The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues. Like in two-photon excited fluorescence (2P), this type of laser scanning microscopy is characterized by an intrinsic 3D sub-micron resolution that is robust upon light scattering and which allows for higher image depth when compared to confocal microscopy5. SHG is a nonlinear optical process in which highly polarizable and non-centrosymmetric structures emit photons at exactly half the excitation wavelength6. Since SHG does not involve population transfer, it significantly reduces phototoxicity and is photobleaching-free7. The emitted light results from the coherent sum of the electromagnetic field generated by every single SHG emitter and thus scales quadratically with the number of aligned molecules sharing the same polarity8. Indeed, adjacent molecules of the same polarity will emit strong SHG signals due to constructive interference while the SHG signal will almost vanish in the case of adjacent molecules of opposite polarity9.Over the years, Second Harmonic Generation (SHG) microscopy has emerged as an effective tool in biology3, only few biological molecules produce detectable SHG signals12. SHG microscopy has been used to image collagen-rich tissues such as tendon13, cornea14, skin15, fascia16 and cartilage17. Furthermore, SHG signals have been obtained from the myosin band in skeletal muscles12. Lastly, SHG signal have been reported in microtubules (MTs)11.Over the years, many groups have demonstrated that exploiting the intrinsic nonlinear optical properties of the sample is a valuable, although challenging approach. In the case of SHG, since the signal is highly specific for dense non-centrosymmetric media19 and plays a role in the organization and function of various cell type22. The polarity of MTs has long been thought to be involved in the assembly of mitotic spindles23, responsible for the segregation of chromosomes during cell division. Indeed, near the centrosomes, the MTs exhibits the same polarity while in the center MTs are antiparallel25. However, the relationship between MTs polarity and spindle dynamics during mitosis remains poorly understood, mainly because of the huge challenge to measure MTs polarity in spindles.MTs are a key component of the cell cytoskeleton, involved in structural support and intracellular transport. Formed as a lattice of tubulin heterodimers, having two different ends, MTs exhibit an intrinsic polarity. This polarity has important biological consequences since it determines the dynamics of polymerization and the directionality of molecular motor movement26, based on electron microscopy to directly visualize individual MTs. However, this technique requires thin and fixed sections and has been so far limited to study few tissue preparations28. Fluorescently labelled plus-end tracking proteins29 and laser ablation30 provide information about MT polarity, but the quantification remains indirect and these techniques are invasive. More recently, SHG microscopy has been proposed as an alternative method to probe, non-invasively, the polarity of MTs in thick living tissues. Individually, MTs are very weak SHG emitters. However, the constructive interference in MTs arrays that have the same polarity results in detectable signals that have been used to visualize MTs in neurons32, cilia12, astroglial filaments33 and mitotic spindles36. Kwan et al. demonstrated that the polarity of MTs can be extracted from the ratio of forward and backward SHG signals32 with a limitation in disordered MTs arrays, such as the mitotic spindle. Lastly, Yu et al. established a very elegant method to measure MT polarity in spindles, based on a combination of SHG and 2 P images of fluorescently labelled MTs36.Several methods have been used to record the polarity of MTs. Currently, the gold standard remains the hook method38, I-SHG is based on the measurement of the phase of the SHG signal. In the past years, its potential for tissue imaging has been demonstrated with different proteins, such as myosin from skeletal muscle39 and collagen from tendon and cartilage41. Having recently solved one of the main drawbacks of I-SHG42, namely the long imaging time, we demonstrate here the possibility to use I-SHG to record the dynamical evolution of MT polarity in mitotic spindles from live zebrafish embryos.Here, we use interferometric SHG (I-SHG) to measure the polarity of MTs in mitotic spindles without requiring any exogenous labelling. Originally developed to characterize non-centrosymmetric crystals2 images were recorded in the forward direction, in about 2 s, using 30 µs pixel dwell-time and typically 200 nm pixel size. The average power after the objective was typically 100–150 mW. Raw data visualization was performed with ImageJ  and image processing was performed with MATLAB (The MathWorks) and Origin 10 (OriginLab) as described below.I-SHG imaging was performed using a custom-built laser-scanning microscope (for a complete description of the setup see ref. ref and Iref (respectively φexp and Iexp) stand for the phase and the intensity of the reference (respectively sample) SHG beams. Finally, the phase image was reconstructed by determining, in every pixel, the reference phase that corresponds to the maximum amplitude. Note that the rotation of the glass plate, used to scan the reference phase, imposed a dead-time between images and that the manual adjustment of the incident polarization and the depth of focus between phase-stack recording, to correct for the slight movement of the spindles result in about 45 s of temporal resolution.I-SHG is based on the interference of the SHG with the reference beam to extract the phase of the signal generated in the sample  of the field of view could be effectively used to perform I-SHG with laser scanning acquisition. While an important limitation to image biological tissues, this was sufficient to measure the phase of SHG signal from mitotic spindles.The use of two galvanometric mirrors to scan the incident angle of the excitation beam on the back pupil of the objective led to a change in the optical path, which is virtually equivalent to rotating the glass plate. Therefore, when scanning the laser to acquire images, the phase difference between the reference and the sample SHG is gradually shifted as we progress further from the center of the interferometric pattern. However, since this phase shift depends only on the excitation geometry, it is possible to calibrate this effectDanio rerio) of the TL line were bred and maintained according to standard procedures43. All experiments were performed in compliance with the guidelines of the Canadian Council for Animal Care and conducted at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM). All protocols were approved by the animal care committee of CR-CHUM (agreement number: N15018PMDz). Embryos were mounted in a 3 cm Petri dish with a 1 cm hole at the bottom sealed with a 170 µm-thick coverslip. Another coverslip was set on top to minimize sample motion during imaging. Temperature in the room was about 20 °C, which slows down the development compared to the standard developmental table44. Embryos were imaged within 4 h after fertilization.Wild-type zebrafish .Using time-lapse SHG microscopy, we imaged the mitotic divisions of a zebrafish embryo within 4 h after fertilization to record the dynamical behavior of mitotic spindles Fig. . The SHGWe then used I-SHG to visualize the change in spindle polarity throughout the course of mitosis. To that end, we acquired phase-stacks at two different times in metaphase and in anaphase Fig. . A mediaTo better visualize these variations of intensity, Fig. 0) the red and green pixels started to segregate, while the phase histogram showed two broad peaks, revealing the opposite polarities in the two poles. Then, at the end of metaphase (t0 + 1 min) and the beginning of the anaphase (t0 + 2 min), the spindles clearly had opposite polarities in the two poles, and the histograms show two narrower peaks. Lastly, at the end of the anaphase (t0 + 3 min), when the spindle uncondensed and the MTs disperse, the polarity was almost random around the two poles and the phase histogram is almost flat.From the reference phase stack, I-SHG allowed us to reconstruct the image of the phase in the sample and to extract the phase histogram Fig. . The gre8 makes it very sensitive of slight change in polarity. Yet, since SHG signals from MTs are rather weak and depend on a wide range of experimental parameters, quantification of structural changes based only on the signal intensity would be subjected to very large uncertainties. Figure 0 + 1 min and t0 + 2 min, the phase distribution appears narrower, revealing a higher degree of polarity alignment of the MTs. In parallel, Fig. The SHG intensity is a good qualitative indicator of structural organization, since its quadratic dependency with oriented molecules0 and t0 + 3 min) correspond to low SHG signal (around 200 s and 380 s), while peaked distributions (t0 + 1 min and t0 + 2 min) are associated with higher intensities (around 260 s and 320 s). As previously shown41, one can quantify this effect, since the width of the phase peaks is related to the ratio of MTs with opposite polarities:β(2)) and N(−β(2)) are respectively the number of MTs with a positive and negative nonlinear susceptibility, or equivalently with one polarity or the other. Therefore, f = 0 or 1 when all the MTs are pointing in the same direction, while f = 0.5 when the MTs are randomly polarized in the focal volume. Note that there is an ambiguity in the definition of the f ratio, which reflects the ambiguity in the sign of the measured polarity. By convention, we attributed the f ratio below 0.5 to the negative phase peak (centered at −π/2) and the f ratio above 0.5 to the positive phase peak (centered at π/2). Determining the absolute sign of the polarity would require measuring the phase of the SHG signal in a reference sample.Altogether, Fig. f ratios calculated from the phase distribution over different time points. As expected, f is closer to 0.5 at the beginning of the metaphase (t0) and the end of anaphase (t0 + 3 min), since the MTs are arranged in a more disorganized array. In turn, at the end of the metaphase (t0 + 1 min) and the beginning of the anaphase (t0 + 2 min), f departs from 0.5, revealing the same polarity of MTs within highly aligned mitotic spindles.Figure 6 and several studies have derived the theory of signal generation by a focused laser in biopolymers36. Assuming that all individual MTs generate equal signals, the SHG is directly proportional to the coherent sum of all the elementary responses. Thus, SHG intensity (ISHG) depends on intrinsic properties of the MTs as well as on their macromolecular arrangement (density and polarity):(2) is the hyperpolarizability and N the total number of MTs in the focal spot. This equation shows that the SHG signal scales quadratically with the excess number of MTs in one direction. Extracting the f ratio from the phase histogram (in Fig. 41.The physics of SHG is well known in Fig.  while siin situ, impeding a proper investigation of these effects. Note that this method is not limited to imaging mitotic spindles but should be directly transferable to offer new insights into MT polarity in other systems such as dendrites11 or neuronal processes28.Since SHG microscopy does not require external perturbation, such as fixation or exogenous labelling, it can be applied in native tissues. Therefore, I-SHG microscopy offers a direct and dynamical method to quantify the degree of polarity of MT arrays in cells. We report here its first use in live cell imaging and show that I-SHG allows measurement of the evolution of MT polarity in mitotic spindles during the first divisions of a zebrafish embryo. It has long been proposed that this polarity plays a role in many cell processes and behaviors such as force transmission, cell migration, protein localization and transport. However, up to now, it has remained highly challenging to image MTs polarity


Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo.Serotonin  is an important regulator of physiological and behavioral processes in both protostomes  and deuterostomes . In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In  The biogenic amine serotonin  is an ancient neuroactive substance and present throughout the animal kingdom. Serotonin plays a key role in regulating and modulating many physiological and behavioral processes in both protostomes and deuterostomes. In humans, malfunction of the serotonergic system has been associated with several impairments and diseases, such as schizophrenia, migraine, depression, suicidal behavior, infantile autism, eating disorders, and obsessive-compulsive disorder , certain behavioral effects have been ascribed to the serotonergic system. In D. melanogaster larvae, serotonin modulates the heart rate (To gain insight into serotonergic function(s), insects are highly attractive models. In comparison with vertebrates and especially mammals, they allow assessing the anatomical distribution, development, and neurophysiological properties of serotonergic neurons with unprecedented inter-individual reproducibility and precision. Applying this experimental strategy, the activity of serotonergic neurons has been related to physiological functions and changes in behavior  has been postulated in D. melanogaster . So far, four 5-HT receptor subtypes have been pharmacologically characterized in  CG1056; , and Dm5CG12073; . These Gnogaster . This obnogaster  and was nogaster . The recellifera  and the prolixus , knowled to 5-HT . Furthernfection . At the behavior .2B receptor. The cDNA encoding Dm5-HT2B was amplified on mRNA extracted from D. melanogaster heads. A cell line was established constitutively expressing Dm5-HT2B. Since 5-HT2B receptors are known to cause inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from intracellular stores, we examined Dm5-HT2B functionality by Ca2+ fluorimetry. The receptor’s pharmacological profile was established after applying concentration series of various agonists and antagonists. In addition to serotonin as the native ligand, 5-methoxytryptamine and 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were very potent agonists. Receptor activity was efficiently blocked by metoclopramide and mianserin. Thus, this study provides important new data regarding the pharmacological characteristics of the fifth 5-HT receptor of the fruit fly.The aim of the current study was to focus on the pharmacological properties of the Dm5-HT+ RNA was prepared from 180 heads of male flies  by using the Micro-Fast TrackTM 2.0 Kit . Synthesis of cDNA employed the AccuScriptTM High Fidelity First Strand cDNA Synthesis Kit . For amplification of the entire coding region of Dm5-ht2b, specific primers were designed based on available sequence information . PCR products were cloned into pGEM-T vector  and subsequently sequenced .Poly(A)ormation : sense pDm5-ht2b (Dm5-HT2B) as “bait.” Values for identity (ID) and similarity (S) were calculated by using the BLOSUM62 substitution matrix in BioEdit 7.1.9. Phylogenetic analysis was conducted as described by D. melanogaster FMRFamide receptor (DmFMRFaR) sequences were used to root the phylogenetic tree.Amino-acid sequences used for phylogenetic analysis were identified by protein-protein Basic Local Alignment Search Tool (BLAST) searches of the National Center for Biotechnology Information (NCBI) database with the deduced amino acid sequence of Dm5-ht2b was generated in pcDNA3.1 vector . PCR was performed with specific primers . PCR products were digested with HindIII and XhoI, and subcloned into pcDNA3.1(+) vector (Invitrogen). Thus, the resulting construct contained the Kozak consensus motif  by a modified calcium phosphate method  to activate Gq proteins was assessed by monitoring changes in [Ca2+]i with the Ca2+-sensitive fluorescent dye Fluo-4 (Invitrogen). Non-transfected HEK 293 cells and cells expressing Dm5-HT2B were grown in minimal essential medium  containing 2% (w/v) UtroserTM G , 1 × non-essential amino acids and 1 × antibiotics/antimycotics) in 96-well plates to a density of ∼3 × 104 cells per well. In this format, each vertical row (=8 wells) of the 96-well plate is incubated with the same ligand concentration. Cells were loaded at room temperature with Fluo-4 as described earlier ]. Plates were transferred into a fluorescence reader  to monitor Fluo-4 fluorescence. The excitation wavelength was 485 nm, and fluorescence emission was detected at 520 nm. Various concentrations of biogenic amines and synthetic receptor ligands were added, once Fluo-4 fluorescence had reached a stable value in each well. The changes in Fluo-4 fluorescence were recorded automatically. Concentration-response curves for putative agonists/antagonists were established in at least two independent experiments with octuplicate determinations (s.a.) per data point. Data were analyzed and displayed by using PRISM 5.0.4 software .The ability of the Dm5-HT earlier  in extra2 receptor from D. melanogaster had been annotated in previous studies (Dm5-ht2b (CG42796) was experimentally proven to encode a functional 5-HT receptor  of 2,715 bp and encodes a protein of 904 amino-acid residues (Dm5-HT2B) with a calculated molecular mass of 99.5 kDa. The hydrophobicity profile according to Figures 1A,B), which is a characteristic feature of GPCRs. The TM segments are flanked by an extracellular N-terminus of 74 residues and an intracellular C-terminus of 26 residues. The Dm5-HT2B receptor contains an extremely long third cytoplasmic loop (CPL3) of 563 residues. We submitted the Dm5-HT2B sequence to Phyre2 .The sequence of a second potential 5-HT studies . Later, receptor . Here, wo Phyre2  and obta2B receptors  and are also present in Dm5-HT2B. Three consensus motifs for potential N-glycosylation (N-X-S/T) are located in the extracellular N-terminus of Dm5-HT2B . A cysteine residue in the C-terminus (Cys892) is a possible site for post-translational palmitoylation. Twenty phosphorylation sites for protein kinase A (PKA), 38 phosphorylation sites for protein kinase C (PKC) and nine phosphorylation sites for protein kinase G (PKG) are present within intracellular domains of Dm5-HT2B . N-glycosylation sites were predicted by NetNGlyc 1.0 Server1 and putative palmitoylation sites were predicted using GPS-Lipid2. Putative phosphorylation sites were predicted by NetPhos 3.1 Server3  and similarity (S) was found with the 5-HT2B receptor of A. mellifera , Dm5-HT2B forms a highly supported cluster with other protostomian 5-HT2B receptors. This protostomian 5-HT2B cluster represents the sister group to deuterostomian 5-HT2 receptors within a monophyletic 5-HT2 receptor group. However, the basal branching of 5-HT-receptor subgroups is not stable and thus has to be subject of future studies.A comparison of the Dm5-HT2B-expressing cells and non-transfected HEK 293 cells were incubated with the biogenic amines dopamine, histamine, octopamine, serotonin, and tyramine . The application of serotonin led to an increase in the fluorescence signal in Dm5-HT2B-expressing but not in non-transfected cells. Neither dopamine, octopamine nor tyramine evoked responses in transfected or non-transfected cells. Histamine, however, caused a rise in Ca2+-dependent Fluo-4 fluorescence in both, Dm5-HT2B-expressing and non-transfected HEK 293 cells. This effect is due to endogenously expressed histamine (H1) receptors in the HEK 293 cell line used in this study . Half-maximal activation of Dm5-HT2B (EC50) was at 2.11 × 10-8 M. In non-transfected HEK 293 cells, a slight increase in the fluorescence signal was observed at the highest ligand concentration applied (10-4 M).To further investigate the pharmacological properties of Dm5-HT2B-expressing cells. For 5-methoxytryptamine and 8-OH-DPAT, concentration series ranging from 10-9 M to 10-4 M were applied and Ca2+-dependent Fluo-4 fluorescence was monitored . Both ligands caused specific responses. The EC50 for 5-methoxytryptamine was 1.05 × 10-6 M. In contrast to serotonin and 5-methoxytryptamine, the concentration-response curve for 8-OH-DPAT did not saturate and, therefore, the deduced EC50 of ≅6.5 × 10-4 M might be taken with some caution.Two potential agonists were tested for their activity on Dm5-HT2B activity. Measurements were performed with increasing concentrations of the antagonists clozapine, cyproheptadine, ketanserin, metitepine , methysergide, metoclopramide, mianserin, prazosin, SB 242084, and spiperone on a background of 10-7 M serotonin.Next, we examined the ability of potential receptor antagonists for impairing Dm5-HT2B-expressing cells, many of the antagonists caused a decrease of the serotonin-induced Ca2+-dependent fluorescence signals. Representative data are shown in Figure 4C. Ligand concentrations that led to half-maximal inhibition of Dm5-HT2B-induced responses (IC50) were determined from the concentration-response curves and are summarized in Table 1. The most effective antagonist on serotonin-stimulated Dm5-HT2B was metoclopramide with an IC50 of 1.78 × 10-8 M. The order of antagonist efficiency (IC50) on the Dm5-HT2B receptor was: metoclopramide > clozapine > cyproheptadine > mianserin > metitepine (Table 1). Two ligands, prazosin and spiperone, also caused a reduction of the cellular response. However, the signals did not reach saturation and, due to solubility problems higher concentrations could not be tested  are present in the third cytoplasmic loop. Interestingly, the Dm5-HT2A receptor is of similar size and contains 869 amino acid residues . A large number of phosphorylation sites to common protein kinases are spread throughout the intracellular loops . Which of these sites participate in receptor desensitization and/or internalization . After heterologous expression of these receptor variants, their signaling properties can be examined and quantified by Ca2+ fluorimetry. Finally, residues in the binding site for serotonin that is formed by the transmembrane segments of Dm5-HT2B are well conserved. Notably, the aspartic acid residue  in TM5 could bind to the 5-hydroxy group of serotonin’s phenyl moiety. Phenylalanine and/or