Of note, it was also observed that atrazine suppressed the transcription of Na+, K+ transfer associated genes (Na+-K+-ATPase subunits) and Ca2+ transfer associated genes  in heart and liver. In conclusion, atrazine induced cardiac and hepatic damage via causing the ionic disorder, triggering the transcription of the ion transporters and leading the histopathological and functional alternations in the heart and liver of quails. This study demonstrated atrazine significantly induced the ionic disorder via decreasing the ATPases activities and disturbing the transcription of the ion transporters.The widely used atrazine has been reported to exhibit extensive ecological hazards. Due to the biological accumulation, atrazine elicits widespread toxic effects on different organisms. However, true proof for the mechanism of atrazine-induced toxicity is lacking. To determine the potential mechanism by which atrazine exerted toxic effects, quails were treated with atrazine  by gavage administration for 45 days. Atrazine significantly increased the histological alterations and serum creatine kinase, lactate dehydrogenase and choline esterase levels. A marked disorder in ionic (Na The most commonly used herbicide, atrazine, has been extensively monitored in the whole world, contaminating plants, soil, water resources , 2. AtraNumerous studies have demonstrated that exposure to atrazine induced oxidative stress, lipid peroxidation and antioxidant depletion -26. In aProper ion concentrations are benefit for a number of physiological processes such as transmembrane ionic balance, membrane potential, pH balance and cell volume to ensure correct functions of the entire body, especially the heart and liver . Indeed,Considering the central role in whole-body osmoregulation and high susceptibility to atrazine attack, ATPase is widely used as a marker for ion regulatory changes , 36. In P < 0.05) in quails of HD group and the liver coefficient increased  in all atrazine-exposed quails, respectively , suggesting an injury in atrazine-exposed quail liver.Regarding the biochemical analyses, the serum LDH was significantly pronounced upon decrease by atrazine at LD group , the serum Na+ didn't show any difference in groups  in all atrazine treated groups compared to Con group on heart and liver. The maximal adverse effects were observed for cardiac and hepatic Na+-K+-ATPase activities after the administration of 500 mg/kg atrazine (P < 0.01).To investigate the effects of Nap Figure . Na+ cons Figure . Similarerum Cl- . The act+-K+-ATPase can alter the activity of Na+-K+-ATPase, we next detected the expression of Na+-K+-ATPase associated subunits transcription. These results revealed a set of Na+-K+-ATPase subunits  in quail heart whose transcription was first increased in LD and then decreased in MD and HD significantly by atrazine treatment, compared to Con group. Additionally, a set of Na+-K+-ATPase subunits  genes was decreased by atrazine in heart, compared to Con group (P < 0.001)  was first increased in LD and then decreased markedly in HD. A set of Na+-K+-ATPase subunits  in quail liver was decreased induced by atrazine treatment  Figure . Inspectt Figure . The effs Figure .2+ disorder in organ injury, we tested not only Ca2+ content, Ca2+-ATPase activity and ATPase subunits transcription, but also calpain, the Na+/Ca2+ exchangers (NCXs) and the Na+/Ca2+-K+ exchangers (NCKXs) associated subunits gene transcription in heart and liver. The cardiac Ca2+ content was significantly increased in LD, MD and HD groups . Similar to alterations in heart, the hepatic Ca2+ content was significantly increased in LD and MD groups . However, we also observed that there were no significant alterations in serum Ca2+ of atrazine-exposed quails  in all atrazine treated groups  in atrazine-treated quails  (P < 0.05)  expression was markedly decreased in MD and HD groups   (P < 0.001)  . Another< 0.001) , compare < 0.05) . Similars Figure , compare< 0.001) . The expp Figure , compare < 0.05) . Moreove) Figure . The hepThe NCXs form the SLC8 family, with NCX1, NCX2 and NCX3 being coded for by the genes SLC8A1, SLC8A2 and SLC8A3 respectively , 40. The2+ content showed a significant decrease in LD and HD groups compared to Con group . Similar to the alterations in heart, a significant decrease in hepatic Mg2+-ATPase activity in atrazine-exposed groups  was also observed. The results of Ca2+-Mg2+-ATPase activity after atrazine exposure were presented in Figure 2+-Mg2+-ATPase activity of atrazine-exposed groups compared to Con group  in heart and liver. These data, in light of previous reports including ours [2+ disorder was increased by disturbing the activities of Mg2+-ATPase and Ca2+-Mg2+-ATPase.Figure ing ours , 30 statP < 0.05), and the results were depicted in Figure PCA for the relevant metabolites was performed with an analysis of variance P < 0.0, and theTo distinguish potential effects of biochemical parameters in quails receiving different doses of atrazine, PCA was performed as an unsupervised pattern recognition method. The PCA scores showed that the dose-related separation within the atrazine-treated animals was quite remarkable in both heart and liver, and the changes in metabolic profiles from the controls to the low- middle- and high-dose groups occurred in a clockwise direction; while clearly separated from the controls Figure . Taken tAtrazine can interfere with the normal operation of the cardiovascular system leading to a broad range of disorders, increased rates of cardiovascular diseases , 42. LivAtrazine had been reported to interfere the growth and development of quails, to disturb its reproductive function, to induce endocrine disruption and to trigger the pathological and genotoxic effects , 11, 45.In this study, significantly ionic disorders and organs injury were observed in the liver and heart of quails. According to previous studies, atrazine can induce pathological effects in multiple tissues , 12. Atr+, K+ homeostasis. Among them the most important one in all living cells is Na+-K+-ATPase, which actively transport K+ into and Na+ out of the cells, responsible for the balance between Na+, K+ intra- and extracellular concentration, maintaining Na+, K+ serum levels [+, K+ disorders are associated with the inhibition of Na+-K+-ATPase [+, which revealed that the decrease of cardiac and hepatic K+ may be a response to the increase of K+ outside the cardiomyocytes and hepatocytes via disturbing Na+-K+-ATPase on quails. The changes of contents of K+ of experimental group in the liver were more obvious than heart's, suggesting that atrazine could disorder K+ content directly via influencing Na+-K+-ATPase activities. Moreover, atrazine caused cardiac, hepatic, nephric dysfunction, activated renin-angiotensin-aldosterone system, boosted levels of aldosterone [+ excretion and Na+ reabsorption. Thus, the contents of K+ in serum drop as well. The decreased Na+-K+-ATPase activity causes retention of Na+ in the cells. These results suggested that atrazine delivered cardiotoxicity and hepatotoxicity in quail via the decreased of Na+-K+-ATPase activity and subunit transcription as well as disorders in K+ levels  use the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+ and maintain Ca2+ homeostasis. NCXs and NCKXs are encoded by SLCs [2+-ATPase associated subunits transcriptions were significantly decreased by atrazine, which may be responsible for the overloading of Ca2+. Moreover, the retention of Na+ in the cells leads to the inhibition of the NCXs expression and the Ca2+ overload in the cells. The further overloading of Ca2+, the increase of Na+ by Na+-K+-ATPase were relieved by NCX. Thus, the changes of Na+ contents were not obvious compared with the K+ contents. As a consequence of Ca2+ influx by atrazine, the transcription of calpain is partially activated. Although the change of the activity of Ca2+-ATPase was extremely obvious in liver, the changes of the NCXs expression were more extremely obvious in heart. Additionally, Unlike Na+ channels, Ca2+ channels are also regulated by neurotransmitters. Several studies had reported that atrazine could activate cAMP [2+ channel, inducing the influx of Ca2+. However, the excretion of Ca2+ is inhibited by Ca2+-ATPase. The overloading of Ca2+ in the hepatocytes can be discharged to the bile by the activation of cAMP. Thus, the changes of the Ca2+ content were not obvious in liver compared with in heart. These findings emphasize the view that the decreased Ca2+-ATPase activity may contribute to the disturb of intracellular Ca2+ homeostasis by increasing Ca2+-ATPase associated subunits and SLCs mRNA levels, leading the organ dysfunction  was purchased from Zhonghe Chemical Limited Company . Birds were housed in cages in an environmentally controlled room (temperature 26 ± 2□ and humidity 50 ± 15%). Temperature and relative humidity were monitored daily. Feed and water were offered ad libitum during the experiment. After one week acclimation, the quails were randomly divided into four groups  aged 18 days and weighted 89.04 ± 3.66 g were purchased from Wan Jia farm in Harbin, China. Chemical atrazine  of 675 mg/kg in the northern bobwhite quail. In this study, all the experiments conducted in animals were in accordance with the guidance of ethical committee for research on laboratory animals.At the end of the experiment, the quails were weighed. Then, the birds were fasted before the day of sacrifice, and their heart and liver were carefully dissected out and weighed, then storing at -80 □ for assays. And the organ coefficient is obtained by the ratio of organ weight to body weight. The blood was collected from the heart of each bird and centrifuged at 3000 rpm for 10 min to obtain the serum. The serum were stored at -80 □ for assays.+, K+, Ca2+, Mg2+, Cl- concentrations and the activities of creatine kinase (CK), lactate dehydrogenase (LDH), choline esterase (CHE), glutamyltranspeptidase (GGT) were measured. The activities or contents of biochemical parameters were detected using the detection kits  by a biochemical auto-analyzer.Blood samples were used to investigate changes in the serum enzymes and concentration of ions considered to be biochemical indicators of hepatic and myocardial enzyme. Serum NaCardiac and hepatic specimens were fixed in 10% buffered neutral formalin and were processed for paraffin wax sectioning. Sections of about 5 μm thickness were stained with hematoxylin and eosin for light microscopy.+-K+-ATPase, Ca2+-ATPase, Mg2+-ATPase and Ca2+-Mg2+-ATPase were determined using the appropriate assay kits  according to the manufacturer's instructions using 10% tissue homogenates [The activities of Naogenates , 30.-ΔΔCt method [Total mRNA was extracted from hypothalamo, pituitary and ovary using RNAout reagent , according to the manufacturer's instructions. First cDNA strand was synthesized using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for quantitative real-time PCR (qRT-PCR)  . The primers for real-time amplification of relative cDNAs were designed using Oligo 7.22 Software  based on the deposited sequences in GenBank and primers used are given in t method , the resP < 0.05, **P < 0.01 and ***P < 0.001. Ranking of genes by degree of differential expression was analyzed with a heat map using the R Programming Language version 3.2.1. In addition, Principal component analysis (PCA) was used as an effective tool for simplifying the information from inter-correlated variables through linear transformation of the original variables into a few principal components. PCA was performed in this work to define the most important parameters, which could be used as key factors for individual variations using the same software. The observed relationships among the parameters were confirmed and quantified according to a Spearman's test. All data was presented as mean ± standard deviation (SD). In addition, Chem Draw Pro (version 15.0) was used for drawing pictures.The data was analyzed with GraphPad Prism 5.1  and SPSS 19.0 software . Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc pairwise comparison. Asterisks (*) indicate statistically significant differences from the control group, *


