Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals . We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses. Collagen is the most abundant protein in the extracellular matrix, and plays a central role in the formation of fibrillar structures in connective tissues23467910However the generalization of this approach is hampered by the fact that histological sections generally exhibit inhomogeneous thickness and poor flatness, so that SHG-based probing with a point-scanning system requires time-consuming 3D scans. In addition, it is difficult to derive a quantitative interpretation of 3D SHG image stacks in terms of collagen density, since the contrast mechanism is coherent while the scanning process is not. One workaround could be to extend the depth-of-field of the microscope by using a weakly focused excitation Gaussian beam to remove the need for an axial scanning131415In this article, we analyze and demonstrate the use of Bessel beam excitation for extended-depth SHG imaging of thick samples. We first describe the production of Bessel beams with adjustable properties in a scanning multiphoton microscope. We then demonstrate extended depth SHG-2PEF imaging of fibrotic mouse kidney biopsy samples. Finally, we analyze more precisely the imaging properties of Bessel beams for SHG and two-photon-excited fluorescence (2PEF) microscopy in terms of signal level and emission directionality as a function of sample geometry. Overall, these results show how Bessel beam excitation can be used to probe collagen accumulation more rapidly and more reliably than the usual Gaussian excitation scheme.et al. in 19871718J0 is the 0-order Bessel function of the first kind, r is the distance from the optical axis in a transverse plane, z is the propagation distance along the optical axis, k is the wave vector and β the angle between the optical axis and the wave vectors forming the Bessel beam  regions in the sample. This is exemplified in We now demonstrate the potential of nonlinear imaging with Bessel beams for rapid probing of fibrotic collagen accumulation in thick histological sections. Using 23 μm × 0.3 μm Bessel excitation, we recorded simultaneous SHG-2PEF images of an entire section of fibrotic mouse kidney . SHG was2 and was assembled from 18 × 19 tiles recorded with 40% surface overlap for simplifying subsequent stitching, and 0.5 μm lateral pixel size. In order to detect both small and large signals without saturation, we used a pixel dwell time of 30 μs. Overall, the total exposure time was here 1 h 17 min for 154 000 000 pixels. We note that this acquisition time could be significantly reduced by using a smaller pixel dwell time  and less overlap. Recording a Gaussian excitation image with similar lateral resolution and depth of field would have typically taken 10 times longer. The fluorescence image (shown in red) reveals the general morphology of the kidney, while the SHG image highlights fibrillar collagen.The entire image spans approximately 5 × 5.5 mmAs discussed in previous paragraphs, this imaging strategy provides data that are simpler to relate to collagen density than when using a 3D scanning scheme, since the Bessel SHG signal is proportional to the square number of in-plane collagen molecules. Note however that the collagen density may be underestimated because of partially destructive interference when collagen fibrils with anti-parallel orientations lie together within the extended focal volume5As explained in the introduction, the application of SHG microscopy for collagen scoring in fibrotic tissue samples requires to record extended-depth volumes because of the non-flatness of tissue biopsy samples and of the heterogeneity of collagen accumulation in fibroses. Our acquisition scheme has therefore practical advantages over the Gaussian-based approach. The acquisition of mosaic such as the one shown in Overall, these data show that Bessel excitation enables an important step towards the development of systematic SHG-based diagnostics. In the next section, we analyze more precisely the physics of Bessel SHG microscopy signals.In order to investigate the physics of SHG with Bessel excitation, we consider in this section 2PEF and SHG signal levels as a function of Bessel beam axial extension, in the case of point-like and extended objects. We first discuss simple scaling arguments and corroborate them with experiments, and in the subsequent section of this article we will discuss more precisely SHG phase matching with Bessel beams.ρz while keeping constant its lateral extension and the average laser power, the energy in the central lobe varies as 1/ρz. Therefore, the signal from a point-like object is expected to decrease as As pointed out earlier, the energy carried by the main lobe of the Bessel beam decreases in proportion to its axial extension: in the case of a Bessel beam25ρz  or coherent (e.g. harmonic generation) contrast mechanism is used. In the case of 2PEF if we make the simplifying assumption that only the main lobe contributes to the excitation, the total signal is proportional to the illuminated volume times the excitation intensity squared and should scale as 1/d sample .1 and ρ2 (z = 0) to be (ρ2)/(ρ1) = 0.50 ± 0.05, and the ratio between integrated intensities at z = 0 taking into account the first 10 secondary rings to be (ρ2)/(ρ1) = 0.7 ± 0.1.To