tryptophan residues in TM6 and TM7  might contribute to π-π interaction with delocalized electrons in serotonin and stabilize the receptor ligand interaction.Although Dm5-HTlization  upon serDm5-ht2b gene in this study, compelling evidence is available from previous studies supporting the general finding that 5-HT receptors are widely expressed in the CNS throughout development of D. melanogaster  or 8-OH-DPAT (EC50 ≅6.5 × 10-4 M). Since the concentration-response curve with 8-OH-DPAT did not saturate, this latter value should be taken with caution. More recently, two 5-HT2 receptors from the honeybee, Am5-HT2A and Am5-HT2B, have been molecularly and pharmacologically characterized using the same heterologous expression system  and that ligand affinity may be influenced by the expression system used. In contrast to Dm5-HT2B, where half-maximal stimulation with 5-methoxytryptamine was at ≅1 × 10-6 M, both honeybee 5-HT2 receptors and the C. vicina receptor displayed EC50 values in the nanomolar range [Am5-HT2A, 7 × 10-8 M; Am5-HT2B, 6.04 × 10-8 M  was less efficacious than that of Am5-HT2A (EC50 = 5.59 × 10-5 M) and Am5-HT2B receptors , we observed three distinct types of inhibition profiles on Dm5-HT2B. Two antagonists caused saturating responses and reduced serotonin-evoked Ca2+-dependent fluorescence to values ≤ 40% of control measurements. With an IC50 of 1.59 × 10-8 M, metoclopramide was more potent than mianserin (IC50 = 1.64 × 10-6 M). Serotonin-evoked cellular Ca2+ responses were reduced to 40 and 25% of control measurements without antagonists by metoclopramide and mianserin, respectively. Responses to clozapine (IC50 = 4.45 × 10-7 M), cyproheptadine (IC50 = 1.58 × 10-6 M), and metitepine (IC50 = 3.56 × 10-6 M) also saturated but all three substances were much less potent inhibitors at the receptor than metoclopramide or mianserin . A maximal reduction to 60% of the serotonin-evoked signal was achieved with clozapine (Table 1). Finally, prazosin and spiperone also reduced serotonin-induced Ca2+-dependent fluorescence in the cell line but the responses did not saturate. From the whole series of antagonists used in the current study, only metitepine has been tested in an earlier study by 2B in HEK 293T cells to investigate the pharmacology of this drug on D. melanogaster 5-HT receptors. Using Ca2+ fluorimetry on individual cells expressing Dm5-HT2B, the authors reported an IC50 of 2 × 10-6 M which is very similar to the value determined in the current study. Interestingly, metitepine has been uncovered as a potent anorectic drug in D. melanogaster larvae  and activates 5-HT4 receptors  and to block the β-adrenergic-like octopamine receptor Octβ3R , however, the concentration required for in vivo experimentation to specifically target this receptor subtype might be kept rather low. In summary, our data may facilitate future behavioral pharmacological studies on the role of Dm5-HT2B in the fruit fly. Such studies would be desirable, since current knowledge on the role of this 5-HT receptor subtype solely depend on the investigation of flies that have been genetically manipulated.In the current study, we identified metoclopramide as the most potent antagonist at the Dm5-HTeceptors . The latCG42244;  in D. meWB designed and evaluated experiments, wrote the paper; DS conducted experiments and evaluated data; SB conducted experiments and evaluated data; MT conducted experiments; AB designed experiments and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptococcus pneumoniae endophthalmitis. Rabbits were treated with alloxan to destroy pancreatic islet cells, or mock-treated with vehicle, and maintained for 10 days before intravitreal infection with S. pneumoniae E353. Clinical scoring of the eyes was performed 24 and 48 hours after infection, followed by euthanasia and vitreous harvest to quantitate bacterial loads. There were no significant differences in clinical scores (P ≥ 0.440) or bacterial loads (P = 0.736), however, 4/12 (33%) of the diabetic rabbits became bacteremic. This finding not only indicates a breakdown in the blood-ocular barrier, but also prompts further investigation into the exploitation of the diabetic eye by the streptococci.Diabetics are at increased risk for eye infections including bacterial endophthalmitis. It is unclear whether the severity of endophthalmitis is greater in these patients due to confounding factors such as pre-existing ocular diseases in some but not others. Therefore, we tested the hypothesis that disease severity and/or bacterial loads would be significantly higher in a Type I diabetic rabbit model of  Pseudomonas, and streptococcal species67Staphylococcus aureus in addition to coagulase-negative staphylococci and viridans group streptococciIndividuals with systemic conditions such as diabetes are more susceptible to infections than healthy individuals. One such example is bacterial endophthalmitis, an inflammatory infection of the interior of the eye. The two major interior cavities of the eye are the aqueous humor and the vitreous humor. Diabetics are susceptible to both the endogenous (metastatic) and exogenous forms of this disease1234134Streptococcal species of the oral cavity are three times more likely to be isolated in post-injection endophthalmitis, where bacteria are introduced into the eye from intraocular injections of therapeutic drugs for other ocular diseases612Streptococcus species as the isolated agent causing endophthalmitisThe Centers for Disease Control and Prevention estimated that 9.3% of Americans have diabetes as of 2014S. pneumoniae endophthalmitis. We hypothesized that clinical scores and/or bacterial loads would be significantly higher for the eyes of diabetic rabbits compared to those of non-diabetic rabbits.Large epidemiological studies of bacterial endophthalmitis have reported general trends toward worse visual outcomes for diabetics versus non-diabetics4Two independent experiments were performed with a starting number of 12 rabbits in each experiment . One of the diabetic rabbits in the first experiment lost more than 20% body weight and was euthanized as a humane endpoint. Two additional diabetic rabbits in the first experiment, and one in the second experiment, became moribund shortly before the 48-hour clinical examination and were euthanized; blood and vitreous were collected from these rabbits to quantify bacterial loads, but 48-hour clinical examinations were not possible. One of the diabetic rabbits in the second experiment had fluctuating blood glucose measurements throughout the study and had a measurement of 231 mg/dL (less than 300) by the time of the infection, and was therefore excluded from the study. Both experiments yielded statistically similar results, and the data from the 2 experiments were combined for analysis .All rabbits had similar blood glucose concentrations prior to the start of the study  with no other apparent colony types. Two of these 3 rabbits were the moribund ones that were euthanized without a final clinical examination. The bacterial loads for the contralateral eyes of these 3 rabbits were 8.167 × 103, 4.333 × 104, and 3.833 × 103 CFU/mL. All of the contralateral eyes in the control group of rabbits were culture-negative. There was no significant difference between diabetic and non-diabetic rabbits in vitreous bacterial loads of the infected eyes  had culture-positive blood resembling S. pneumoniae on blood agar with no other apparent colony types. Two of the blood samples produced colonies that were too numerous to count at the highest dilution plated, translating to greater than 108 CFU/mL in the blood. The other 2 blood samples had 1.267 × 105 and 1.50 × 102 CFU/mL. These culture-positive rabbits included the 3 moribund rabbits. All of the control rabbits had culture-negative blood.The diabetic rabbits appeared in good health (with the exception of the one that lost more than 20% of its starting weight) until after the vitreous infection with The major findings of this study were that clinical endophthalmitis scores and vitreous bacterial loads were not significantly different between diabetic and non-diabetic rabbits. However, one-third of the diabetic animals became bacteremic. This latter result is likely due to a weakened blood-ocular barrier which is common for diabetes2223S. pneumoniae utilizes glucose as a primary carbon source for growth26S. pneumoniae is also able to utilize mannose, galactose, and N-acetylglucosamine. Each of these sugars can be processed to yield substrates that are necessary for the glycolytic pathway25The study described herein used an acute diabetes animal model in which the rabbits were infected a relatively short time after inducing Type I diabetes. Our choice of this acute model was based partly on the knowledge that We verified that the diabetic rabbits had elevated glucose concentrations in the vitreous (≥600 mg/dL compared to approximately 185 mg/dL in control rabbits) at the time of infection, and reasoned that the bacteria would be more metabolically fit in that high-glucose environment. Our results, however, suggest that increased glucose in the vitreous does not have a significant effect on disease severity or bacterial growth. Overall, the vitreous environment regardless of glucose concentration appeared to provide an optimal medium for bacterial fitness, however, the specific components of the vitreous enabling bacterial growth are unknown.To examine whether chronic diabetes results in more severe endophthalmitis, long-term maintenance of diabetic animals would be warranted. Likewise, examination of endophthalmitis in Type 2 diabetes with or without metabolic disease would require a separate model system than that described here. Age, sex, and animal strain should also be considered to investigate correlations between diabetes and development of bacteremic infections from exogenous endophthalmitis.Streptococcus pneumoniae E353, a capsule type 6 A human endophthalmitis strain, was kindly provided by Regis P. Kowalski . Frozen stocks of E353 were routinely cultured for isolation on sheep’s blood agar  for 18–24 hours at 37 °C and 5% CO2. Isolated colonies were inoculated into Todd Hewitt (BactoTM) broth containing 0.5% yeast extract (THY) and then incubated for approximately 18 hours at 37 °C and 5% CO2. These 18-hour cultures were then diluted 100-fold in fresh THY and incubated until the optical density at 600 nm reached 0.23, which corresponded to approximately 1 × 107 colony-forming units per mL (CFU/mL) as determined by previous growth curve analysis. Serial 10-fold dilutions of this logarithmic-phase subculture were prepared in sterile PBS  and the third dilution (corresponding to a target of 102 CFU per 10 μL) was used for inoculation. Serial dilutions were also plated on blood agar to verify purity and quantity.ad libitum. Enrichment included plastic chew toys, jingle bells, plastic balls, daily petting, and hay as a treat. Animal procedures were reviewed and approved by the University of Mississippi Medical Center’s Institutional Animal Care and Use Committee. All animal experiments were performed in