Herein, I consider the properties of the parathyroid Ca2+ sensing mechanism, the identity of the Ca2+ sensor, the intracellular biochemical mechanisms that it controls, the manner of its integration with other components of the PTH secretion control mechanism, and its modulation by other nutrients. Together the well-established, recently elucidated, and yet-to-be discovered elements of the story constitute the past, present, and future of the parathyroid and its calcium-sensing receptor (CaSR).Parathyroid hormone (PTH) defends the extracellular fluid from hypocalcemia and has powerful and well-documented actions on the skeleton and renal tubular system. To achieve a satisfactory stable plasma calcium level, the secretion of PTH, and the resulting serum PTH level, is titrated carefully to the prevailing plasma ionized Ca To prevent uncontrolled elevations in plasma calcium concentration in response to PTH, a molecular feedback mechanism mediated by the extracellular Ca2+ ion concentration (Ca2+o) suppresses PTH secretion from the cells of the gland  we now know as the calcium-sensing receptor (CaSR). I go on to describe how studies of this receptor in these cells have led to deep understandings of parathyroid function in health and disease and new approaches to therapies for various disorders of calcium metabolism and parathyroid function.The parathyroid gland elaborates a peptide hormone, parathyroid hormone (PTH) whose primary role is to prevent and/or reverse acute hypocalcemia. It achieves this by: mobilizing calcium from stores in bone; stimulating renal Cain vivo by intravenous infusions of calcium salts to induce hypercalcemia or Ca2+ chelators such as citrate or EGTA to induce hypocalcemia provoke rapid negative and positive changes in the serum PTH concentration respectively  also suppressed PTH secretion although Mg2+o was less potent than Ca2+o. Finally, increases in PTH secretion were observed in response to the β-adrenergic agonist isoproterenol that were partially reversed by the β-adrenergic antagonist propranolol  exogenous, GPCR-stimulated and (ii) spontaneous PTH secretion. This follows because pertussis toxin disabled Ca2+o- and divalent/tervalent cation-induced suppression of dopamine-stimulated PTH secretion  and ERK1/2 downstream of an extracellular Ca2+ sensing GPCR (see below).The results suggest the existence of an extracellular Ca2+ sensor was subsequently identified in populations of bovine parathyroid cells loaded with the cell-permeant Ca2+-sensitive fluorophore fura-2AM. The cells exhibited robust intracellular Ca2+ transients in response to elevated Ca2+o suggesting the action of a PI-PLC coupled GPCR that senses increases in Ca2+o  of parathyroid PI-PLC, suppressors of GPCR-stimulated cAMP accumulation, and inhibitors of PTH secretion . In this case, the successful cloning of the novel class C GPCR that is now referred to as “the calcium-sensing receptor” relied on its high degree of sensitivity to Gd3+, which was used to identify “active” pools of mRNA for further separation and purification. Once cloned, the receptor was readily expressed not only in Xenopus oocytes but also various mammalian cell lines including HEK-293 cells and was found to exhibit sensitivity not only to divalent inorganic cations including Ca2+ and Mg2+, and tervalent inorganic cations including Gd3+ but also to organic cations including the antibiotic neomycin . With the identification of two variants of FHH arising from mutations of two other genes, Gα11 and AP2S (see below), the major form of FHH that arises from mutations of the CaSR has been recently renamed FHH1.Deactivating, typically heterozygous, mutations of the CaSR gene in FHH result in impaired or disabled Ca2+o-mediated feedback control of PTH secretion along with skeletal demineralization and pathological fractures  arises from unrestrained CaSR activation on the contraluminal membrane of the thick ascending limb, which disables NKCC2-dependent NaCl reabsorption , which exhibits hypocalcemia, suppressed serum PTH levels, hypocalciuria, hyperphosphatemia, and ectopic mineralization and cataracts  at the extreme C-terminal end of the VFT domain, immediately prior to the start of the Cysteine-rich domain. Mice with this genotype exhibited a condition comparable to NSHPT in which homozygotes were normal at birth but exhibited severe growth retardation and markedly reduced muscle power in the days after birth  into the mature, fully processed mRNA  or stimulated (−202 to 791) PTH secretion  has entered clinical practice for the treatment of patients with secondary hyperparathyroidism on hemodialysis 2+- or L-amino acid-induced cellular or tissue responses  have been developed. These agents, in general, bind in the HH domain and suppress CaSR signaling. For this reason, they have proved useful in assessing the role of the CaSR in Ca2+ ions, the CaSR also responds promiscuously to L-amino acids of various classes  inflammatory cytokines including IL-1β, IL-6 and TNFα . Consistent with these findings, inactivating and activating mutations of the human Gα11 gene have been shown respectively to underlie variant forms of FHH (FHH2) and ADH  was present in both the inactive and active structures and three of which were only identified in the active structure and, thus, may act to stabilize it. Interestingly, no Ca2+ binding site was located in the closed (active) form of the agonist-binding cleft in the structure reported by Geng et al., which was occupied instead by the amino acid L-Trp  as well as Ca2+ ions and that Ca2+ stabilizes the active state, whereas Pi stabilizes the inactive state have potentially important implications for understanding parathyroid function since elevated Pi concentrations stimulate PTH secretion  subject to control by CaSR heterodimers with metabotropic glutamate receptors .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We develop a clustering algorithm to automatically group pixels with similar microstructural features. μMAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (μMAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. μMAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply μMAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors  Since its organization changes in pathological processes9, it can be exploited as an early diagnostic marker in experimental and clinical medicine11. Collagen, myosin and microtubules in cells and tissues13 can be non-invasively imaged also in-vivo by Second Harmonic Generation (SHG) microscopy, a nonlinear coherent optical process where two incident photons of frequency ω are converted into a single photon of exactly twice the frequency, 2ω.Collagen is the prominent constituent protein of tissues, providing them structural support and protection19, the polarization dependence of the SHG response (P-SHG spectrum) can provide the molecular and supramolecular structure of different organs and tissues27. P-SHG microscopy is a promising and efficient diagnostic tool in the identification of cancer types23 and other pathological conditions such as ischemia, skin pathologies, fibrosis and aging31. In fact, P-SHG imaging, in different experimental implementations39 and with the support of various theoretical models of the fibrils organization, can be exploited to obtain pixel-by-pixel maps of the fibril orientation41, of the protein pitch angle42 and of the nonlinear susceptibility tensor components46. Although usually a time consuming non-linear fitting of the P-SHG spectrum of each pixel is exploited, substantial speed-up in the analysis has been recently obtained through 1D Fast Fourier Transform of the P-SHG curves26.A part from the morphological information extracted from intensity-based SHG imaging50, allowing to extract quantitative information on a model parameter at a pixel-by-pixel level.Starting from a similar Fourier Transform (FT) approach, we build here a phasor analysis of the P-SHG images that acts at a global level and identifies automatically clusters of homogenous microstructure. The phasor approach has been extensively applied to graphically analyze spectra on a variety of techniques from fluorescence lifetime imaging to MRIF) and of the susceptibility anisotropy parameter ). μMAPPS retrieves maps of both model parameters in few hundreds of ms to few seconds, by working on two coupled phasor spaces. Moreover, instead of manual and subjective clustering procedures exploited in other phasor approaches53, we develop an automated clustering algorithm, working simultaneously on the two phasor spaces, to back-project onto the original image the Regions of Interest (ROIs) characterized by similar θF and γ values. In this way we are able to automatically single out patches sharing similar micro-structural properties.We extend this global analysis by proposing a new method, named Microscopic Multiparametric Analysis by Phasor projection of P-SHG (μMAPPS), that provides micro-structural information (at pixel level) of the collagen architecture in tissues, both in terms of the mean orientation angle of the fibril  directly in the phasor spaces and by measuring geometrical features of this distribution. Data denoising can be performed in the phasor space without affecting the spatial resolution of the original data. More important, by assuming a molecular model for the SHG response, the microstructural parameters retrieved graphically are not biased by the initial parameters values and constrains, contrary to the usual non-linear fitting procedure.ex-vivo experiments on mouse-tail tendons54.In the following a description of the μMAPPS core is presented together with its validation against simulations and in-vivo experiments.The application of μMAPPS is then illustrated in the extraction of the described microscopic parameters in fixed and explanted tumor samples (biopsies). Finally, the robustness of our method has been tested in L. The maximum intensity projection of the stack provides the overall disposition of the collagen in the sample  of a point in the θp-plot are the first DFT of the normalized P-SHG curve, L (0 ≤ θL < 3π/2) with resolution Δθ. We define the frequency More in details, the coordinates  in the γp-plot  are analyzed in order to discard the background contribution.Only P-SHG spectra above a threshold  describes the molecular anisotropy and the scale factor k includes the absolute intensity of the SHG signal as affected by the setup parameters. Equation (∞) or hexagonal (C6) plus Kleinman symmetries .The parameter γ > 0  and wit0 term added to Equation  vector and all vectors spanning the reference curve  was modEquation ), probab26, which is equivalent to our Equation F and γ parameters, avoiding the algebraic solution proposed in ref.26 to determine γ, and exploiting the denoising opportunities of the phasor approach.It should be noted that our graphical method, by acting on a double phasor space, goes beyond the measure of the phase of the first Fourier component of the P-SHG spectrum as done in ref.F. Non ideal data sampling  results in the shift of the points from the ideal reference curve in the γp-plot. This is due to the fact that we compute the point coordinates  on the P-SHG spectrum shifted by an angle that is the nearest sampled θF value estimated from the θp-plot. Moreover, two P-SHG spectra