experimentally validate these predictions, we recorded SHG and 2PEF signals from point-like and extended objects using Bessel beam excitation . We used1 and ρ2 . We charAs a model for point-like objects, we chose samples smaller than the axial extent of our excitation beams, namely 1 μm diameter fluorescently-labelled polystyrene beads for 2PEF and 150 nm diameter KTP nanoparticles for SHG. As models for axially extended 2PEF and SHG samples, we recorded signal from a liquid bodipy solution sealed between two glass coverslips and from potato starch granules, respectively.S(ρ2)/S(ρ1) was 0.50 ± 0.03 in the case of small objects for both 2PEF and SHG cases. These measurements are consistent with the measured squared intensities of the excitation beams and the predictions given in i.e. the SHG signal in this case increased with beam extension). These observations confirm that, in contrast with fluorescence, harmonic generation with Bessel beams occurs coherently over the extent of the beam, so that large SHG signals may be observed from large objects despite reduced excitation intensity. These results suggest that the extended depth of field properties of Bessel beams may be used for rapidly probing nonlinear properties of thick samples when using coherent signals. In the next section, we will analyze more specifically the signal generation mechanisms for SHG imaging.As summarized in kθ between the driving and harmonic fields in a direction θ is given by the geometrical projection of k(2ω) − 2k(ω) along that direction. If we consider the case of a non-dispersive medium for simplicity, with Bessel excitation characterized by an angle β ) . A model sample geometry was considered, corresponding to the simple case of a semi-infinite slab with configurable thickness transverse to the propagation axis. The sample was considered to be a monocrystal characterized by a unique tensor element x the polarization direction of the incident beam. 272829To provide a more complete picture of the emission directionality, we performed numerical simulations of SHG emission directions by a sample of different thicknesses illuminated by a Bessel beam. The simulations followed the approach described inWe experimentally validated these predictions by recording transmission SHG images with spatially filtered emission directions . We recoi.e. a coherent nonlinear process. Considering collagen imaging, we have shown that strong Bessel SHG signals are observed in dense collagen regions owing to coherence over the axially extended probe volume, despite the reduction in excitation intensity involved by beam shaping. When combined with spatially filtered detection and 2D scanning, this technique is an effective means for probing collagen accumulation in histological sections. This approach has two advantages compared to the usual imaging scheme relying on 3D scanning of a tightly focused Gaussian beam.Bessel beam excitation is an effective approach for increasing the depth-of-field while maintaining lateral resolution in point scanning microscopy. In this work we have analyzed and demonstrated the use of Bessel beam excitation specifically for SHG imaging, Firstly, the Bessel SHG signal is proportional to the square number of collagen molecules with parallel orientation within the focal plane assuming constant molecular polarity within the excitation volume, which seems to be verified for fibrotic tissues. Signals therefore have a simpler relation to collagen density than when using a 3D scanning scheme. A meaningful map of the fibrillar collagen content in fibrotic samples can be deduced from the Bessel SHG image, opening the way to fibrosis scoring. In addition, our analysis of the contrast mechanisms involved in Bessel SHG microscopy shows how spatial filtering of the detection should be used to select the signal corresponding to coherent extended depth of field emission. We note however that the quadratic relation between SHG and collagen density is lost in samples where collagen fibril polarity is heterogeneous at the submicron scale2 due to their poor flatness. Faster imaging and 2D scanning are advantageous as they mitigate issues associated with mechanical stability and 3D image stitching. We also note that the Bessel acquisition scheme could be further optimized for fast scanning of histological samples by using a more efficient phase modulator such as a static diffractive phase maskSecondly, as also illustrated in this work, the acquisition time needed to probe a histological section can be significantly reduced with Bessel excitation. An extended depth of field is needed to probe histological sections over several mmBessel excitation appears to be well suited for rapidly probing non-flat thin samples such as most tissue biopsy samples while preserving lateral resolution. Overall, this study paves the way to the development of systematic SHG-based assays for large-scale physiopathological studies.Imaging was performed on a lab-built multiphoton point-scanning microscope constructed around an inverted frame (Olympus IX-70). The output beam from a Titanium-sapphire oscillator  or an optical parametric oscillator  was sent though a variable beam expander  and then shaped into an annular intensity distribution using a reflective liquid crystal spatial light modulator  and a lens . The zerTo measure the excitation