accordance with the tenets of the Association for Research in Vision and Ophthalmology.Twenty-four 2 kg specific-pathogen-free female New Zealand white rabbits (strain code 571) were purchased from Charles River . Rabbits were socially housed and given standard chow and drinking water Two independent experiments with 12 rabbits per experiment were done. For each experiment, 5 rabbits were assigned to the control group and 7 rabbits were assigned to the diabetic group. More rabbits were chosen for the diabetic group to account for possible attrition in this group.et al.Induction of Type I diabetes was done similarly to the method of Wang Alloxan-treated rabbits have been reported to develop severe hypoglycemia within the first few days after injectionet al.Blood glucose measurements were taken twice daily for the following 3 days, and then once daily for another 7 days. During this time, diabetes was managed by administration of subcutaneous pharmaceutical-grade insulin  diluted in sterile filtered PBS once daily in the following quantities: 1 U/kg for blood glucose of 350–400 mg/dL, 2 U/kg for 400–500 mg/dL, or 3 U/kg for 500–600 mg/dL. Body weights were also recorded every other day during this time. Based on the study of Wang S. pneumoniae E353 was injected into the vitreous humor of the anesthetized eye of each rabbit at a target concentration of 102 CFU in a volume of 10 μL. Clinical examination was done 24 and 48 hours after infection by an observer who was masked to the grouping of each rabbit. Clinical severity scores were assigned according to previously published methods30et al.Each rabbit was anesthetized with a subcutaneous injection of 35–50 mg/kg ketamine and 2–10 mg/kg xylazine, and topical 0.5% proparacaine was applied to one eye. 10 CFU/mL with standard deviations of 0.5, at an alpha level of 0.05 (5% probability of Type I error). All quantitative data are expressed as the mean ± standard deviation. Following a one-way analysis of variance to detect overall differences, pairwise comparisons were done using a two-tailed t-test. All comparisons were between control animals and diabetic animals at a given time point. P-values and effect sizes (Cohen’s d) were recorded.Sample sizes were determined a priori to provide a minimum of 90% power based on the pre-experimental assumptions that (1) mean clinical scores would differ by 8 with standard deviations of 5, and (2) mean bacterial loads would differ by 2 logHow to cite this article: Benton, A. H. et al. Exogenous Streptococcus pneumoniae Endophthalmitis in Diabetic Rabbits. Sci. Rep.7, 46196; doi: 10.1038/srep46196 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium  A transient rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is decoded by a complex network of downstream components and is involved in the response to both abiotic and biotic stresses12+]cyt is one of the first responses to microbial elicitors, forming a common part of the plant defense response342+]cyt have also been observed in response to wounding caused by chewing insects, such as lepidopterans62+ signals in response to live biotic threats that cause damage to only a few cells has not been explored. The green peach aphid Myzus persicae is a hemipteran insect that represents a significant threat to world agriculture82+ efflux from the extracellular space has been observed in leaves infested with M. persicae2+ signals while M. persicae feed from leaves using a fluorescent Ca2+ biosensor, with both aphids and GCaMP3 offering novel tools with which to dissect the role of Ca2+ during biotic interactions.Calcium Ca+ is one 2+-selective microelectrodes were formerly used to measure [Ca2+] in plants112+ and emit light, allowing for un-paralleled opportunities to study Ca2+ dynamics in both cells and whole tissues. Ca2+ biosensors can be injected as dyes or stably produced upon the introduction of the biosensor coding sequence into the genome of the organism via transformation . The latter offers the major advantages of being easily expressed in live tissue and capable of subcellular localizationAequorea victoria (jellyfish) was the first genetically encoded Ca2+ biosensor deployed in plants2+] fluxes in response to various stimuli, including temperature1718202+] fluxes in individual cells and from tissues with poor sensor expression difficultCa2+ biosensors that can fluoresce has complemented aequorin by allowing for detailed subcellular and tissue-level analysis of Ca2+ dynamics. One of the most common fluorescent biosensors are the fluorescence resonance energy transfer (FRET)-based Cameleons. FRET Cameleons are composed of two proteins, typically CFP and YFP, which are brought into close contact by the conformational change induced by the binding of Ca2+ to a calmodulin domain in the CFP-YFP linker region. This contact allows the transfer of energy from CFP to YFP, and the resulting change in the fluorescence of these fluorophores allows for the accurate quantification of [Ca2+] through the calculation of the ratio of the fluorescence signals from the two fluorophores2+ signals in plants and to resolve these to the cellular level2425The development of Ca2+ biosensors has been the development of highly sensitive single-fluorophore (single-FP) biosensors. Single-FP biosensors consist of a single circularly permutated GFP linked to a calmodulin and M13 peptide, with Ca2+ binding to calmodulin, resulting in a water-mediated reaction between calmodulin and GFP so as to protonate GFP and increase fluorescent yield27282+] as simply as FRET sensors, they are superior for the analysis of the temporal and spatial dynamics of Ca2+ signals52+ affinity, and signal-to-noise ratios313233Arabidopsis thaliana (henceforth referred to as Arabidopsis) to measure Ca2+ fluxes in response to ATP, chitin, and the bacterial elicitor flg22. This study also demonstrated that the R-GECO biosensor outperformed the FRET Cameleon YC3.6 in terms of maximal signal change and signal-to-noise ratioA recent breakthrough with fluorescent GFP-based CaCauliflower mosaic virus 35S promoter. The genetic tools available for Arabidopsis research allow for the detailed molecular analysis of the Ca2+ signals measured by GCaMP3. In addition, the GCaMP3 biosensor can be visualized under a fluorescence microscope rather than a costlier confocal system. This protocol allows for whole-tissue imaging, essential when conducting experiments with live biotic stresses. The experiment is designed such that detached leaves from 35S::GCaMP3 plants are floated in water, to prevent insect escape and to restrict feeding to a specific tissue. The method outlined in this paper therefore allows for the analysis of leaf Ca2+ dynamics during feeding by M. persicae, resulting in the characterization of a novel plant signaling response. This method can also be adapted to work with other biotic stresses, such as additional insect species and microbial pathogens, and with other plant tissues, such as roots.Because of the ease of use, high fluorescent yield, and high temporal resolution that can be achieved with GCaMP biosensors, GCaMP3 was genetically encoded in Arabidopsis under the 35S::GCaMP seeds using three 75% ethanol washes, 1 min per wash, and plate them on 100 mm2 square plastic plates with ¼-strength Murashige and Skoog (MS) medium On day 1, sterilize Stratify the seedlings in the dark for three days at 8 °C to obtain synchronous germination.On day 4 of the experiment, move the GCaMP seedlings to a controlled environment room (CER) at 23 °C, with a 16 h light and 8 h dark photoperiod.M. persicae to 5-week-old soil-grown Arabidopsis plants using a moist artist's paint brush (size 2 or 4) (grown in a CER at 22 °C with 10 h light and 14 h dark photoperiod).On day 11 of the experiment, add 15 adult Cage the aphids on the plant by placing clear plastic tubing (10 cm x 15 cm) around the plant and cap with a plastic lid.Leave the aphid-infested plants for 24 h in a CER at 22 °C with a 16-h light and 8-h dark photoperiod.M. persicae using a paintbrush. NOTE: Insects can be seen by eye on the plant. This gives a population of nymphs with a similar developmental age. Roughly 1 nymph per adult will be produced during this period.On day 12 of the experiment, remove all adult Leave the infested plants in a lower-temperature CER at 16 °C, with an 8 h light and 16 h dark photoperiod, to prevent the nymphs from increasing in size too rapidly.35S::GCaMP3 seedlings from the CER and detach the largest leaf from each plant using a pair of sharp scissors.On day 19 of the experiment, remove the Using a pair of tweezers, place the detached leaf into the well of a 96-well plate containing 300 µL of distilled water, abaxial surface facing up. Repeat for additional leaves .35S::GCaMP3 Leaf Assay. Figure 1: The largest leaf from each 16 day-old 35S::GCaMP3 Arabidopsis seedling is detached and floated on 300 µL of distilled water in a 96-well plate. Photo taken the day after detachment. Please click here to view a larger version of this figure.Cover the plate in clear plastic wrap and aluminum foil and leave at room temperature overnight to allow the stress of the detachment to subside.On day 20 of the experiment, remove the 96-well plate from the aluminum foil and transfer it to a fluorescence stereomicroscope. Configure the stereo fluorescence microscope to excite GFP with a 450-490 nm light and to capture the fluorescent emission between 500 and 550 nm.Collect the aged aphids from the colony established on day 11 by removing the plant from the soil and placing it in a transparent box with a lid. Keep the insects contained in the box while conducting the experiment.Place the 96-well plate under the microscope. Alter the light exposure until the GCaMP3 fluorescence can be clearly visualized in the veins of the detached leaves. Keep the exposure constant for all experiments; for the current protocol, a 1-s exposure was used with a gain of 3.5 .Adjust the magnification and zoom of the microscope such that 4 wells can be observed in one frame; for the current protocol, a 7.8X magnification and a focus of -127.833 mm was used.Transfer one aphid to a detached leaf under the microscope using a moist paint brush. Leave an adjacent leaf untreated as a control, but lightly touch it with the paintbrush to mimic the touching that occurs during aphid transfer. Remember to place the plastic wrap back on top of the 96-well plate during microscopy to prevent the insect from escaping.Begin the fluorescence recording of the leaves in pairs (1 aphid-treated and 1 untreated) by clicking "start experiment" in the built-in microscope software . Record e.g., tagged image file format, TIFF).After 50 min, stop recording by clicking on "stop experiment" and remove the aphid from the leaf. Save the fluorescence measurements as image files  and convert them to 32 bits by clicking on "Image" > "Type" > "32-bit;" this allows for the conversion of the images into heatmap videos (see step 7).Discard the samples in which the aphids do not settle in one location for more than 5 min by viewing the insect movement under the microscope. NOTE: This is often the