characterized by θF values differing by a multiple integer of Δθ (and γ1 = γ2), have similar projections on the γp-plot reference curve. In fact, exactly the same (θF ≤ θL < θF + π/2) portion of identical P-SHG spectra are DFT analyzed in this case.The μMAPPS core has been thoroughly validated against numerical simulations. μMAPPS is able to tackle with nonideal experimental conditions such as the laser polarization sampling Δθ and the noise  and θ2 = θF + π/2 and 0 < γ2 < 1 may fall on the same position on the γp-plot reference curve. In these cases, the experimental P-SHG spectrum is then directly compared to the model  and θF = θ2, γ = γ2 . The solution with the minimum chi-squared value from the experimental P-SHG is selected. This procedure can be avoided if the type of collagen  is a priori known.Finally, μMAPPS allows us to solve efficiently also an intrinsic degeneracy of the model (Equation ). As shoFor tissue analysis, it is essential to identify pixels characterized by similar microstructural parameters. Instead of manually (and somehow arbitrary) selecting different areas in the phasor plot and map the corresponding pixels back onto the morphological image, we develop here a data clustering approach (please refer to Supplementary Note 5).F − γ space. It first identifies the putative clusters’ centers according to a maximum density approach56 over both the θF and γ parameters. Each element which is not a putative center is assigned, by an iterative procedure, to the cluster for which the element-center distance is the lowest. The validation of the clustering algorithm on P-SHG simulated noisy spectra  as in Equation , with low anisotropy values (γ < 2). Regions that correspond in the morphological image to crimped features . Over the whole image in Fig. 54 and the distribution of the fibrils angle is peaked at θF ≅ 116° with two secondary components at θF ≅ 80° and at θF ≅ 155°. A more quantitative analysis of the spatial in-homogeneity of the tissue can be obtained starting from the application of our clustering algorithm to the two coupled phasor spaces. Figure F> = 113° ± 7° and <γ> = 1.7 ± 0.2.As highlighted in Fig. C = 2° and γC = 0.1, Figs. 57 . The average SHG signal from collagen reveals thin wavy fibers in the ECM of tumor samples explanted 7 days after inoculation, as shown in Figs. C = 5° and γC = 0.2; ET, minimum number of elements in a cluster with respect to the total number of analyzed pixels, =2%).Alterations in the ExtraCellular Matrix (ECM), whose prominent constituent is collagen, are correlated to tumor development: the increased collagen deposition in some tissues facilitates tumor initiation and progressionC = 2° and γC = 0.1), leading to parameters in excellent agreement with μMAPPS results. This successful validation not only demonstrates that μMAPPS is able to obtain microscopic parameters in agreement with fitting-based algorithms, but also that it is a versatile method that can afford deviations from the theoretical model described by Equation . Two exemplary images of a tumor at day 7 and 12, out of 30 FOVs from 3 biopsies, are shown in Figs.  7 Figs.  and at d 7 Figs. . The γ d 7 Figs.  computed 7 Figs. . The wid 7 Figs. , while i 7 Figs. , variablDifferences in the microscopic organization of the investigated samples can be also extracted by a global analysis of the shape of the cloud of points within the θp-plot Figs. . AlthougC = 30° and γC = 1 ET = 10%) in order to evaluate the mean Cluster Elements Ratio  and to single out the presence of a main cluster. At day 12 the <CER> is 0.54 ± 0.14, while < CER > = 0.14 ± 0.14 at day 7, indicating the presence of a major cluster at day 7. Clusters obtained with these large cutoff parameters are reported in Figs. We further characterize the tumor biopsies by applying the clustering algorithm. First, we assumed large clustering parameters , obtaining the results summarized by the cumulative distributions of Fig. Small scale heterogeneity in the tissue can be singled out by looking for clusters with few elements. To this purpose, we clustered the data set with more tight conditions  in the tumor ECM in-vivo. A ROI . The Gaussian fit of the anisotropy distribution , are shown in Fig. in-vivo  are present, suggests that our algorithm can be effectively extended to more complex situations taking into account axial polarization component, experimental errors and deviations from the present theoretical model40. With respect to existing P-SHG analysis routines29, μMAPPS provides a fast and intuitive representation of the structural content of the sample by working globally on the image and by grouping the pixels through non-spatial features they share with other pixels.μMAPPS is based on the Fourier projection of the P-SHG stacks of images onto two concatenated phasor plots from which, by a simple geometrical construction and the assumption of a molecular model, we can extract distributions of the collagen fibrils angle, θin-vivo) have been selected for a first validation of the method. A more in depth study has been performed in tumor biopsies at two stage of progression by exploiting also geometrical features of the phasor plots and our clustering algorithm.We applied here μMAPPS to characterize the microscopic behavior of both organized tissues, such as tendons, and of the more complex tumor ECM architecture. In this regard, three different tissue preparations  and the CER factor in the cluster analysis, are two examples. For example, the biopsies at day 7 (less ordered) differ substantially from those taken at day 12 (more uniform population of clusters), notwithstanding the fact that we retrieved the same anisotropy distribution at the two time-points.F and γ values within a selected threshold) and to obtain other quantitative global characterization of the microscopic architecture of tissues that can be amenable to define scores for the automatic recognition of pathologies both ex-vivo and in-vivo. We believe therefore that our method could perform fast microstructural analyses that assist or replace the histo-pathological evaluation and could open future applications for in-situ diagnosis of pathologies and diseases. Efforts are ongoing to develop cluster-based machine learning algorithms with diagnostic capability of tumors.In summary, our clustering algorithm, working on a phasor-based analysis of the P-SHG images, allows to automatically back-project data in the original image highlighting areas that share the same microscopic behavior  mounted on an upright optical microscope , coupled to a tunable fs-pulsed Ti:Sa laser . A high working distance objective  has been employed to both excite and collect the emitted signal. The backscattered second harmonic signal, primed by 800 nm wavelength, has been steered to a home-built non-descanned unit, filtered by a short-pass 670 nm filter  and by a 400/20 nm band-pass filter , and collected by a photomultiplier tube . The entire microscope is surrounded by a custom made thermostatic cabinet in which the temperature is kept at 37 °C  for 2 and 600 × 600 μm2). The following excitation laser power, measured before the scanning-head, has been exploited: Pexc = 10 mW for normal mouse-tail tendon, Pexc = 20 mW for mouse-tail tendon in case of collagenase-digestion . Regarding tumors, Pexc = 50 mW for histology sample analysis, while it was variable in the range 50–60 mW for biopsies and in-vivo experiments. Higher power (above 60 mW) have been used only during z-scan imaging deep within the tumor. P-SHG data have been acquired in the range 10–15 μm below the tumor surface. Care must be taken in the proper laser power selection in order to avoid tissue damages. Each image is the result of 3–5 Kalman average scans (512 × 512 pixels) and has been acquired in 3.4–5.6 s. For in-vivo P-SHG experiments in tumors, a sequence of 6 z-planes has been acquired along the z axis at 1 μm inter-distance for each incident laser polarization in order to be able to post-process the images in case of z movements. In this case 2 Kalman average scans have been acquired for each angular step. Specific corrections to the μMAPPS algorithm for birefringence, polarization cross-talk or diattenuation36 were not applied to our P-SHG data, collected from tissue surface (10–15 μm below the surface).Sequences of images of single z plane have been acquired by rotating the laser polarization from 0° to 180° in steps of 5° in order to obtain a rotation of the laser polarization from 0° to 360° in steps of 10°. Each image has a resolution of 512 × 512 pixels. The acquired field of view is described in the images captions , each cluster is encoded with different monochromatic LUT with an intensity scaling as the pixel integrated P-SHG signal.F used for performing the clustering analysis of the experimental data are reported in the caption of the corresponding figures. Moreover only clusters with a number of elements higher with respect a threshold (ET), computed as a percentage of the total number of analyzed pixels, have been considered and reported in the figures captions. Only P-SHG spectra above a threshold (TNL) are analyzed in order to discard the background contribution.The cutoff values for γ and θL for the fit procedure, the signal related to each pixel of the P-SHG stack (belonging to the considered cluster) is divided for its maximum intensity and then the average of these signals is computed.In order to extract, for each cluster, the mean SHG signal as a function of θMeasure tool of ImageJ .In the θp-plot, the ratio between the major and minor axis of the ellipse enclosing the points, AR, has been extracted by means of the in-vivo image acquisition have been manually post-processed and realigned by means of ImageJ and Photoshop software.All the polarization-dependent analysis based on the phasor approach, the θ and γ plots, maps and histograms have been obtained by means of a custom designed C++ based software. The program Origin  has been employed for the polarization-dependent SHG curves fitting. All the acquired images have been visualized and linearly contrast-adjusted using ImageJ . Images affected by xyz drift or to other movements during All mice inoculated with the murine tumor cell line B16, housed under specific pathogen‐free conditions, were female on a C57bl/6 background for at least 12 generations and were used at 7–9 weeks of age. All mice, inoculated with the tumor cell line CT26, were female on a BALB/c background for at least 12 generations and were used at 7–12 weeks of age. The mice were kept in a pathogen‐free conventional animal house facility. The animal house is run by professional employees fully equipped with state‐of‐the‐art instrumentation in order to maintain the standard of animal welfare at the maximum levels. All mice were housed in individual, ventilated cages with 12 h light/dark cycles with food and water ad libitum. Experiments were performed using protocols approved by the Institutional Animal Care and Use Committee of the University of Milano‐Bicocca and by the Italian Ministry of Health or were performed under Boston Children’s Hospital IACUC approved protocols.Mouse-tail tendons have been harvested from sacrificed C57bl/6 background mice at 8 weeks of age. The tendons have been cut into strips 3–4 cm in length and placed in a Phosphate Buffered Saline (PBS) solution at pH = 7.4 before visualization under the two-photon microscope. Then, the samples have been mounted in a chamber made of two cover-slips sealed with silicone and filled with PBS.2 1 mM/CaCl2 5 mM, at a concentration of 5 mg/ml. Mouse-tail tendons have been incubated in the collagenase solution for 1 h at 37 °C; for SHG visualization the samples have been placed in a chamber filled with PBS solution. SHG