point spread functions (PSF), we added a characterization module29We note that implementing a telecentric arrangement for the scanning system, i.e. ensuring that the two scanning mirrors are conjugated with the objective back aperture, is important to minimize aberrations and PSF tilting during the scan. With the 25× objective, this configuration resulted in a usable field of view larger than 400 μm where Bessel excitation was properly shaped and parallel to the optical axis .We also note that, as in the case of Gaussian excitation, the accuracy of collagen quantification may be affected by the orientation of collagen fibrils within the excitation volume, and that this effect can be mitigated by using circularly polarized excitation.For spatially filtering the SHG signal in wave vector space, we used the collecting condenser diaphragm or a blocking mask located just after the condenser. We note that this spatial filtering should be implemented in Fourier space, so that this configuration for the blocking mask is slightly suboptimal. In practice, we found that an acceptable trade-off between filtering and lateral field-of-view is typically obtained when blocking the smallest emission angles over two-thirds of the excitation NA.t the amplitude of the conical phase and tx the amplitude of the additional linear phase along the x direction.The SLM used in this study could generate maximal phase shift slightly larger than 2π radians. Larger shifts were obtained through phase aliasing between 0 and 2π. As described in previous studiesIn terms of light efficiency, the reflectivity of the LCoS-SLM used here is 60% (resp. 70%) at 850 nm (resp. 1100 nm), and the overall efficiency of our beam shaping system is 23% (resp. 25%) at 850 nm (resp. 1100 nm).NA objective et al.x:To calculate field distributions at the focus of a high x, E0 can be written as:ω0 is the waist of the Gaussian beam illuminating the SLM and f the focal length of the projecting lens.The field in the back aperture plane of the microscope objective is related to the field in the SLM plane by a Fourier transform. Therefore, if the SLM is illuminated by a Gaussian beam linearly polarized along θB describes the Bessel beam focusing angle and Δθ its angular spread. These parameters are linked to NAB and ΔNAB by the following relations:To simulate SHG using Bessel beams, we used a simplified model for Bessel beams that is easier to relate to excitation numerical aperture. Following the approach described in3. The nonlinear polarization induced near focus was calculated as a function of the sample geometry assuming a simplified second-order tensorial response:The excitation field near focus was discretized on a 201 × 201 × 251 grid with voxel size of 35 × 35 × 70 nmr in the focal region and propagated to a position V spans the excitation volume and I the identity matrix.The harmonic field originating from all positions Emission diagrams were calculated over a sphere of radius R and the projection of the diagrams were displayed on a 2D plane whose axes are given by dimensionless variables R with the cone angle of the objective lens:Finally, the total radiated SHG power was calculated by integrating the far field intensity on the sphere of radius Renal fibrosis was induced in wild-type mice using the AGBM model (anti-glomerular basement membrane glomerulonephritis)All experimental animal protocols were performed in accordance with guidelines of the European Community (L358–86/609EEC), and were approved by the Institut National de la Santé et de la Recherche Médicale and local Ethic Review Board at Paris Descartes University .KTP (KTiOPO4) nanoparticles were produced as a colloidal suspension following the process previously described inHow to cite this article: Vuillemin, N. et al. Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation. Sci. Rep.6, 29863; doi: 10.1038/srep29863 (2016).


The nTiO2 were aged for 0, 1, 3 and 6 d in media with varying ionic strengths (Milli-Q water: approx. 0.00 mmol/L and ASTM: 9.25 mmol/L) in the presence or absence of natural organic matter (NOM). Irrespective of the other parameters, aging in Milli-Q did not change the acute toxicity relative to an unaged control. In contrast, 6 d aged nTiO2 in ASTM without NOM caused a fourfold decreased acute toxicity. Relative to the 0 d aged particles, nTiO2 aged for 1 and 3 d in ASTM with NOM, which is the most environmentally-relevant setup used here, significantly increased acute toxicity (by approximately 30%), while a toxicity reduction (60%) was observed for 6 d aged nTiO2. Comparable patterns were observed during the chronic experiments. A likely explanation for this phenomenon is that the aging of nTiO2 increases the particle size at the start of the experiment or the time of the water exchange from <100 nm to approximately 500 nm, which is the optimal size range to be taken up by filter feeding D. magna. If subjected to further agglomeration, larger nTiO2 particles, however, cannot be retained by the daphnids’ filter apparatus ultimately reducing their ecotoxicological potential. This non-linear pattern of increasing and decreasing nTiO2 related toxicity over the aging duration, highlights the knowledge gap regarding the underlying mechanisms and processes. This understanding seems, however, fundamental to predict the risks of nanoparticles in the