majority of samples, and up to 100 treatments may be required to find 30 samples exhibiting successful settling.Set the measurement scale to pixels  and the time frame to the same time interval as used during the microscopy by clicking on "Image" > "Properties."Using the cursor, place a region of interest (ROI) around the area of tissue for GFP analysis. NOTE: In the current protocol, a circular ROI 50 pixels (0.65 mm) in diameter was used at 3 locations of interest: the aphid feeding site (Fs), a systemic region on the midrib of the leaf (Sm), and a systemic region adjacent to the midrib  , and edit the size using "Edit" > "Selection" > "Specify." See ue," Sl) . Create i.e., the same region of the leaf as where the aphid fed on the treated leaf)  .Figure 2: Analyzing GCaMP3 Fluorescence Under the Microscope. Left: untreated control leaf. Right: aphid-treated leaf. The aphid feeding site is indicated with an arrowhead. Scale bar = 1 mm. (A) Bright field image. Magnification: 7.8X, focus: -127.833 mm, exposure: 1 s. (B) GFP image showing the ROIs used for the quantitative analysis of fluorescence. Fs = feeding site, Sm = systemic midrib, Sl = systemic lateral tissue. Magnification: 7.8X, focus: -127.833 mm, exposure: 1 s. The GFP was excited using a 450 to 490 nm metal halide lamp, and fluorescent emission was captured between 500 and 550 nm. Please click here to view a larger version of this figure.Use the Time Series Analyzer plugin by clicking "Plugins" > "Time Series Analyzer" to analyze the raw fluorescence values (F) in the ROI over time. Add the ROI of interest ("Add [t]"). Making sure that the ROI is selected, select "get average;" this will display a table of F values for each frame in that ROI.Copy this data into a spreadsheet.Calculate the area of the feeding site signal by first selecting the region using the "freehand selection tool." Outline the maximal GFP signal and then calculate the area of this shape by clicking "Analyze" > "Measure."Calculate the speed of the feeding site signal using the MTrackJ plugin by clicking "Plugins" > "Tracking" > "MTrackJ."Click on the "Add" button and then click the cursor at the center of the signal when it is first visible. Click again on the edge of the signal at its point of furthest spread. Click "measure" to calculate the speed of the signal.Define the point of aphid settling by playing through the image frames from the microscope in Fiji. NOTE: The time of settling is the frame during which the aphid stays in a single location for 5 min or more. The duration of settling events can also be calculated from the images in Fiji.0)/F0, where F0 is the average baseline fluorescence calculated from the average of F over the first 60 frames of the recording before the aphid settled. Repeat for the untreated control.Normalize the F data (ΔF/F) according to the equation ΔF/F = .Plot the normalized GFP fluorescence (ΔF/F) over time for that ROI in the treated and untreated leaf. Discard samples (both leaves) if the untreated control shows large CaRepeat until at least 20 - 30 viable samples have been analyzed per genotype.Convert the 32-bit image files into heatmaps using the NucMed_Image LUTs plugin by clicking "Plugins" > "NucMed" > "Lookup tables." In the lookup tables menu, select "blue green red" to convert the images to heat maps.Enhance the contrast of the heatmap to highlight the aphid-elicited GFP signals using "Process" > "Enhance contrast" > "Adjust saturated pixels %."e.g., 5 s).Add time information using the Time Stamper plugin by clicking on "Plugins" > "Time Stamper". Set the "starting time" at "0" and the "interval" based on the time interval used for the microscopy .Export this video as an audio video interleaved (AVI) file. NOTE: This video can then be edited further in other software packages . It is also possible to observe secondary increases in GFP fluorescence after the initial peak in some experiments ( Table 1); therefore, 25 - 30 replicate samples should be averaged for quantitative analysis. Visualization of the area and speed of the feeding site [Ca2+]cyt elevation should reveal a signal of around 110 µm travelling at 6 µm/s (Table 1). In addition, no [Ca2+]cyt elevations should be detected systemically within the leaf upon aphid treatment, either in the systemic midrib  of normalized GFP fluorescence (ΔF/F) around the feeding site of a 35S::GCaMP3 leaf. The traces display 5 min before settling until 25 min post-settling. Control = equivalent location on an untreated 35S::GCaMP3 leaf. Right: representative stereomicroscope image of [Ca2+]cyt elevation seen around an aphid feeding site on a 35S::GCaMP3 leaf. The GFP fluorescence is color-coded according to the inset scale. The aphid id outlined in white and the feeding site indicated with an arrowhead. Magnification: 7.8X, focus: -127.833 mm, exposure: 1 s. The GFP was excited using a 450 to 490 nm metal halide lamp, and the fluorescent emission was captured between 500 and 550 nm. (A) An example of a large aphid-induced [Ca2+]cyt elevation. (B) An example of an average aphid-induced [Ca2+]cyt elevation. (C) An example of no aphid-induced [Ca2+]cyt elevation. Please click here to view a larger version of this figure.2+]cyt  Elevation and Aphid Behavior Parameters during 35S::GCaMP3 Imaging. Table 1: [CaParameters calculated from viable samples in which settling of >5 min occurred.(A) Speed of the visible signal from the point of initiation to the furthest point of spread. (B) Duration of the initial settling events used for the analysis of fluorescence. (C) Length of time between the beginning of imaging and the first aphid settle. [Ca2+]cyt elevation data previously submitted to The Plant Cell .2+ Signals Systemic to the Feeding Site. Figure 4: GCaMP3 Cannot Detect Aphid-elicited CaRepresentative traces (n = 30) of normalized GFP fluorescence (ΔF/F) in locations systemic to the aphid feeding site in 35S::GCaMP3 leaves. The traces display 5 min before settling until 25 min post-settling. Control = equivalent location on an untreated 35S::GCaMP3 leaf. (A) ΔF/F in the systemic midrib region. (B) ΔF/F in the systemic lateral tissue region. Please click here to view a larger version of this figure.Video 1: GCaMP3 Florescence Over time as an Aphid Feeds. The GFP fluorescence is color-coded according to the inset scale. The location of the aphid feeding site is indicated with an arrowhead. Left: 35S::GCaMP3 leaf exposed to an M. persicae adult. Right: A 35S::GCaMP3 no-aphid control leaf. Please click here to view this video. (Right-click to download.)2+ signaling during a biotic stress such as insect feeding. It demonstrates that one of the first plant responses to such threats is a localized [Ca2+]cyt elevation around the feeding site of the insect. Through the use of mutants, this method will allow for the the molecular and physiological characterization of such signals, which was not previously possible. A critical step in this protocol is to ensure that the detached leaves are not excessively disturbed during the detachment process (step 3.2) or when transferring insects to the leaves (step 4.5). Given that the current protocol provides a relative measurement of [Ca2+]cyt rather than an absolute concentration, it is vital that the microscope settings are kept constant throughout the experiment. There is also the potential for human bias during the selection of ROIs and the analysis of the data, and as such, it is recommended that the experiments are conducted double-blind.The method described in this paper allows for the real-time analysis of plant Ca2+]cyt during biotic stress with this protocol. First, the use of a single fluorophore with a high fluorescent yield allows the imaging to be conducted on a stereomicroscope, which is less costly than using a confocal microscope. The use of a single fluorophore also makes data collection and analysis simple, as there is just one measurement to record. In addition, the use of a stereomicroscope allows for the imaging of entire leaves, which is essential given that many biotic interactions, including plant-aphid interactions, occur on a large spatial scale. The high temporal resolution of image capture possible with GCaMP3, based on the rapid disassociation of Ca2+ from the sensor after binding23 (in preparation). The detached leaves also ensure that the insect feeds from a pre-defined location, allowing for the analysis of Ca2+ dynamics before, during, and after feeding. This protocol also ensures that leaves of similar developmental stages are used for analysis.There are several significant advantages of measuring [Ca2+]cyt, such as changes in cellular pH, motion, or the expression level of the biosensor. These issues are not encountered with FRET Cameleons during FRET, as the transfer of energy from CFP to YFP only occurs upon Ca2+ binding. Other conditions that alter the fluorescent properties of the individual fluorophores are unlikely to mimic the opposing changes in intensity of CFP and YFP, and the ratiometric calculation that is used inherently normalizes the measurements for many of these other optical artifacts232+]cyt more reliable with FRET Cameleons. Consequently, GCaMP3 is best used as a biosensor to measure relative [Ca2+]cyt, although it is still sufficient to detect and characterize biological phenomena in plants5,(in preparation). Therefore, it is essential to use controls to show that the observed effect is due to Ca2+, including Ca2+-related genetic mutants(in preparation) or pharmacological Ca2+ channel inhibitors such as La3+. Importantly, single-FP biosensors typically display a greater fluorescent yield and greater dynamic range  than FRET Cameleons5The main disadvantage of this protocol originates from the use of a non-ratiometric biosensor. With single-FP biosensors, variation in GFP emission may result from experimental variables other than [Ca2+]cyt elevations are discarded (step 6.3). Such transients are most likely the result of stress induced by the microscopy. Indeed, blue light is known to elicit Ca2+ signals3839402+]cyt elevations21252+]cyt elevations4344M. persicae, the insects do not settle on the leaves in several samples. This could be a result of wound-elicited defense in the detached leaves46M. persicae is governed by three photoreceptors, including one with a peak sensitivity of 490 nmDuring the execution of this protocol, it is possible that some issues will arise that require troubleshooting. For example, it is recommended that samples in which the control (untreated) leaf displays large [Ca2+ signaling in a range of novel biotic interactions between plants and other species.The protocol outlined in the current paper gives new insights onto the molecular understanding of plant-insect interactions and the plant response to biotic stress. It allows for the visualization of one of the first plant responses to insect feeding and facilitates further investigations through the use of the considerable Arabidopsis genetic resources available. In addition, this protocol allows for the use of live organisms, as opposed to extractsThe authors have no conflicts of interest to declare.