polarization-dependent images have been acquired at the surface of the mouse-tail tendons in order to eliminate or reduce effects due to refractive-index-induced spherical aberrations.For collagenase-digestion, highly purified collagenase has been employed (Collagenase VIII Sigma-Aldrich C2139) in the buffer Hepes-NaOH 10 mM pH 7.4/NaCl 150 mM/KCl 5 mM/MgCl2) related to 4 tendons of different mice have been analyzed.10 FOVs , 2 mM l‐glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin. Cells at the confluence of 70% were collected for the mice injection.CT26 cell line , a BALB/c mouse colon carcinoma, was obtained from Mario Colombo .in vivo and ex-vivo B16 tumor analysis, C57bl/6 mice were inoculated in the deep derma in the left flank with the tumorigenic dose of B16 tumor cells (2 × 106) at Day 0. For the in vivo experiment, at day 7 mice were anesthetized I.P. with a Ketamine/xilazine cocktail (0.1 mL/20 g mouse), the derma was cut off and the tumor exposed and analyzed with two-photon microscopy. For the ex-vivo experiments at day 7 and day 12, mice were euthanized, the tumor collected and placed in PBS solution before polarized-SHG analysis. For the ex-vivo (biopsies) experiments, tumors have been placed in a chamber made of two cover-slips sealed with silicone and filled with PBS solution.For the 4) at Day 0. Explanted tumors at Day 7 were embedded in OCT freezing media (Biooptica). Sections (5 μm) were cut on a Cryostat, adhered to Superfrost Plus slide (Thermo Scientific), and then imaged under the two-photon excitation microscope.For the CT26 tumor analysis, BALB/c mice were inoculated in the deep derma in the left flank with the minimal tumorigenic dose of CT26 tumor cells  was used to evaluate the welfare of the mice. Generally, mice did not present signs of distress given the short period of time between tumor injection and killing.2) for each analyzed sample .Regarding experiments in histology fixed sections, during our analysis we acquired five field of views  for each analyzed tumors (n = 3) both at day 7 and at day 12 after tumor inoculation.Regarding biopsies we acquired 10 FOVs . Means between two groups were compared with two-tailed Student’s t-test. The value of P ≤ 0.05 was considered statistically significant. The degree of significance was assigned as follows: *for P ≤ 0.05, **for P ≤ 0.01, ***for P ≤ 0.001, and ****for P ≤ 0.0001.The data that support the findings of this study are available from the corresponding authors on reasonable request.http://fisica.mib.infn.it/media/homepages/biofisica/bicocca-hepth/index.html.The custom-written C++ software will be made available online at Supplementary Information


We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca2+]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca2+]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways.In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNF Mucociliary clearance (MCC) is a critical defense mechanism as it removes microbes and contaminants from the airway. The epithelium of the upper and lower airways system is composed of ciliated cells, whose ciliary beating removes the mucus layer that covered the epithelium. The MCC velocity is determined by ciliary beat frequency (CBF) and viscosity of the mucus layer . Bacteriα) is a proinflammatory cytokine produced primarily by cells of hematopoietic origin, including myeloid lineage such as monocytes and macrophages i through the activation of the transient receptor potential vanilloid 4 channel, which produces an increment of [Ca2+]i by the release of this ion from intracellular stores at lower viscous load  or the entry of calcium from extracellular space at high viscous load  i) were determined using a spectrofluorometric technique described previously i increment associated to the effect of TNFα on CBF response to increase viscous loading, cultures were previously incubated for 48 hours with TNFα (10 ng/mL). Then cultures were treated with either Thapsigargin 2.5 μM  for 30 min or Gadolinium 100 μM  for 5 min, previous to the change in viscosity of the medium. Thapsigargin was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 0.1%. At this concentration, DMSO had no effect on CBF.To determine the source i was measured in cultures exposed to 10% dextran and pretreated for 48 hours with TNFα. A slight increase in [Ca2+]i was observed after viscosity of the cultures was changed, which was significantly higher in cultures pretreated with TNFα  compared to cultures treated only with TNFα (17.03 ± 1.13%) or to controls (9.28 ± 3.4%), when the viscosity of the medium was increased (10% dextran) , when the viscosity of the medium was changed  was observed compared to control cultures (9.28 ± 3.4%). Similar decrease of CBF was observed in cultures pretreated with TNF Figures .α, a proinflammatory cytokine, produced a significant decrease in CBF response to changes in viscous loading (10% dextran), but has a positive effect, since we observed an increase in [Ca2+]i that may prevent the MCC collapse. Although, the changes in CBF observed after increasing the viscosity in the presence of TNFα represent a decay of up to 30% of the CBF with respect to basal CBF, it is well established that MCC velocity is directly dependent on CBF and that small changes in CBF have a substantial effects on MCC effectiveness i that it was significantly augmented after incubation with TNFα for 48 hours. In airway smooth muscle cells, it has been shown that TNFα increased [Ca2+]i by modifying mitochondrial calcium concentration i observed after TNFα incubation could protect cells from a worse cilia slowing and the recovery of CBF to basal ciliary activity, after viscous loading.In this study, cultures exposed to an increased viscosity (10% dextran) produced a slight increment in i, we monitored changes in CBF in cultures exposed to 10% of dextran and TNFα in the presence of an inhibitor of calcium uptake (Thapsigargin) or a calcium channel blocker (Gadolinium). Our results showed that Ca2+ from the cytosolic reservoir and the extracellular influx contribute to maintaining the augmented levels of [Ca2+]i, to prevent a further decrease of CBF induced by the additive effect of viscous loading and TNFα. The combined action of TNFα and 10% dextran on CBF on ciliated cells from adenoid tissue seem to simulate the effect of high range viscosity (20% dextran), where the autoregulatory mechanism depends mainly on extracellular calcium influx i i , 43, 44.α has a negative effect upon the response of human nasopharyngeal pediatric airway ciliated cells to viscous loading. However, TNFα incubation also has a beneficial effect, since it induces an increment in [Ca2+]i that contributes to maintaining the effectiveness of MCC in the upper respiratory tract. Further studies are required to advance our understanding of the mechanism of intracellular calcium homeostasis affected by TNFα, in order to prevent the collapse of MCC observed in chronic airway diseases.In conclusion, our findings suggest that, during acute inflammation, where mucus viscosity and secretion of cytokines in airway are increased, TNF


Results indicated that translocation kinetics were modulated by the state of oligomerization as expected for varying Stokes’ radii of the participating proteins. Following depolymerization of the microtubules and the actin filaments we found that Ca2+ induced membrane accumulation of PKCα was independent of the filamentous state of the cytoskeleton. Fusion of PKCα to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKCα-cytoskeleton interactions under resting conditions. Redistribution following spatially restricted photoconversion showed that the mobility of the fusion protein was independent of the state of the cytoskeleton. Our data demonstrated that in living cells neither actin filaments nor microtubules contribute to PKCα’s cytosolic mobility or Ca2+-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell’s cytoskeleton contributes to cPKC’s dynamics. This was addressed by analyzing the recruitment behavior of PKCα while manipulating the cytoskeleton. Photolytic Ca In general, after maturation and priming PKCα localizes in the cytosol with the pseudosubstrate domain occupying its kinase cavity thus silencing its kinase activity4. When intracellular Ca2+ increases, two Ca2+ ions bind to the C2 domain of PKCα molecule. This dramatically changes the affinity of the C2 domain to the inner leaflet of the plasma membrane from a repelling state in rest to an attraction state, resulting in the translocation of PKCα protein from the cytosol to the plasma membrane7. Upon C1 domain mediated binding to diacylglycerol (DAG) the configuration of PKCα changes substantially, leading to an extraction of the pseudosubstrate domain from its kinase catalytic core, relief from inhibition, and initiation of the kinase activity of PKCα9.As a member of the conventional Protein kinase C (cPKC) subfamily PKCα serves as a critical intracellular signal translator, transferring Ca2+-C2 domain binding and protein-membrane association are very transient in vitro11. In line with that, we have reported a very fast association and dissociation process of PKCα to/from the plasma membrane by UV-flash photolysis of caged-Ca2+ and a caged-Ca2+ buffer, respectively7. This indicates that Ca2+ unbinding and cPKC membrane dissociation are very fast2. We and others have shown previously that PKCα translocation from the cytosol to the plasma membrane readily follows intracellular Ca2+ oscillations14. This raises the question how the PKCα proteins translocate from the cytosol to the plasma membrane during such short time periods. Michael Schaefer and colleagues revealed the existence of a transient subplasmalemmal depletion zone of PKCα during its Ca2+-induced plasma membrane accumulation. They interpreted this finding in favor of a diffusion-limited distribution process instead of active transport15. Primarily only in the proximity of the membrane the rapid PKCα association with the inner leaflet of the plasma membrane via the Ca2+ bridge may result in a directional movement that initially depletes the subplasmalemmal cytosol, generating a gradient from the subplasmalemmal space towards the perinuclear cytosol. Later, diffusion equilibrates this gradient, which results in directed cPKC translocation from the cytosol to the plasma membrane. Such a notion, however, still mainly relies on computational simulations and is lacking direct experimental evidence.This highlights the central role of translocation to the plasma membrane for PKCα activation. It has been reported that Ca17. In multiple types of mammalian cells it has been reported that cytoskeletal components are either associated to PKCα19 or might be involved in PKCα activation20. Moreover, active PKCα might also modulate cytoskeleton structure23. These reports suggest a rather intimate relationship between an intact cytoskeleton and PKCα.In addition, there is a substantial body of evidence that discusses the direct involvement of the cytoskeleton in this dynamic translocation process. Apart from scaffolding tasks cytoskeletal filaments such as actin filaments and microtubules are engaged in a variety of intracellular transport processes, signal transduction, and cell movements2+ mediated translocation in the absence and presence of intact actin filaments and microtubules.Here we employed high speed life cell confocal microscopy to investigate the process of PKCα redistribution under resting conditions and during Ca2+ dependent translocation from the cytosol to the plasma membrane2. To characterize this translocation process kinetically we loaded PKCα-eGFP expressing HEK293 cells with a caged Ca2+ compound (NP-EGTA) and rapidly photo-released