field.During their aquatic life cycle, nanoparticles are subject to environmentally driven surface modifications (e.g. agglomeration or coating) associated with aging. Although the ecotoxicological potential of nanoparticles might be affected by these processes, only limited information about the potential impact of aging is available. In this context, the present study investigated acute (96 h) and chronic (21 d) implications of systematically aged titanium dioxide nanoparticles (nTiO The enormous production of engineered nanoparticles is suggested to contribute trillions of dollars to the global economy . This hiDaphnia magna was used as model species to assess for both acute and chronic effects, while titanium dioxide nanoparticles  served as model nanoparticles. This selection was motivated by (i) their frequent application in various products [D. magna) in an unaged form  as w as wDaphrm  with and without NOM and subsequently assessed for their acute toxicity (immobility). Based on these data, four scenarios were selected for the chronic studies, which showed particularly strong alterations in ecotoxicity. Accordingly, nTiO2 were aged for 0 or 3 d in ASTM with or without NOM prior to the evaluation of their respective chronic effects  on daphnids. Both experimental setups were supplemented by particle size characterization before (acute toxicity tests) but also during the exposure (chronic) period.It was hypothesized that higher ionic strength , which i2/g. Using this powder, a dispersant and additive free, size homogenized, stable suspension of ~90 nm was obtained by stirred media milling , and the resulting suspension was subsequently centrifuged  in order to remove residual coarse material. Prior to its application the stock suspension (2 g nTiO2/L) was analysed for its particle size distribution (intensity weighted) using dynamic light scattering , which revealed a mean particle size of 87±1 nm (polydispersitiy index: 0.10–0.26). Moreover, before the start of each acute toxicity test and thus directly after (max. 3 min) the nTiO2 aging process  was provided as powder by Crenox GmbH (Germany), exhibiting an advertised primary particle size of 6 nm and a surface area of approximately 230 mtrometry  was usedDaphnia magna  were kept in permanent culture within a climate controlled chamber  at 20±1°C with a 16:8 h (light:dark) photoperiod . Thereby, groups of 25 organisms were cultured in 1.5 L of reconstituted hard freshwater (= ASTM) according to the ASTM International standard guide E729 [Desmodesmus sp. (200 μg C per organism).ide E729 . The medide E729 ) and was2 were aged for different time intervals, i.e., 0, 1, 3 or 6 d (acute toxicity tests) and 0 or 3 d (chronic toxicity tests). Either Milli-Q water (solely used for the nTiO2 aging prior to the acute toxicity tests) or ASTM (as part of both: acute and chronic toxicity tests) was used as aging medium. The first represents a medium without ions  and the second a comparably high ionic strength  took place in 15 mL centrifuge vials with a nominal concentration of 1.00 g nTiO2/L. In contrast, the nTiO2  aging in ASTM with and without NOM was accomplished in a 500 mL glass vessel. Independent of the medium, each aging process was performed in total darkness (excluding photoactivation of nTiO2 to avoid the oxidation of NOM during aging) on a horizontal shaker . Prior to toxicity testing all aged and unaged suspensions were vortexed for 30 seconds ensuring a homogenous distribution of nTiO2 , measured amounts of the aged and unaged nTiO2 stock suspension were added to ASTM (without NOM), resulting in a series of nominal nTiO2 concentrations with 0.0 (= control), 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L. Subsequently, daphnids were carefully transferred to 50 mL of each treatment. In contrast, for experiments with nTiO2 aged in ASTM with and without NOM, juvenile daphnids were directly placed in the aged and unaged nTiO2 ASTM suspensions, which were evenly distributed in 50 mL volumes (using concentrations from 0.00 (= control) to 128.00 mg nTiO2/L). All acute toxicity tests were conducted at 20±1°C with a 16:8 h (light:dark) photoperiod .During all acute toxicity tests, groups (n = 4) of five juvenile (<24 h) daphnids each were exposed for 96 h to different concentrations of 0, 1, 3 or 6 d aged nTiOline 202 , during 2 using ASTM with and without NOM as aging medium. The aging duration was selected based on the outcome of the acute experiments, where nTiO2 aging for 3 d (ASTM with NOM) displayed an increased toxicity compared to unaged (0 d) nTiO2 in the presence of NOM. In detail, ten daphnids (<24 h) were individually placed in 50 mL of aged and unaged nTiO2 and fed daily with Desmodesmus sp. (from 50 to 100 μg C/organism with increasing age). Dead animals as well as released offspring were counted and removed daily. The test medium was renewed three times a week, while adult daphnids were carefully transferred using plastic pipettes. Dissolved oxygen (median: 7.8 mg/L) and pH (median: 8.2) fulfilled the requirements of the guideline [2; UVA, 0.109 W/m2; UVB, 0.01 W/m2).Each chronic toxicity test was conducted according to the OECD guideline 211 . Brieflyuideline  . Therefore, immobilization data of each acute toxicity test was corrected for control mortality (never exceeding 20%) using Abbott's formula. Subsequently, adequate