WFS1 mutations, but recessive mutations in a second gene, CISD2, have been described in a small number of families with Wolfram syndrome type 2 (WFS2). The defining diagnostic criteria for WFS2 also consist of optic atrophy and diabetes mellitus, but unlike WFS1, this phenotypic subgroup has been associated with peptic ulcer disease and an increased bleeding tendency. Here, we report on a novel homozygous CISD2 mutation  in a Moroccan patient with an overlapping phenotype suggesting that Wolfram syndrome type 1 and type 2 form a continuous clinical spectrum with genetic heterogeneity. The present study provides strong evidence that this particular CISD2 mutation disturbs cellular Ca2+ homeostasis with enhanced Ca2+ flux from the ER to mitochondria and cytosolic Ca2+ abnormalities in patient-derived fibroblasts. This Ca2+ dysregulation was associated with increased ER-mitochondria contact, a swollen ER lumen and a hyperfused mitochondrial network in the absence of overt ER stress. Although there was no marked alteration in mitochondrial bioenergetics under basal conditions, culture of patient-derived fibroblasts in glucose-free galactose medium revealed a respiratory chain defect in complexes I and II, and a trend towards decreased ATP levels. Our results provide important novel insight into the potential disease mechanisms underlying the neurodegenerative consequences of CISD2 mutations and the subsequent development of multisystemic disease.Wolfram syndrome (WS) is a progressive neurodegenerative disease characterized by early-onset optic atrophy and diabetes mellitus, which can be associated with more extensive central nervous system and endocrine complications. The majority of patients harbour pathogenic  WFS1 mutations, but recessive mutations in a second gene, CISD2 (CDGSH iron-sulfur domain-containing protein 2), have been described in a few patients with Wolfram syndrome type 2 . Unlike WFS1, patients with WFS2 have been reported to develop bleeding intestinal ulcers and defective platelet aggregation, in the absence of diabetes insipidus and psychiatric disorders , also known historically as DIDMOAD , is an autosomal recessive disorder characterized by the association of diabetes mellitus and early-onset optic atrophy, which can occur in varying combinations with diabetes insipidus, sensorineural deafness, renal tract abnormalities or neuropsychiatric disorders ,2. The misorders .CISD2 gene is located within a region on human chromosome 4q where a genetic component for human longevity has been mapped through a comparative genome analysis of centenarian siblings  in a patient with a “classical” WS phenotype marked by childhood-onset insulin-dependent diabetes mellitus and progressive visual failure secondary to optic atrophy, but without peptic ulcers or defective platelet aggregation as reported previously in affected CISD2 mutation carriers. We provide experimental evidence implicating dysregulation of Ca2+ homeostasis and disturbed ER-mitochondria interactions as key pathophysiological determinants that ultimately contribute to the development and progression of CISD2-mediated disease.Here, we report the identification of a novel homozygous A 45-year-old Moroccan man (V3) was referred to the neurology clinic for the investigation of progressive cognitive and gait disturbances. He was born at term from consanguineous parents. His father (IV1) had late-onset type 2 diabetes mellitus and his older sister (V1) developed insulin-dependent diabetes mellitus at the age of 8 years old with severe diabetic retinopathy. At the age of 40 years old, she suffered from a cerebrovascular accident and she passed away following an acute lower limb peripheral arterial occlusion complicated by a severe infection. The proband was diagnosed with insulin-dependent diabetes mellitus at 8 years of age. He developed progressive visual impairment from the age of 33 years old, and this was initially ascribed to diabetic retinopathy. He had two episodes of hypoglycaemic coma at the age of 38 and 41 years old, and a few months after the second episode, he developed neurological impairment with progressive cognitive disturbances, apathy, gait instability, urinary incontinence and dysphagia. At his baseline assessment in the neurology clinic, neurological examination showed cerebellar ataxia, myoclonic tremor, dysarthria, dysexecutive syndrome and a pseudobulbar affect. Brain MRI showed generalised cortical and cerebellar atrophy, and bilateral optic nerve atrophy. We also observed loss of the normal hyperintense signal of the pituitary gland on T1-weighted sequence although the patient did not present with features of diabetes insipidus . OphthalWFS1, but CISD2 sequencing revealed a novel homozygous variant, c.215A > G (p.Asn72Ser), in exon 2. His healthy mother (IV2) was heterozygous for this variant whereas his healthy sister (V2) was wild-type (http://lovd.euro-wabb.org/home.php?select_db=WFS1), dbSNP (http://www.ncbi.nlm.nih.gov/sites/), EVS (http://evs.gs.washington.edu/EVS/), and ExAC (http://exac.broadinstitute.org/). The c.215A > G variant was predicted to be “disease causing” based upon in silico analysis with SIFT (http://sift.jcvi.org/) and Mutation Taster (http://www.mutationtaster.org/). Protein modelling indicates that the Asn residue at position 72 is located within a random coil region of the cluster-binding domain. The p.Asn72Ser substitution is predicted to alter the interactions necessary for the stabilization of the cluster-binding domain, which in turn affects the redox and functional properties of the CISD2 protein , all of which have been shown to affect mRNA splicing  was used to induce IP3R-dependent ER Ca2+ release and the ensuing Ca2+ transients were quantified in the mitochondrial and cytosolic compartments with the Rhod-2-AM and Fluo-4-AM dyes, respectively  mutation induced significantly elevated ER stress levels unlike the WFS1 patient.The patient’s fibroblasts also exhibited a larger lumen with a swollen appearance in some areas , indicatassessed . There wElectron microscopy did not reveal marked ultrastructural abnormalities in the patient’s mitochondria, which demonstrated numerous, thin, well-defined cristae running perpendicularly to the mitochondrial longitudinal axis, and with a regular pattern of parallel organization . MitochoSpectrophotometric analysis of the patient’s fibroblasts cultivated in glucose medium revealed no respiratory chain deficiency . PolarogWFS1 and the only other causative gene that has been identified to date in a few isolated cases worldwide is CISD2  and the corroborative evidence we have provided all point towards its pathogenicity, namely: (i) co-segregation of this variant with affected disease status in a consanguineous family; (ii) the high degree of evolutionary conservation of the Asn amino acid residue located at position 72 in the functionally important cluster-binding domain of the protein; (iii) the absence of the c.215A > G variant in normative SNP and exome databases and; (iv) in silico evidence predicting a disease causing effect based on the induced conformational change of the CISD2 homodimeric protein model. Recessive CISD2 mutations have been only described so far in five families from Jordan and Italy  of WS patients carrying pathogenic WFS1 mutations, which in turn resulted in calpain activation . The activation of the UPR