Ca2+ by application of a short high-energy UV-flash. While this maneuver resulted in a quasi-instantaneous increase of the cytosolic Ca2+ concentration , dimers (e.g. Katushka) and tetramers (e.g. DsRed2)26. Such PKCα fusion proteins were expected to reveal substantially altered translocation behavior, independent of whether active transport processes do contribute to the translocation process. HEK293 cells expressing either of the PKCα fusion proteins were loaded with NP-EGTA and protein translocation was characterized following a UV-flash induced Ca2+ increase by fitting the plasma membrane fluorescence upstroke to an mono-exponential equation. Typical results for eYFP, Katushka and DsRed2, are displayed in Fig. 2+ dependent translocation of PKCα was sensitive and also suggested diffusion to be a major contributor to this redistribution process.To verify and investigate the sensitivity of our approach in characterizing the Ca2+ photolysis assay together with manipulations of the cytoskeleton. Directional, active transport along the actin cytoskeleton critically depends on the availability of polarized filaments (F-actin) that can be visualized by expressing the actin binding protein Lifeact-GFP27 , nocodazole or Cyto-D and subjected them to the UV photolysis of Ca29. In order to verify whether and to what degree a functional PDZ ligand motif might actually unveil PKCα-cytoskeleton interactions we used a N-terminally fused florescent protein of PKCα, eYFP-PKCα. Interestingly, we observed that the time constant of membrane aggregation of eYFP-PKCα following the UV photolysis of Ca2+ was slightly prolonged when compared to that of PKCα-eYFP  with an intact PDZ-ligand motif for possible interactions with the cytoskeleton, similar to the approach introduced in Fig. 2+ triggered PKCα translocation were not altered in the absence of functional cytoskeleton filaments, thus strongly suggesting that active transport along the cytoskeleton is not contributing to the fast, directional Ca2+ triggered translocation of PKCα to the plasma membrane.These data indicated that the kinetics of Ca2+ conditions. In order to address this, we made use of the photo-convertible fluorescent protein Dendra230. In its resting state Dendra2 shows green fluorescence that can be switched to red upon photoconversion with blue light. We employed a focused UV laser (spot size around 3 µm in diameter) to cause photoconversion of the PKCα-Dendra2 fusion protein in a spatially restricted manner and followed the diffusion of the photoconverted protein in the presence and absence of a functional cytoskeleton. PKCα-Dendra2 expressed readily in HEK293 cells  is the photolysis assay sensitive enough to detect different diffusion rates at all and (ii) is the photolysis assay reliable enough to yield quantitative information about the translocation process. Interestingly, we observed a substantially slower translocation of the PKCα-tetramer when compared to the PKCα-monomer  fusion proteins as globular proteins, we would rather refer to them as “two balls on a stick” and thus oligomerization of the FP part will result in rather unpredictable topologies of the resulting protein complex. Moreover, these data not only further reassured a majorly diffusion-driven mechanism as the principal mechanism for PKCα translocation, but also strongly indicated that there is no additional carrier molecule involved in this translocation process.Thus, for a given concentration gradient, the speed of protein flux is mostly determined by the hydrodynamic radius of the protein, also called Stokes’ radius, which correlates with the molecular mass of soluble globular proteinsmer Fig. , which i31. Using pharmacological approaches, we successfully depolymerized the actin filaments and the microtubules, and analyzed possible effects such interventions might have for the relative speed of PKCα translocation. Because there are no significant differences of the photolytic release of Ca2+ (data not shown) and no significant viscosity changes of the cells following Cyto-D or Nocodazole treatment35, the results from the experiments clearly indicated that neither actin filaments nor microtubules were directly responsible or involved in Ca2+-dependent plasma membrane translocation of PKCα. C-terminal fusion proteins do obscure possible PKCα-protein interactions via PKCα’s PDZ-ligand motif and we thus also confirmed that N-terminally fused fluorescent proteins depicted the same properties. Their translocation behaviors were also independent of functional actin filaments or microtubules. Interestingly, the N-terminally fused PKCα variant displayed a significantly slowed down translocation speed. This behavior could either be attributed to a slightly increased hydrodynamic radius of the protein or to a slightly hindered and therefore slower plasma membrane interaction. One should keep in mind that the N-terminus of PKCα contains the C-domains responsible for Ca2+- and lipid-dependent membrane interaction.Beside the hydrodynamic radius, the absolute temperature and the viscosity of the medium are also important determinants in simple diffusion. In living cells, at least during the course of an experiment, the temperature can be seen as constant and is therefore not relevant. The viscosity of the medium, in this case the intracellular cytosol, is one important variable19, or in cardiomyocytes20. Moreover, activated PKCα has the capability to regulate the structure of the cytoskeleton23. Spatially restricted illumination of photoconvertible fluorescent proteins in living cells provides an ideal approach to study the diffusion process of PKCα in the cytosol and investigate such a dynamic process under the resting condition31. As discussed above, the Stokes’ radius of proteins is the key factor determining the diffusion coefficient in living cells. Because photoconversion only changes the fluorescent protein’s inner core in the vicinity of the chromophore, the typical barrel structure will not be altered significantly and thus the Stokes’ radius will remain constant. Considering the value of the Stokes’ radius to be 45.2 Å for YFP fused PKCα and 22.7 Å for free YFP15, similar Stokes radii are expected for PKCα-Dendra2 and free Dendra2. The differences in our measured decay time constants for those proteins , plasmids were transfected into HEK293 cells according to the vendor’s recommendations. Cells were investigated 48 hours after transfection.HEK293 cells were cultured and transfected as previously described2, 1.8 mM CaCl2, 10 mM glucose, 10 mM HEPES adjusted to pH 7.35 with NaOH. All compounds used were of research grade. Cytochalasin D and Nocodazole were purchased from Sigma .All experiments were conducted at room temperature (20–22 °C) in Tyrode’s buffer comprising: 135 mM NaCl, 5.4 mM KCl, 2 mM MgCl38. The full-length of human PKCα N-terminally fused to eYFP was constructed into the plasmid pEYFP-C by PCR with following primers, 5′ GCCTCGAGTGGCTGACGTTTTCCCGGGC and 5′ GCAGATCT TTATACTGCACTCTGTAAGATGG. The human PKCα without the last three amino acids (PKCα PDZ)29 was N-terminally fused to eYFP (into the plasmid pEYFP-C) by PCR with following primers, 5′ GCCTCGAGTGGCTGACGTTTTCCCGGGC and 5′ GCAGATCTTTACTGTAAGATGGGGTGCAC.The wild-type human PKCα protein was fused with eGFP, eYFP or, DsRed2 at the C-terminus in the pCDNA3 plasmid as described previously26. The plasmid encoding the red fluorescent protein Katushka, pTurboFP635-N was a gift from Prof. Dmitriy M. Chudakov25. Human PKCα protein fused to Katushka or photoconvertible fluorescent protein Dendra2  was achieved at the C-terminus employing the following primers, 5′ ATCTCGAGGCCCTTGGGACCATGGCT and 5′ AGAATTCCTACTGCACTCTGTAAGATG.The plasmid encoding the monomeric variant of YFP, mYFP, was a gift from Prof. Alexandra Newton27. The plasmid encoding eGFP-α-tubulin was purchased from Clontech .The plasmid encoding Lifeact-GFP was a gift from Prof. Wedlich-SöldnerAll plasmids were confirmed by sequencing.When HEK293 cells on coverslips reached about 40% confluency, they were fixed in 4% paraformaldehyde (in PBS) for 10 min. After a wash step with PBS, the fixed cells were permeabilized with 0.1% Triton X-100 (in PBS) for 10 min. After a PBS wash, 5% BSA in PBS was used for blocking unspecific binding for 20 min at room temperature. For actin filament staining, 0.5 μM ATTO-647N phalloidin  was employed in 1% BSA of PBS at room temperature for 1 h. Anti-α-tubulin  was employed for microtubules’ staining in 1% BSA of PBS at room temperature for 2 h. The polyclonal secondary antibody ) was left on the cells for 1 h (at room temperature). After washing with PBS for 3 times, the cells were mounted in ProLong Gold Antifade reagent and visualized by confocal microscopy.All confocal images from living cells were based on an inverted microscope  using an oil immersion objective . The microscope was attached to a fast 2D-kilobeam array scanner  that simultaneously scans 2500 parallel laser beams across the specimen and projects the resulting fluorescence images on two spectrally separated EMCCD-cameras . For excitation of eGFP, eYFP, Katushka and DsRed2, we employed respective solid-state lasers; eGFP excitation with a 491 nm laser , eYFP excitation with a 514 nm laser  and collected fluorescence emission with 515 mm long-pass filter . Katushka and DsRed2 were excited with a 561 nm laser  and fluorescence emissions were collected with a 570–675 nm band-pass filter . The entire setup was integrated and controlled through VoxCellScan software .2+ were performed as described earlier7. Briefly, cells were loaded with 7.5 μM NP-EGTA-AM  for 30 min. Ca2+ was photo-released by a bright UV flash . Images were recorded at 10 fps with 256 × 256 pixels’ images or at 71 fps with 256 × 30 pixels’ images by Infinity-3 system  described above.Experiments using UV flash photolysis of caged CaSpatially restricted conversion of Dendra2 was achieved by a 379 nm UV laser  focused to a spot of approx. 3 µm in diameter. In order to avoid cross talk of the conversion and imaging, we electronically placed the UV excitation pulse between two successive images (illumination duration 300 μs). Unconverted Dendra2 was illuminated with a 491 nm laser  and detected through a 497–550 nm band-pass filter  while the photoconverted Dendra2 was detected by illumination with a 561 nm laser  and fluorescence detection through a 570–675 nm band-pass filter. Images (128 × 128 pixels) were recorded at 10 fps.After the experiments, the resulting image series were transferred into a large-scale image database system running OMERO 5.02  for long-term storage. Images were processed either in MatLab (see below) or in ImageJ. To obtain fluorescence over time plots, the fluorescence information from regions-of-interest were averaged, saved and imported into Igor software .0), for which the fluorescence at any given time point (F) was divided by the resting fluorescence (F0) to account for different dye loading and/or expression of the fluorescent proteins and their distribution in subcellular compartments  and Canvas Draw 3.0 .Statistical analysis of the data was performed in Prism 6 software . After testing for a normal distribution of the data points  the data were analyzed with an unpaired t-test. Bar graphs depict mean ± SEM unless stated otherwise. Statistical significance was defined as follows; (*)p < 0.05; (**)p < 0.01; (***)p < 0.001. N numbers give the number of cells analyzed, whereas the number of experiments depicts independent experiments (different passage or cover slip).Supplementary Information


