability effect of VEGF is due to increased Src activity, independent of Akt1.21 These studies further informed us the important function of Akt1 to inhibit FoxO and β-catenin transcriptional activities by preventing its nuclear entry, thus protecting the tight junctions via preserving the expression of 20-member claudin family of proteins.14 Such an effect of Akt1 on tight-junction protein, claudin-5, in particular, was also previously reported upon VE-cadherin overexpression in endothelial cells.23 The latter study also reported cooperation between FoxO and β-catenin in mediating the transcriptional repression of claudin-5. This enabled restoring claudin-5 expression in endothelial cells by treating with either one of the FoxO or β-catenin inhibitors.14 In the current study, inhibition of β-catenin resulted in the reversal of claudin-5, ZO-1 and ZO-2 in endothelial tight junctions, and blunted the enhanced ability of PC3 and DU145 cells to migrate through the shAkt1 compared to shControl HLECs indicating the involvement of β-catenin in the process.An important gap in our knowledge on Akt’s role in vascular permeability vs vascular normalisation in tumours was the mechanisms leading to its differential regulation by various vascular permeability modulators. For example, vascular permeability inducers such as VEGF and TNFα19 GSK-3 is known to phosphorylate β-catenin,18 thereby dislodging it from the adherens junction and translocating it to the nucleus.19 Apart from this, cytosolic β-catenin levels are also determined by the Wnt signalling pathway.41 The ability of both transcriptional inhibitor (ICG001) and Wnt signalling inhibitor (IWR-1) to suppress human DU145 cell invasion in vitro and murine RM1 prostate cancer cell metastasis in vivo through the Akt1-deficient endothelium via reduced nuclear translocation of β-catenin and restoration of tight-junction protein expression revealed the importance of total nuclear pool of endothelial β-catenin on prostate cancer metastasis. In prostate cancer cells, the effect of GSK-3 on β-catenin phosphorylation is also dependent on Src-mediated phosphorylation of its tyrosine 218 residues.19 On the other end, reduced Akt1 activity in endothelial cells is associated with increased Src activity.21 In our study, shAkt1 HLECs exhibited increased GSK-3 tyrosine 218 phosphorylation, increased β-catenin phosphorylation, and its nuclear translocation compared to shControl cells indicating the role of GSK-3 in the process.Another important candidate downstream of Akt1 activation resulting in β-catenin nuclear translocation is the glycogen synthase kinase-3 (GSK-3).8 Such a novel role of Akt1 has also recently been shown in breast, liver and non-small cell lung cancers.45 Unlike endothelial cells, Akt1 deletion in prostate cancer cells although activated by GSK-3, the resultant increase in β-catenin phosphorylation led to its proteolytic degradation.8 Hence, we believe that, although inhibition of β-catenin resulted in the prevention of prostate cancer metastasis to the lungs in VE-Cad-CreAkt1 mice, long-term treatment with β-catenin inhibitors may promote epithelial-to-mesenchymal transition specifically in cancer cells and hence metastasis. Nevertheless, this study demonstrates for the first time that Akt1 suppression in endothelial cells will promote prostate cancer metastasis involving β-catenin-mediated suppression of tight-junction proteins such as claudin-5, ZO-1 and ZO-2.Although the effect of β-catenin inhibition on suppressing lung metastasis in VE-Cad-CreAkt1 mice would appear to be a promising strategy to prevent prostate cancer metastasis, our experience with regard to the role of Akt1 in advanced prostate cancer informs us to be more cautious with such an approach. In a very recent study investigating the stage-specific role of Akt1 in early and advanced prostate cancer, we identified that although Akt1 is essential for the oncogenic transformation and tumour growth; its suppression promotes metastasis.Supplemental Figure


Inhibition of the tankyrase enzymes (TNKS1 and TNKS2) has recently been shown to induce highly dynamic assemblies of β-catenin destruction complex components known as degradasomes, which promote degradation of β-catenin and reduced Wnt signaling activity in colorectal cancer cells. AXIN1 and AXIN2/Conductin, the rate-limiting factors for the stability and function of endogenous destruction complexes, are stabilized upon TNKS inhibition due to abrogated degradation of AXIN by the proteasome. Since the role of AXIN1 versus AXIN2 as scaffolding proteins in the Wnt signaling pathway still remains incompletely understood, we sought to elucidate their relative contribution in the formation of degradasomes, as these protein assemblies most likely represent the morphological and functional correlates of endogenous β-catenin destruction complexes. In SW480 colorectal cancer cells treated with the tankyrase inhibitor (TNKSi) G007-LK we found that AXIN1 was not required for degradasome formation. In contrast, the formation of degradasomes as well as their capacity to degrade β-catenin were considerably impaired in G007-LK-treated cells depleted of AXIN2. These findings give novel insights into differential functional roles of AXIN1 versus AXIN2 in the β-catenin destruction complex. APC gene [The Wnt signaling pathway orchestrates multiple developmental and adult homeostatic processes, whereas aberrant activation of the pathway underlies numerous human diseases such as cancer . β-catenAPC gene . RecentlAPC gene , 6. ConsAPC gene . IntriguAPC gene –10. ThesIn the current study, we have compared the role of AXIN1 with AXIN2/Conductin in the formation of degradasomes and degradation of β-catenin induced by the TNKSi G007-LK in colorectal cancer cells (SW480), as their relative contribution in the Wnt signaling pathway still remains incompletely understood. Surprisingly, we find that AXIN1 is neither required for G007-LK-induced degradasome formation nor for G007-LK-induced degradation of β-catenin, despite its significant