is thought to mediate the reduced insulin content in these cells and Wolframin could act upstream of the UPR, likely in an effort to maintain ER function under protein folding stress. Further studies will be necessary to analyse the effects of Ca2+ homeostasis dysregulation in cell lines established from patients harbouring other CISD2 mutations. However, it is conceivable that ER stress induces membrane expansion through UPR-mediated activation of lipid biosynthesis  and CISD2 (NM_001008388.4) were amplified with intronic primers by using standard conditions. Primers and PCR conditions are available on request. PCR products were purified with an Illustra ExoProStar enzyme , processed with a BidDye® Terminator Cycle Sequencing kit  and analyzed on an ABI 3130 XL automated sequencer (Applied Biosystems).The coding exons of CISD2 were sequenced using primers in the 5’UTR (5’-AGCTTGGCCAGAGCGGA-3’) and 3’UTR (5’-AACCAAATGCAGTTTGGAAGG-3’). Measuring ER stress was performed by qRT-PCR using TaqMan Gene Expression Assays : XBP1 (Hs00231936_m1), CHOP (Hs0035 8796_g1), BIP (Hs00607129_gH) and HPRT (Hs99999909_m1). qRT-PCR reactions were run in duplicate on a LightCycler 480  and assays were repeated four times. Fibroblasts from 2 healthy subjects and one WS patient were used as controls. The results were analysed by students t-test.Total RNA was extracted from fibroblasts by using the Trizol reagent according to the manufacturer’s instructions and treated by DNAse I . cDNA synthesis was performed using the Kit Transcriptor first strand cDNA Synthesis . The 3 exons of Enzymatic spectrophotometric measurements of the OXPHOS respiratory chain complexes and citrate synthase were performed at 37 °C on patient’s fibroblasts according to standard procedures . ProteinPolarographic studies on fibroblasts of intact cell respiration and digitonin (0.004%) permeabilized cells mitochondrial substrate oxidation were carried out as previously described . Protein50 µg of total protein extracts, obtained as previously described , were seet al.  was downloaded. Swiss-Pdb Viewer 3.7 software was used to generate the ribbon diagram of the homodimeric CISD2 complex and analyze the structural and conformational disruptions induced by the c.215A > G (p.Asn72Ser) For ultrastructural analysis, cells were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer, rinsed in 0.1 M cacodylate buffer, post-fixed for 1h in 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 M cacodylate buffer to enhance the staining of membranes. Cells were rinsed in distilled water, dehydrated in alcohols and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope mounted with a Morada Olympus CCD camera.2+, respectively. 4 µM Fluo-4-am was loaded at 37 °C for 15 min or 8 µM Rhod-2-am was loaded at room temperature for 30 min. Cells were washed twice with PBS and incubated in Tyrode’s solution  for 15 min. Dynamic measurements of cytosolic and mitochondrial Ca2+ were performed at room temperature with a Nikon A1R inverted confocal microscope equipped with a x60, 1.40 NA oil objective in resonant scanning mode at 3.7fps for 5 min. After 1 min  histamine (100 µM) was added to evoke IP3R dependent ER Ca2+ release and the Ca2+ transient was monitored. Image analysis was performed in Fiji, the background was subtracted and Ca2+ levels were calculated as (F-Fo)/Fo where F indicates fluorescence and Fo indicates basal fluorescence.Fibroblast cell lines were seeded at 100,000 cells/dish on glass bottom dishes . Fluo-4-am (ThermoFisher) and Rhod-2-am (ThermoFisher) were used to evaluate cytosolic and mitochondrial Ca2+ levels. Cells were transiently transfected with D1ER ∼48 h before imaging in opti-MEM (ThermoFisher) with GeneJuice Transfection Reagent (Merck Millipore) and 2 µg of plasmid DNA . Cells were washed twice with PBS and imaged in HBSS without phenol red or Ca2+ and supplemented with 5 mM glucose and 25 mM HEPES (Sigma-Aldrich) at pH 7.4. Live cell imaging was performed at room temperature with a Nikon A1R inverted confocal microscope equipped with a x60, 1.40 NA oil objective. The filters used were a 457 nm excitation filter and two emission filters: 482 nm for CFP and 540 nm for YFP. The FRET to CFP ratio was calculated by ratioing the emission at 540 nm and 482 nm.The ER targeted fluorescence resonance energy transfer (FRET) based probe D1ER was used to measure ER Ca2+ levels were determined using the ratiometric Ca2+ sensitive fluorescent dye Fura-2-am at basal and following thapsigargin induced ER Ca2+ depletion. Fibroblast cell lines were seeded at 40,000 cells/well on black 96 well optical bottom microplates (ThermoFisher) the day before the experiment. Cells were loaded with 5µM Fura-2 (ThermoFisher) at 37 °C for 1 h 30 min, then washed twice with PBS and incubated for a further 30 min. Fluorescence was monitored on a VarioskanTM LUX multimode microplate reader at emission wavelength 510 nm following alternate excitations at 340 nm and 380 nm in HBSS without phenol red or Ca2+. ER Ca2+ depletion was triggered by the addition of 1 µm thapsigargin. To calibrate the Fura-2 probe, Rmin was determined by the addition of 2 µM ionomycin (ThermoFisher) and 3 mM EGTA (Sigma-Aldrich), and Rmax was determined by the addition of 2 µM ionomycin (ThermoFisher) and 10 mM CaCl2 (Sigma-Aldrich). The standard equation was used to calculate cytosolic [Ca2+]: [Ca2+] = Kd x Q x [(R-Rmin)/Rmax-R], where R is the 340/380 ratio, Rmin is the 340/380 ratio under Ca2+ free conditions, Rmax is the 340/380 ratio under Ca2+ saturating conditions, Q is the ratio of emission intensity at 380 nm for Ca2+ free and Ca2+ saturated Fura-2 and Kd was assumed to be 225 nM.Cytosolic CaFibroblast cell lines were seeded at 100,000 cells/dish on glass bottom dishes. GFP-Sec61β was transiently transfected to detect the ER. Cells were transfected ∼24 h before imaging in opti-MEM (ThermoFisher) with GeneJuice Transfection Reagent (Merck Millipore) and 1 µg of plasmid DNA. Mitotracker red  was loaded at 37 °C for 30 min to detect mitochondria. Cells were washed twice with PBS and imaged in MEM without phenol red (ThermoFisher) supplemented with 25 mM HEPES (Sigma-Aldrich). Live cells were imaged at room temperature with a Nikon A1R inverted confocal microscope equipped with a x60, 1.40 NA oil objective. Sixty nine Z-stacks were acquired across the cell at 0.11 µM increments using a high speed piezo Z stage. All image analysis were performed in Huygens Essential Software (SVI). Images were deconvolved and a 3D-reconstructed. Colocalisation of the ER and mitochondria signals was quantified using the Manders’ coefficient. ER total volume and the length and volume of each mitochondrial fragment was analysed in Huygens Object Analyser.ATP levels were measured using the CellTiter-Glo Luminescent Assay (Promega) according to the manufacturer’s instructions. Fibroblast cell lines were seeded at 40,000 cells/well on 96 well white microplates (Thermo Scientific). The cells were incubated for 1 h 30 min at 37 °C in either glucose  or, 2-Deoxy-D-glucose . Following incubation, 90 µL buffer was removed and 100 µL of CellTiter-glo reagent was added to each well. Following 25 min incubation in the dark with shaking, luminescence was measured on a Luminoskan Ascent (Thermo Scientific) with a 1000 ms integration time. Luminescence signal was normalized to mg of protein using the Bradford assay.