This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α‐synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high‐throughput assay based on measuring the extent of aggregate‐induced Ca Methods to detect membrane disruption in bulk solution, based on measuring dye leakage, are valuable for high μm concentrations of toxic species,2+ ions. The entry of a small number of Ca2+ ions into such small volumes leads to a significant change in concentration, enabling high resolution measurements to be made. For example, if a single Ca2+ ion enters an individual vesicle with a diameter of 200 nm, the concentration of Ca2+ will change by approximately 400 nm, which can readily be detected using highly sensitive fluorescence microscopy techniques. We designed our assay to achieve an optimal combination of sensitivity and dynamic range by preparing vesicles of an appropriate diameter encapsulated with the optimal concentrations of the Ca2+ binding dye, Cal‐520 . We immobilized thousands of single vesicles with a diameter of 200 nm containing dye molecules on the glass coverslides and imaged the Ca2+ influx into hundreds of individual vesicles per field of view using total internal reflection fluorescence microscopy  and biotinylated POPC (at a ratio of 100:1) filled with the Ca2+ at a concentration of 1.26 mm and imaged them using TIRFM . Typically, we imaged 16 fields of view (on a 4×4 grid) for each coverslide using a computer‐controlled automatic microscope stage, allowing the measurement of identical areas of the coverslide . We then added a solution containing the species of interest (e.g. protein aggregates) and re‐imaged the identical fields of view. If Ca2+ entered any given vesicle, an increase in the localized fluorescence intensity could be detected . The fluorescence signals, before and after the addition of aliquots of the solution, were stable with time  and hence the changes in the localized fluorescence can be attributed to species that disrupt the vesicles. To quantify the Ca2+ influx into individual vesicles, we added the cation transporting ionophore, ionomycin , which allows Ca2+ to enter the vesicles to saturation. The maximum fluorescence intensity of each vesicle was then measured  and the percentage of Ca2+ influx was calculated. This approach corrected for the fact that the number of Cal‐520 dye molecules in a vesicle is not constant, owing to variations in the efficiency of dye encapsulation2+ influx for individual vesicles and then averaged over all the vesicles imaged , and its addition to the vesicles resulted in no detectable increase in fluorescence, indicating that the monomeric protein does not induce Ca2+ influx . We detected an increase in the localized fluorescence due to the influx of Ca2+ resulting from the interaction of protein aggregates with the lipid bilayer. The aliquots taken from the aggregation reaction mixture had to be diluted 200 times to avoid saturation of individual vesicles with Ca2+, highlighting the sensitivity of our method. Note that oligomers of the Aβ peptide have been reported to be stable upon dilution2+ influx into individual vesicles to remain constant for up to 2 h after dilution . We also tested aliquots corresponding to time points of an aggregation reaction at the plateau phase (t3), by which time most of the monomeric protein had been converted into fibrils.2, although greater than that of aliquots at t1.We then incubated 2 μ2+ influx we employed size exclusion chromatography to separate species of different size2). We then probed the fluorescence changes arising from Ca2+ influx caused by the different fractions .To characterize the dynamic range and sensitivity of this assay we measured the dependence of the Ca2+ ions. This approach has potential value for screening inhibitors in the search for effective therapeutic agents. We here studied the ability of the extracellular chaperone clusterin,2+ influx using aliquots taken from the Aβ42 aggregation reaction at time t2. We then diluted this solution containing pre‐formed oligomers to a concentration of 10 nm Aβ42  and added 200 nm clusterin or 200 nm Nb3, respectively, both of which have been reported to bind to the Aβ peptide.2+ influx while incubation with an anti‐GFP antibody, as a control that does not bind to the Aβ peptide, had no detectable effect. These results are consistent with previous reports that clusterin2+ influx into neuronal cells.2+ influx by half; we obtained values of 0.1±0.02 nm and 18±3.4 nm for clusterin and Nb3, respectively  of individual vesicles similar to that described above for Aβ42.2+ influx induced by recombinant Aβ42 aggregates in the presence of a complex biological environment, such as CSF, which contains a multitude of different proteins and other macromolecules.2 and added them at varying concentrations to the human CSF of one individual . We observed an increase in the measured Ca2+ influx with Aβ42 concentrations of 100 pm and above. These results demonstrate that our method is sufficiently sensitive to measure the Ca2+ influx caused by protein aggregates at low concentrations in the presence of a complex human biofluid. Only small volumes of CSF were used in each experiment, 15 μL, allowing multiple measurements on a typical sample of CSF with a volume of 1 mL.We also examined the degree of Ca2+ influx. It also allows the extent of Ca2+ influx caused by aggregates in a sample to be quantified, which serves as a measure of the ability of aggregates to disrupt membrane integrity. The method also enables the effectiveness of reagents to reduce aggregate‐induced Ca2+ influx to be measured and can be applied directly to other proteins, such as α‐synuclein, and also to complex biofluids. The misfolding and aggregation of proteins has been associated with approximately fifty misfolding diseasesIn conclusion, we have demonstrated that our single‐vesicle assay identifies oligomers as the form of protein aggregates in the complex aggregation mixtures of Aβ42 that cause CaThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3β) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis . In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (The online version of this article (10.1007/s12015-017-9762-0) contains supplementary material, which is available to authorized users. Perturbations in the bone marrow microenvironment in acute myeloid leukemia (AML) favor the selective progression of leukemia over normal hematopoiesis . AlthougMesenchymal stromal cells (MSCs) are central to the integrity of the bone marrow microenvironment as they give rise to all cell types of the marrow stroma, including adipocytes, osteocytes and fibroblasts . Recent Exosomes are microvesicles formed from intracellular endosomes in MSCs and other cell types. Microvesicles encapsulate signaling molecules such as receptors, chemokines, mRNA and microRNA (miR) . Bioacti7 cells per T-75 flask. The emergence of plastic-adherent MSCs was followed for 3–6 weeks in a humidified tissue culture incubator  with fresh medium changes performed every 7–10 days. MSCs were characterized by flow cytometry, in accordance with criteria established by the International Society for Cellular Therapy (ISCT), [Cells were obtained from filters following normal bone marrow harvests and bone marrow aspirates from newly diagnosed AML patients, in accordance with the Ottawa Health Sciences Network Research Ethics Board (see Table  (ISCT),  using th (ISCT), .Passage 4 MSCs were expanded to 90% confluency (as described above) in complete growth medium containing exosome-free FBS . Exosome-free medium was used to ensure that no contaminating exosomes were present from bovine serum. Conditioned medium  was obtained after 48 h from confluent cultures and kept frozen at − 80 °C. Exosome extraction of CM was performed by SBI Biosciences using ExoQuick-TC reagent and miRNA extraction using SeraMir kit . Quality and quantity of obtained RNA was verified using Agilent Bioanalyzer Small RNA Kit prior to generation of libraries using NEBNext Multiplex Small RNA Kit . Next generation sequencing was performed using Illumina HiSeq 2500 platform with 100 bp paired end runs . Sequencing data for all samples was normalized to picograms of miR.p < 0.05) or 2) had a relative level of more than 1.5-fold difference between AML- and control-derived MSC exosomes (p < 0.05). Interaction networks were built using Ingenuity Pathway Analysis (IPA) (version 24390178) which queries the Ingenuity Pathway Knowledge Base for interactions between each candidate miR and all molecules stored in the Knowledge Base. Only findings related to the human species were considered. We further inquired about the effect of the candidate miRs on the direction of change in expression of the connected genes within the network using the Molecule Activity Predictor (MAP) tool. This in silico approach enabled us to visualise the overall effect of the candidate miRs on their interactome.Putative candidate miRs were selected as either those that: 1) were significantly more or less abundant  of ± 1.5 in gene expression with adjusted p-value of < 0.05).We next enquired whether the prediction models derived from the in silico analyses performed using IPA could be validated with the Gene Expression Omnibus (GEO) database repository at the National Center for Biotechnology Information (NCBI). Using the GEO2R interactive web tool (NCBI) that allows the identification of genes that are differentially expressed from two groups of samples, we analyzed GEO datasets of AML samples and CD34-selected healthy controls , derived from 3 GEO datasets  were then determined from AML samples and CD34-selected healthy controls. These data sets were identified through a search of GEO datasets containing AML samples with concomitant healthy controls using systematic gene expression analysis.−) BM cells were obtained using the EasySep™ Human Progenitor Cell Enrichment Kit with Platelet Depletion to enrich for stem and progenitor cells (HSPCs). Lin− cells were directly stained for CD34+ (Clone 4H11) cell surface marker and sorted for CD34+ cells using the Beckman Coulter MoFlo sorter. RNA was isolated  from Lin−CD34+ cells and rDNase treated (Qiagen). RNA was converted to cDNA using SuperScript II (Invitrogen), according to manufacturer’s instructions. cDNA was used for qPCR using primers listed in Supplemental Table Mononuclear cells were isolated from bone marrow aspirates of patients with AML and healthy controls as described above. Lineage-negative (Lin Clone 4H1 cell suMSCs used in these experiments to derive exosomes were previously characterized and reported . The ideKnown targets of the five candidate miRs were identified as described in the “n = 69) and healthy donor CD34-selected cells . The characteristics of the three GEO datasets are summarized in Table Validation of the predicted change in expression of the target genes was investigated by interrogating gene expression datasets of marrow-derived CD34-selected cells from patients with AML and controls identified through a systematic search of the GEO repository. Three datasets were identified and gene expression data were analyzed for each of the known targets in AML-derived samples . Increased miR-26a-5p was predicted to inhibit 4 molecules indirectly. Only reduced expression of GSK3β, however, was validated in GEO-GSE17054 with a 0.33-fold change in relative expression . Reduced levels of miR-339-3p in AML-derived MSC exosomes was predicted to increase the expression of 14 potential targets through a loss of direct inhibition and 3 molecules were validated in GEO-GSE30029. Expression of KRBA2 increased 5.0-fold , RRBP1 increased 3.9-fold , and HIST2H2BE increased 5.1-fold  in AML samples compared to controls. miR-425-5p was predicted to interact with 23 different targets as a direct inhibitor. While levels of gene expression were predicted to increase in leukemia cells for all identified potential