upregulation after prolonged TNKS inhibition (24 h). In contrast, the formation and function of degradasomes were considerably impaired in G007-LK-treated SW480 cells depleted of AXIN2. In addition, new synthesis of AXIN2 was required for degradasome formation upon G007-LK incubation. Taken together, our results imply that AXIN2 is more important than AXIN1 in both initiation of degradasomes and turn-over of β-catenin upon TNKS inhibition.The following reagents were used: rabbit anti-AXIN1 C95H11), rabbit anti-AXIN2 (76G6), ; mouse anti-β-catenin (BD Transduction Laboratories); mouse anti-active β-catenin (clone 8E7) (Millipore); mouse anti-β-Actin (Sigma-Aldrich); Hoechst (Invitrogen); G007-LK [, rabbit 5’-GGTGTTGGCATTAAAGGTG-3’ [5’-GAGATGGCATCAAGAAGCA-3’ [siRNA oligonucleotides were from GE Healthcare Dharmacon. All siRNA transfections were performed using RNAiMax (Invitrogen) according to the manufacturer's protocol and 50 nM siRNA oligonucleotide per well. Non-targeting siRNA used as negative control was from Dharmacon (D-001810-01). For siRNA-mediated depletion of AXIN1 and AXIN2, the following targeting sequences were used: AXIN1: AGGTG-3’ ; AXIN2: AAGCA-3’ .The SW480 cell line was purchased from American Type Culture Collection (ATCC). Upon receipt, cells were frozen and individual aliquots were taken into cell culture, typically for analysis within 15 passages. Cells were grown in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Testing for mycoplasma contamination was performed every sixth week. For inhibition of TNKS activity, cells were treated with 0.5 μM G007-LK for indicated time points. Dimethyl sulphoxide (DMSO) was used as a control. For inhibition of proteasomal activity, cells were treated with 10 μM MG132 for 6 h, either alone or in combination with G007-LK. mRNA translation was inhibited by adding 25 μg/ml cycloheximide (CHX) for up to 6 h, either alone or in combination with G007-LK. The SW480 cell line stably expressing GFP-TNKS1 has been described previously [® Imager system (LI-COR Biosciences) was used to scan all blots. Protein bands were quantified using the Odyssey software.Cells were rinsed in PBS and lysed in Laemmli lysis buffer  glycerol, 0.01% bromophenol blue, Dithiothreitol (DTT)). Equal amounts of whole cell lysate were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad Laboratories) and blotted onto PVDF membranes (Millipore). Immunodetection was performed with IRDye-conjugated secondary antibodies (LI-COR Biosciences). The OdysseySW480 cells stably expressing GFP-TNKS1 were treated with G007-LK or a combination of G007-LK and MG132 and then imaged on a Deltavision live cell microscope . To cover the whole cellular volume, stacks were acquired. One stack was acquired before addition of the inhibitors (timepoint "0"), afterwards images were acquired every 10 minutes for 6 h. After deconvolution, sum intensity projections were made. Image analysis was done in ImageJ/Fiji  to countCells were grown on coverslips and fixed in paraformaldehyde. Images were automatically taken using the Olympus ScanR system with an UPLSAPO 40×/0.95 objective. All images were taken with the same settings and below pixel saturation. The Olympus ScanR analysis program was used to measure the average number of G007-LK-induced GFP-TNKS1 puncta. Typically between 2000 and 10000 cells were analyzed per condition in each experiment.Student`s t-test was done on ScanR and Western blot quantifications to examine statistical significance.APC tumor suppressor gene [The colorectal cancer cell line SW480 is commonly used as a model for Wnt-dependent cancers due to a mutation in the sor gene . TNKS insor gene . Degradasor gene . Surprissor gene . Concurrsor gene . To invesor gene , 10). Tisor gene . InteresAXIN protein levels are regarded as the rate limiting step for the stability and function of endogenous β-catenin destruction complexes . The forAXIN2 transcription [Taken together, our findings indicate that formation of degradasomes during the initial 6 h of TNKSi treatment requires a stabilization of AXIN2 protein levels. We further propose that the immediate formation of degradasomes (<30 min) observed in live-cell imaging upon combined G007-LK and MG132 treatment is due to a temporary stabilization of AXIN2 protein levels when inhibiting the proteasome and/or a delay in the MG132-mediated repression of cription . HoweverAXIN2 by complexing predominantly with the TCF/LEF family of transcription factors [AXIN2 initiates a negative regulatory feedback loop upon activation of the Wnt pathway [AXIN2 mRNA transcription, but on the other hand mediates decreased PARsylation and degradation of AXIN2 protein, we sought to further investigate this delicate regulation of AXIN proteins stability upon TNKS inhibition. To elucidate the contribution of AXIN2 new synthesis, we investigated to which extent degradasomes are formed in G007-LK-treated cells after inhibiting mRNA translation with cycloheximide (CHX). SW480 cells expressing GFP-TNKS1 were treated with CHX and G007-LK for 6 h, either alone or in combination, and then investigated by high-throughput image acquisition using the Olympus ScanR microscope  experiments demonstrated that β-catenin-mCherry was rapidly turned over in the G007-LK-induced degradasomes. Taken together, these findings provided a direct mechanistic link between degradasome formation and reduced Wnt signaling in colorectal cancer cells . To furtin vivo . Howevert ligand , which it ligand –31. ThusIn conclusion, we have studied the individual contributions of AXIN1 and AXIN2 in the formation of TNKSi-induced degradasomes and degradation of β-catenin. Taken together our results reveal a surprisingly differential role of AXIN1 and AXIN2. AXIN2 stands out to be the most important player in TNKSi-induced degradasome formation and β-catenin degradation in SW480 colorectal cancer cells. However, the functional role and relative contributions of AXIN1 versus AXIN2 during steady-state turnover of β-catenin in APC wild-type cells remain to be investigated.