Binding of rigor-type myosin S1 to actin-tropomyosin-troponin, particularly at saturating Ca2+, produces activation of myosin ATPase activity in excess of that seen in the absence of the regulatory proteins. The binding energy of S1 can overcome the inhibitory activity of troponin  occurs at low free Ca2+ binding to TnC opens a hydrophobic patch to which the switch region of TnI can bind . The level of activation of ATPase activity at saturating Ca2+ is determined by the amount of M state formed in its equilibrium with the C state. The major state formed with Δ14 TnT containing actin filaments at low Ca2+ is likely to be the C state as the B state cannot form. The C state is also stabilized by a hypertrophic cardiomyopathy causing mutation, R146G TnI. The R146G mutation in TnI gives relative stabilization to the C state at low Ca2+ and the C state is highly stabilized at saturating Ca2+(Mathur et al., Ca2+ and destabilize the M state at saturating Ca2+. Table Studying natural mutations and modifications of troponin has given muscle researchers insights into the regulation of contraction. Long term deviations from the normal distribution of B, C, and M states of regulated actin seem to lead to progressive cardiac dysfunction. The last 14 residues of human cardiac TnT are critical for controlling the equilibria among the B, C, and M states of regulated actin; they stabilize the B state at low Ca2+ by interfering with tropomyosin movement into the actin groove. At low Ca2+, the C-terminal region of TnT could participate in holding tropomyosin away from the actin groove. The C-terminal 14 residues of TnT could also potentially alter the pathway of transmission of information from Ca2+ binding to TnC through the events leading to tropomyosin repositioning. Deciphering the mechanisms of action of the C-terminal region of TnT may lead to new therapies for cardiac disorders.The C-terminal region of TnT might function by directly affecting movement of tropomyosin on the actin surface. The C-terminal region could destabilize the active M state at saturating CaAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.Funded by NIH grant AR44504 and the Brody Brothers Grant to JC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system. FBN1 heterozygous (FBN1 Het) rabbits faithfully recapitulated the phenotypes of MFS, including muscle wasting and impaired connective tissue, ocular syndrome and aortic dilation. Moreover, skin symptoms, lipodystrophy, growth retardation and dysglycemia were also seen in these FBN1 Het rabbits, and have not been reported in other animal models. In conclusion, this novel rabbit model mimics the histopathological changes and functional defects of MPL syndrome, and could become a valuable model for studies of pathogenesis and drug screening for MPL syndrome.Various clinical differences have been observed between patients with the  Summary: A novel genetically engineered rabbit model of MPL syndrome, generated by CRISPR/Cas9-mediated mutation of FBN1, mimics the histopathological changes and functional defects of MPL syndrome seen in the clinic. FBN1 gene lead to a wide range of diseases, such as Marfan syndrome (MFS), Weill–Marchesani syndrome, acromelic dysplasias, stiff skin syndrome and Marfanoid-progeroid-lipodystrophy (MPL) syndrome (FBN1 gene (Gene ID: 100350931) encodes fibrillin-1, a major component of 10-12 nm diameter microfibrils. These microfibrils are found in the periphery of elastic fibers and elastin-free bundles, including the ciliary zonule, the elastic fibers in connective tissue , noncalcium-binding domain, TGF-β-binding protein-like domain, hybrid domain, proline-rich domain, N-terminal domain and C-terminal domain  into rabbit zygotes. The heterozygous rabbit model recapitulates muscle wasting, ocular syndrome, aortic dilation and lipodystrophy of MPL syndrome in the clinic, and shows decreased assembly of microfibrils in the extracellular matrix. As such, it will be a valuable model for studies of pathogenesis and drug screening.FBN1, two sgRNAs targeting the C-terminus of fibrillin-1 were designed . The mutation detection results showed that 26 of the 28 (93.0%) newborn pups carried the mutation . An off-target assay showed that the CRISPR/Cas9 system did not induce undesirable off-target effects in the FBN1 mutant rabbits . These results demonstrated that mutations in FBN1 can be achieved via the CRISPR/Cas9 system with high efficiency in rabbits.The C-terminus of fibrillin-1, which contains a glucogenic hormone, correlates with lipodystrophy in MPL patients . In ordedesigned A. To genFBN1 mutation and characterize the phenotypes of FBN1 Het rabbits, body weight, mortality rate and histopathology were compared between the WT and FBN1 Het rabbits. As shown in FBN1 Het rabbits exhibited apparent progeroid facial features, including thin face, eyelid anomalies, slender limbs, enophthalmus and bent ears, characteristics identical to the progeroid symptoms seen in human MPL syndrome (FBN1 Het rabbits (P<0.05). As expected, a significantly high mortality rate was observed in FBN1 Het rabbits . The results revealed that FBN1 mutations were detected in all 18 F1 rabbit pups, and the frame-shift mutation was determined in 12 pups .In order to obtain a sufficient number of rabbits for detailed phenotypic characterization of MPL syndrome, a sexually mature F0 founder  was mated with a WT female and the genotypes were determined by PCR and T-cloning (FBN1 Het rabbits (Table S2). The results showed that the phenotypes of MPL syndrome – including bent ears, thin face and eyelid anomalies , growth retardation and high mortality  – were seen in the F1 generation rabbits (+/−2bpFBN1), but not in the in-frame mutated rabbits . Thus, our data confirm that the phenotypes of MPL are dominantly inherited in the FBN1 Het rabbits.We further examined the heritability of MPL syndrome in the F1 FBN1 allele with premature termination codons has been widely reported in the clinic . These results were also confirmed by western blotting, gray-scale analysis and immunohistochemical analysis (IHC) of the ciliary body at the protein level (P<0.01). The results suggested that reduced expression mediated by NMD contributes to generation of MPL syndrome in FBN1 Het rabbits.The reduced expression of the e clinic . In addiic   . These rFBN1 mutations . Weigert staining and statistical analysis confirmed an age-related decrease in elastic fibers in thoracic and abdominal aortas of the FBN1 Het rabbits (P<0.05). These results suggest that the FBN1 Het rabbits faithfully reproduce the aortic symptoms of MPL syndrome.Aortic dilatation is the main clinical feature of MPL syndrome . Moreoveutations . To examcontrols A. Moreovcontrols A,B  mutation than in patients with the in-frame mutation (FBN1 Het rabbits (FBN1 Het rabbits (FBN1 Het rabbits compared with those in WT controls (P<0.01). These results suggest muscle wasting in the FBN1 Het rabbits.Reduced muscle mass occurs in patients with mutations of the BN1 gene . Furthermutation . In this rabbits D. Weight rabbits E. The recontrols F,G . In addition, the results of H&E staining showed a paucity of marrow cells and bone marrow adipocytes in the FBN1 Het rabbits compared with the WT controls  were isolated and used for weight comparisons, H&E staining and Oil Red O staining. The results showed significantly decreased adipose tissues in the FBN1 Het rabbits compared with the WT controls (P<0.001). H&E and Oil Red O staining showed no significant pathologic changes in brown and white adipose tissues in the FBN1 Het rabbits (FBN1 Het rabbits compared with those in the WT controls (P<0.05). These results confirm that the PTC mutation in the highly conserved region of C-terminus of FBN1 induces lipodystrophy in rabbits  functioning in the activation of the G protein-cAMP-PKA pathway, results in rapid glucose release into the circulation  failed to differentiate adipocytes, which was thought to be related to congenital lipodystrophy in MPL syndrome  were maintained at the Laboratory Animal Center of Jilin University. All experiments involving rabbits in this study were performed in accordance with the Animal Care and Use Committee guide from Jilin University.New Zealand rabbits  according to the manufacturer's instructions. PCR primers for the sgRNA target sites are listed in Table S3. PCR products were gel extracted and cloned into pGM-T vector (Tiangen). At least ten positive plasmid clones were sequenced and analyzed using BLAST  and DNAMAN (https://www.lynnon.com/pc/framepc.html).Mutation detection was performed as previously described . Genomichttp://crispr.mit.edu/). The PCR products were subjected to T7EI assay  were predicted using the CRISPR Design Tool (EI assay  and SangFBN1 Het and WT rabbits was extracted using TRNzol-A reagent (Tiangen) according to the manufacturer's instructions and reverse-transcribed to cDNA using a FastQuant RT Kit (with gDNase) (Tiangen). Primers for quantitative PCR (qPCR) are listed in Table S3. qPCR was performed using an ABI PRISM 7500 system . The 2−ΔΔCT method was used to determine FBN1 gene expression, which was normalized to the amount of GAPDH mRNA. All experiments were repeated three times for each gene, and data are expressed as mean±s.e.m.Total RNA from the ear skin of FBN1 Het rabbits and WT controls were euthanized by injection with pentobarbital sodium. The lungs, eyes, aorta, quadriceps, thigh skin, ears and adipose tissue were fixed with 4% paraformaldehyde for 24 h and processed for paraffin embedding before sectioning. The tibiae and knee joints were subsequently decalcified in 15% EDTA for 2 weeks, followed by dehydration in increasing concentrations of ethanol . The tibiae and knee joints were then washed with xylene and embedded in paraffin (paraffin . Slides paraffin . Oil Redparaffin .FBN1 Het rabbits was cut into 0.5 mm3 pieces. The pieces were then digested with 1 mg/ml type I collagen solution (Sigma-Aldrich) for 3 h. Skin fibroblasts were transferred to 24-well plates and grown for a further 3 days in Dulbecco's modified Eagle medium (DMEM) with 10% serum. Then, immunofluorescence was performed as previously described .Transmission electron microscopy was performed as previously described . The FBNFBN1 Het and WT rabbits were anesthetized at 1 month of age and heart blood was collected. Serum samples were extracted through precipitation and centrifugation. Triglyceride (TG) levels were detected using a triglyceride test kit . Total cholesterol (TC) levels were detected using a TC test kit (Ri Yuan). Serum low density lipoprotein (LDL) levels were measured using an LDL test kit (Ri Yuan). Serum high density lipoprotein (HDL) levels were measured using an HDL test kit (Ri Yuan). Heart blood from the FBN1 Het and WT rabbits was collected after 12 h fasting and 1 h feeding. Serum insulin levels were measured with an insulin ELISA kit  following the manufacturer's instructions. Serum glucagon levels were measured with a glucagon ELISA kit (IBL) following the manufacturer's instructions. FBG tests and GTTs were carried out as previously described (escribed .FBN1 Het rabbits, were recorded daily and used for the survival analysis. Body weights were recorded weekly.Analysis of body weight and survival time was performed as previously described . The deaFBN1 Het and WT groups were isolated and weighed at 2 months of age. Thirty myofibers of quadricep were used for analyses of cross-sectional area. The diameters and lengths of rabbits’ tibiae and femurs were recorded and analyzed. All data are presented as mean±s.e.m., and at least three individual determinations were used in all experiments. Data were analyzed with the Student's t-test using GraphPad Prism software 6.0. *P<0.05 was considered statistically significant.Gastrocnemius, quadriceps and adipose tissues from the scapular and groin of