targets of reduced levels of miR-425-5p, only expression of APOBEC3A was significantly different and was decreased (opposite direction than predicted) with a 0.2-fold change in expression . The molecules with significant differential gene expression in AML CD34-selected cells compared to controls that were predicted by the miR profiling are summarized in Table Increased miR-101-3p was predicted to inhibit the expression of 8 potential targets (1 directly and 7 indirectly). Only reduced expression of EZH2 was validated in GEO-GSE30029 with a 0.33-fold change in expression relative to controls  and GSK3  were reduced in all 5 AML-derived samples compared with controls and the mean expression of KRBA2 , RRBP1 , HIST2H2BE  were all increased  and exaThe release of factors by MSCs, including the release of microvesicles, may allow MSCs to alter the biology of malignant cells occupying niches within the marrow microenvironment. For instance, exosomes from MSCs in patients with multiple myeloma contributed to tumor growth in mice injected with cells from myeloma cell lines whereas mice treated with exosomes from normal MSCs demonstrated inhibition of tumor growth . The impWhile the expression of microRNAs was not different in the parent MSCs in our study, differential packaging of microRNAs into exosomes has been reported previously , 20 and Future studies that enrol greater numbers of patients will be needed to gain insight regarding any potential therapeutic targets arising from our work and also to understand if differentially packaged microRNAs in MSC-derived exosomes can provide a potential biomarker panel to screen MSCs in patients treated for AML to assess whether the microenvironment has normalized. We hypothesize that normalization of the microenvironment is required for sustained remission of AML. Persistence of MSC-derived signalling abnormalities may perpetuate inhibition of normal hematopoiesis and the continued competitive advantage of leukemia cells for stem cell niches in the marrow.Limitations of our study are worth mentioning. Preparing MSC-derived exosomes and RNA sequencing is laborious and a larger sample size is needed to corroborate our findings in separate populations, including various subtypes of AML with different molecular and cytogenetic abnormalities, in older patients with AML, patients with secondary AML, and patients with refractory AML. Moreover, the effect of treatment and transplantation on MSC-derived exosomes remains to be studied. There are variations in technical approaches to the isolation of exosomes although the use of the approach applied in our study has been associated with high purity compared to other methods in a recent study . While pIn summary, MSC-derived exosomal microRNA represents a potential mechanism for influencing gene regulatory networks in AML. Candidate miRs were identified with corroborating changes in gene expression of known targets in AML samples. Our work provides justification for pursuing additional studies to understand better the microenvironment-induced changes in AML.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 24 KB)Supplementary material 2 (DOCX 104 KB)Supplementary material 3 (DOCX 27 KB)Supplementary material 4 (DOCX 94 KB)


It integrates information about external Ca However, the CaR is also a factor in other more common pathologies that include chronic kidney disease6, cancer7, cardiovascular pathologies11, and Alzheimer’s disease12. For a complete survey of CaR’s function in molecular physiology and pathology, readers are referred to some of the many recent reviews on the topic13.Alteration in the activity or function of the extracellular Ca2+ext, the primary ligand for the CaR, changes in tissue spaces. Ca2+ is, however, just one of the many activators of this fascinating receptor; the CaR is “built” to interact with a dizzying array of other orthosteric agonists and also allosteric modulators that influence the receptor’s response to calcium ions (We will first address when and how Caum ions . These eum ions . However2+ levels (~1.1–1.3 mM) are under stringent homeostatic control exerted by organs such as the parathyroid glands, bone, renal system, and intestine14. Nonetheless, local fluctuations in Ca2+ext levels have been identified and characterized in the restricted volume of interstitial fluids bathing cells of many tissues15. The amplitude and shape of these Ca2+ext fluctuations is thought to represent an autocrine/paracrine form of cell-to-cell communication. Pharmacological agents directed at the CaR therefore work upon a complex backdrop of changing external [Ca2+]. This has the potential to markedly affect the way in which a drug  acts on the receptor in any given moment. Knowledge about these local fluctuations in calcium remains, arguably, among the most significant barriers to fully understanding CaR pharmacologyin vivo.Systemic Ca2+ext changes, e.g. a) intracellular Ca2+ signaling events, b) Ca2+ extrusion via discharge of calcium-enriched granules, and c) synchronous opening of voltage-operated Ca2+ channels. Below, we describe briefly how these extracellular microdomains can be measured and give examples of how they are generated.Many factors are believed to participate in the generation of physiologically relevant Ca2+ext changes in close proximity to the plasma membrane have been hampered by the lack of proper experimental tools to physically access these restricted compartments in intact tissues and by difficulties in measuring [Ca2+] fluctuations against the background of mM Ca2+ concentrations normally present outside the cell. Although many different experimental approaches have been proposed to quantify Ca2+ext fluctuations in a number of diverse tissue models, each of them presents limitations with regard to either sensitivity or spatial resolution. For example, Ca2+-sensitive small molecule fluorescent indicators have proven useful to visualize the temporal/spatial dynamics of Ca2+ext changes, since they provide sensitivity, time resolution, and access to limited spaces24. However, these methods require experiments to be performed in non-physiological conditions such as low or nominally free Ca2+ext because of the relatively high Ca2+ affinity of the available fluorophores. For example, Tepikin and Petersen introduced the droplet technique27 to reliably quantify Ca2+ext changes induced by active Ca2+ extrusion through the plasma membrane Ca2+-ATPase (PMCA) of acinar cells. Fluo-3 was used to characterize changes in Ca2+ext in small clusters of exocrine gland cells maintained in a tiny droplet of solution covered with oil to prevent evaporation, but this method could only be used under Ca2+-free media conditions on account of the high affinity of the Ca2+ indicator.Historically, real-time measurements of Ca2+-selective microelectrodes extensively to directly record the profile of changes in Ca2+ext in the restricted domains of different experimental tissue models following Ca2+-mobilizing stimulation38. As described further below, we also used Ca2+-sensitive microelectrodes to measure real-time Ca2+ext changes induced by the glucose-dependent discharge of Ca2+-rich insulin granules39. Ion-sensitive microelectrodes present certain advantages. First, measurements of Ca2+ex changes under physiological conditions are allowed owing to the availability of Ca2+-sensitive resins with affinities in the μM and mM range. In addition, it is possible to record Ca2+ext changes for hours without technical drawbacks such as the bleaching of fluorescent indicators. However, this approach requires a high level of patience and expertise and samples only one small region of the tissue, so it is not amenable to high-throughput measurements. Moreover, it is difficult to execute in many tissue types. This is an arena in which further developments would be welcome.We, as well as others, have used Ca2+ signaling events.Intracellular Ca Cells facing restricted diffusion spaces can experience Ca2+ext fluctuations during intracellular Ca2+ signaling events as a result of activation of Ca2+ efflux (e.g. by PMCA and/or Na+/Ca2+ exchanger) and influx (e.g. by store-operated channels [SOCs]) across the plasma membrane. The genesis of significant Ca2+ext microdomains requires either differential dynamics or polarized asymmetry of Ca2+ influx/efflux mechanisms43. For example, we found that stimulation with Ca2+-mobilizing agonists resulted in substantial local increase in Ca2+ext at the luminal face and a comparable depletion at the serosal aspect of gastric acid-secreting cells38. An increase in [Ca2+] in the gastric gland lumen is due to activation of Ca2+-ATPase, which is highly expressed at the apical membrane of these cells, where it co-localizes with CaR38.2+ extrusion via discharge of calcium-enriched granules.Ca Very high Ca2+ concentrations have been measured within secretory granules46. For example, insulin granules from rat insulinoma have a granular concentration of Ca2+ between 60 and 120 mM47. Therefore, one can assume that exocytotic events may generate consistent increases in Ca2+ext. Recently, we showed that the stimulation of insulin secretion by high glucose and other secretagogues resulted in late elevation of Ca2+ext within rat insulinoma (INS-1E) β-cell pseudoislets, as measured with Ca2+ microelectrodes39. Ca2+ extrusion via Ca2+-enriched granules has also been proposed for a number of different cell types that undergo exocytosis such as salivary gland cells22, bovine adrenal medullary cells48, neurohypophyseal nerve endings49, and sea urchin eggs50.2+ channels.Synchronous opening of voltage-operated Ca Excitable cells have, in addition to the above-mentioned mechanisms, a variety of voltage-dependent Ca2+ entry pathways that might impact Ca2+ext during their physiological activity53. In the central nervous system, synchronous opening of voltage-gated Ca2+ channels (VGCCs) can stimulate significant reductions in Ca2+ext55. Pumain and Heinemann recorded Ca2+ext reductions from a basal level of 1.25 mM to as low as 0.08 mM in rat neocortex following the application of excitatory amino acids54. In cardiac muscle, transient depletions in Ca2+ext by about 200 μM were measured during a single heartbeat16. In mouse islets of Langerhans and INS-1E pseudoislets, glucose stimulation induced a reversible and significant depletion in Ca2+ext by about 500 μM as a consequence of VGCC-mediated Ca2+ influx across the plasma membrane39.58. When GPCRs are coupled to multiple G-proteins in the same cell type, as is the CaR, the old dogma hypothesized that they activate each of the downstream signals equally, without preference for any one pathway61.In the classical (yet oversimplified) view, upon interaction with a G-protein-coupled receptor (GPCR), ligands stabilize preferred conformational state(s) that in turn activate distinct subsets of G-protein-mediated downstream signaling pathways62. This will depend not only on the signaling toolkit of the cell in which they are expressed but also on numerous other factors63, such as the localization of the GPCRs, the duration of stimulus for GPCRs working in non-equilibrium conditions, the downstream signaling protein level (i.e. involvement of different effectors able to shape diverse Ca2+ and cAMP microdomains and kinetics), and the specific agonist/modulator activating the receptor64. Also, it has been shown that GPCRs traffic through subcellular compartments such as the nucleus65, mitochondria66, and endosomes68, where they are capable of initiating specific signaling pathways.In the past few years, several studies have painted a more complex scenario, in which receptors, existing in multiple active states, can specifically trigger selected pathways at the exclusion of others69. The development of new technologies, such as microscopy techniques and probes to follow receptor trafficking70 and to assess in real time subcellular signaling dynamics72 as well as biased signaling73, has been essential for such advances and will certainly continue to promote novel and exciting discoveries in this field.In this context, pharmacological studies have shown that ligands are able to bias the signaling of their GPCRs towards specific intracellular responses and/or are capable of crossing cell membranes, thus activating or rescuing intracellular GPCRs (by acting as molecular chaperones)74. The internalized receptor can be degraded by the lysosome or recycled to the cell membrane. Importantly, both β-arrestin and internalized receptors can initiate signaling.In the classical life cycle of GPCRs, the newly synthesized receptor is inserted into the endoplasmic reticulum and, after folding, is transported through the cis-Golgi/Golgi/trans-Golgi, where it goes through further post-translational changes. Then the mature protein, packaged in small vesicles, undergoes insertion into the cell membrane. If misfolded, the protein is degraded by the proteasome. Upon binding, ligands stabilize preferred conformational state(s) of the receptor that initiate intracellular signaling. The process is terminated via receptor internalization mediated by GPCR kinase (GRK) phosphorylation and β-arrestin(s) recruitment75. The intensity of signaling is thus related to the quantity of GPCRs expressed on the cell surface and accessible for ligand stimulation. This is also true for the CaR, as recently demonstrated by Brennan and colleagues76.It is well established that the fine balance among maturation, internalization, recycling, and degradation can influence the net amount of cell surface receptor level and thus represents a mechanism for the cell to regulate receptor sensitization and modulate the strength of signal transduction81. Both early and recent studies have highlighted that two hallmarks of the CaR are the negligible functional desensitization and the existence of a significant amount of CaR in intracellular membranes. Early studies indicated, both by western blotting or immunohistochemistry83, that CaR immunoreactivity reflected a predominantly intracellular, core-glycosylated form. It is now becoming clear that such an observation is not a mere artifact but is strictly related, and even of functional importance, to the complex and mutual interaction between CaR trafficking and signaling. In fact, both minimal desensitization and high levels of intracellular CaR can be explained by the model of agonist-driven insertional signaling (ADIS)84.Relevant advancements in the knowledge of the key players involved in CaR biosynthesis and trafficking have been achieved in the last ten years85.The process of ADIS depends upon the regulated release of mature CaR proteins from a large intracellular pool located in the endoplasmic reticulum and Golgi/post-Golgi vesicles. The rate of CaR plasma membrane insertion increases as a function of the concentration of CaR agonists and/or allosteric modulators, while the receptor already at the plasma membrane undergoes constitutive endocytosis without substantial recycling. Importantly, and predictably, in this model, CaR signaling can be dynamically regulated by the trafficking of intracellular CaR to the plasma membrane through an agonist-dependent modulation of the net amount of CaR at the plasma membrane. This has implications in both health and disease2+ (or any other ligand), the other class of CaR agonists is represented by allosteric modulators, which after binding to different sites alter the receptor conformation and, as a consequence, affect receptor responses to orthosteric ligands. This action can be exerted in a positive  or a negative  directi69.Interestingly, a number of recent reports have shown that allosteric modulators can act as pharmacoperones. Pharmacoperones  are membrane-permeant ligands  that reach the misfolded protein at the site of its biosynthesis and trafficking (most frequently the endoplasmic reticulum) and, by stabilizing the receptor structure, rescue the protein to the cell surface88. While an early study reported the synergistic effect of acute treatment with L-phenylalanine and NPS R-467 on CaRs with inactivating mutations89, Breitwieser and colleagues first showed that overnight treatment of HEK293 cells expressing loss-of-function mutant CaRs with the calcimimetic NPS R-568 rescued plasma membrane expression and signaling in 50% of the mutations examined87. Similar results were obtained by other groups, although the authors did not investigate the cell surface expression of CaR after NPS R-568-mediated signal rescue91. Interestingly, the capability of the calcimimetic NPS R-568 to rescue CaR activation without altering the cell surface expression of the mutant proteins was shown in a recent study92, suggesting a mutant-specific effect of this drug as a pharmacoperone.Breitwieser's group has published a number of interesting papers highlighting the capability of CaR allosteric modulators to function as pharmacoperones94. They showed that calcimimetics, including the only calcimimetic approved in the clinic , effectively rescue trafficking and signaling of CaR mutants exhibiting a loss of cell surface expression. They also found that the calcilytic NPS 2143 effectively promotes trafficking of CaR mutants to the cell membrane while negatively modulating CaR signaling95. This is in contrast to other studies with NPS 2143 showing a reduced86 or unchanged92 effect on the expression of diverse CaR gain-of-function mutants, suggesting that a mutant-specific pharmacoperone effect also exists for NPS 2143.Relevant findings in this area have also been provided by Leach and colleaguesin vitro96. More recently, the new quinazolinone-derived calcilytics were shown to be effective in attenuating enhanced calcium signaling in mutations causing BS and ADH97. NPS 2143 was also found to correct signaling defects in HEK293 cells transfected with Gα11-mutated proteins causing ADH2 and uveal melanoma98. Very interestingly, the effectiveness of both old (i.e. NPS 2143)99 and new (i.e. JTT-305/MK-5442)100 calcilytics was recently assessedin vivo in mouse models harboring ADH gain-of-function CaR mutations.The potential of calcilytics for patients with activating CaR mutations has been further examined11. Biased signaling 102 represents a general, albeit only recently appreciated, signaling characteristic of GPCRs58. It refers to the ability of different ligands to stabilize distinct receptor conformations and preferentially direct GPCR signaling towards a specific set of pathways while excluding/reducing others.Recent reports suggest that the therapeutic potential of new classes of CaR modulators, as well as the pathophysiological role of endogenous agonists, could be further improved by exploiting the phenomenon of biased signaling103. The existence of ligand- and tissue-specific effects in the signaling pathways activated by the CaR, although not precisely quantified, is traceable in a vast number of papers published throughout the years. In fact, in many cases, biased signaling at the CaR might have been underestimated owing to the use of single assays for the evaluation of CaR signaling outputs  or the low number of CaR agonists and modulators tested.This concept, while greatly complicating the scenario of GPCR signaling, opens up new perspectives in the design of smart and tissue-specific drugs2+ext via Gq signaling while suppressing Gi-mediated signaling104. In other examples, Bruce and colleagues reported differential effects of CaR agonists on Ca2+ dynamics in isolated acini and interlobular ducts of rat pancreas105. Ziegelstein showed that in human aortic endothelial cells only spermine was able to induce intracellular Ca2+ release and nitric oxide production, whereas Ca2+ext, Gd3+, and neomycin were ineffective106. Furthermore, Smajilovicet al. demonstrated a concentration-dependent vasodilatation in rat aorta with the addition of cinacalcet, whereas the agonists neomycin and Gd3+ were ineffective107.A peculiar case of biased signaling at the CaR was observed in response to an allosteric autoantibody isolated from a patient with acquired hypocalciuric hypocalcemia. The antibody potentiated the effects of Ca110. By exploring the effect of 12 orthosteric CaR agonists on inositol -trisphosphate (IP3) accumulation, cAMP inhibition, and ERK1/2 phosphorylation in HEK293 cells stably transfected with rat CaR, Thomsen and colleagues110 revealed that Ca2+ext is biased towards cAMP inhibition and IP3 accumulation, while spermine shows a strong bias towards ERK1/2 phosphorylation. Also, this study demonstrated for the first time that ERK1/2 is partially activated through the recruitment of β-arrestin by the CaR. The same group also obtained interesting results concerning strontium ranelate, currently used in the clinic for the treatment of osteoporosis109. As previously suggested by Chattopadhyay and colleagues111, and contrary to the results obtained by Coulombe112, Sr2+ was shown to bias CaR signaling towards ERK1/2 in rat medullary thyroid carcinoma 6–23 cells. Also, in rabbit osteoclasts, while both Sr2+ and Ca2+ produced stimulation of PLC and translocation of NF-kB, in contrast to Ca2+ext, Sr2+ signaling was independent of the IP3 pathway and induced apoptosis via PKC activation113.On these bases, in the last three years, an increasing number of reports have focused on the physiological and pathological role of biased signaling exerted on the CaR by its physiological agonists and pharmacological modulators as well as on mutation-dependent alterations in such bias. A key contribution to this field comes from the group of Bräuner-Osborne116. These authors analyzed the effect of calcimimetics and calcilytics on a number of CaR mutations115 , R,R-calcimimetic B and AC-265347 biased CaR signaling towards pERK1/2 and IP1 accumulation. This finding may explain the preference of R,R-calcimimetic B and AC-265347 for the suppression of PTH release versus the stimulation of calcitonin secretionin vivo.Potential clues towards the search for a calcimimetic with low/no effect on calcitonin was hinted at in a very recent paper116. Further, Jenny Yang’s lab has published several papers about the potential Ca2+ binding sites and their relevance for related diseases124. Recently, these authors solved the first high-resolution crystal structure of the ECD of human CaR bound with Mg2+117. Of note, a high-affinity tryptophan derivative was found in the crystal structure of the CaR that seems to play a role in potentiating the function of the receptor117. These studies represent important progress in the field, since they provide new insights into the structural basis of human diseases arising from CaR mutations. Ultimately, the subtle differences in modulator binding sites revealed by structural studies may be exploited to design drugs able to elicit distinct signaling outcomes and thus be effective on specific mutations (patient-specific drugs) and/or on tissue-specific signaling pathways (tissue-specific drugs).Recent work explored the structural requirements for bias and allostery mediated by old and new classes of positive and negative allosteric modulators of the CaR2+] in extracellular microdomainsin vivo.In this scenario, a fundamental challenge for future research will be to set up methodological tools to validate these latest pharmacological advances in more physiologically relevant models, such as primary cells or animal models. It also remains to be seen how the functional effects of these drugs are altered in the complex landscape of changing [Ca