dose-response models were fitted to these data  among the different aging processes were statistically compared also using confidence interval testing [2 concentrations (2.00 or 6.00 mg/L). For all statistical analyses and figures the statistical software environment R for Windows [For each chronic reproduction test, the cumulative mean offspring (after 21 d) was calculated separately (considering each treatment and aging process). Afterwards, differences in effect sizes  at 2.00 mg/L of 3 d aged nTiO2 in ASTM without NOM when compared to the respective control, no reproductive implications became evident for the same concentration using ASTM with NOM as aging medium  relative to all other scenarios  and thus revealed comparable 96-h EC50 values for Daphnia's immobility , nanoparticles aged in ASTM (aging medium of high ionic strength) showed a significant shift in their toxicity with aging duration. In particular, acute as well as chronic toxicity of nTiO2 aged in ASTM without NOM decreased with increasing aging duration  and hence reduction of the nTiO2 concentration in the water column  when when2 ag2 at the initiation of the bioassay did not change with aging duration in Milli-Q with NOM medium  and the , which expects stable or decreased toxicity of nanoparticles with aging duration and thus elevating agglomeration and sedimentation [Irrespective of the general tendency of NOM to reduce the ecotoxicity of nTiOarticles  and 3B. –0.64 μm  ‒ which –0.64 μm ,41; Fig  the gut ,43 may h the gut  ultimategae  limigae  and NOM levels (being present in nature)—on the ecotoxicological potential of nTiO2 in particular and nanoparticles in general. Moreover this may also account for ultraviolet irradiation, which likely potentiates negative effects [Daphnias’ filtering apparatus.Results of the present study thus showed that the aging duration as well as the properties of the medium (in terms of ionic strength and NOM content) alter the nTiO effects . OverallS1 Fig50 value together with its 95% confidence interval.The mean mortality for each treatment is indicated by an open circle, while the filled circle represents the EC(PDF)Click here for additional data file.S2 Fig50 value together with its 95% confidence interval.The mean mortality for each treatment is indicated by an open circle, while the filled circle represents the EC(PDF)Click here for additional data file.S3 Fig50 value together with its 95% confidence interval.The mean mortality for each treatment is indicated by an open circle, while the filled circle represents the EC(PDF)Click here for additional data file.S4 Fig50 value together with its 95% confidence interval.The mean mortality for each treatment is indicated by an open circle, while the filled circle represents the EC(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigDaphnia in the respective nTiO2 treatment—the concentrations are indicated in the legend of each figure. (A) Animals exposed to nTiO2 aged for 0 d in ASTM without NOM. (B) Animals exposed to nTiO2 aged for 3 d in ASTM without NOM. (C) Animals exposed to nTiO2 aged for 0 d in ASTM with NOM. (D) Animals exposed to nTiO2 aged for 3 d in ASTM with NOM.Different lines represent response of (PDF)Click here for additional data file.S7 Fig2 aged for 0 d in ASTM without NOM (○). (B) Animals exposed to nTiO2 aged for 3 d in ASTM without NOM (□). (C) Animals exposed to nTiO2 aged for 0 d in ASTM with NOM (●). (D) Animals exposed to nTiO2 aged for 3 d in ASTM with NOM (■). Asterisks denote statistical significant difference relative to the respective control; p < 0.05 (*), p < 0.01 (**). NA indicates a not assessable reproductive output due to 100% mortality of adult daphnids in the respective treatment.(A) Animals exposed to nTiO(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Growing evidence suggests that these differences arise from effects of sex steroid hormones on processes involved in intracellular Ca2+ homeostasis. This review considers how myocardial contractile function is modified by testosterone, with a focus on the impact of testosterone on processes that regulate Ca2+ handling at the level of the ventricular myocyte. The idea that testosterone regulates Ca2+ handling in the heart is important, as Ca2+ dysregulation plays a key role in the pathogenesis of a variety of different cardiovascular diseases. A better understanding of sex hormone regulation of myocardial Ca2+ homeostasis may reveal new targets for the treatment of cardiovascular diseases in all older adults.The incidence of cardiovascular disease rises dramatically with age in both men and women. Because a woman’s risk of cardiovascular disease rises markedly after the onset of menopause, there has been growing interest in the effect of estrogen on the heart and its role in the pathophysiology of these diseases. Much less attention has been paid to the impact of testosterone on the heart, even though the levels of testosterone also decline with age and low-testosterone levels are linked to the development of cardiovascular diseases. The knowledge that receptors for all major sex steroid hormones, including testosterone, are present on individual cardiomyocytes suggests that these hormones may influence the heart at the cellular level. Indeed, it is well established that there are male-female differences in intracellular Ca Cardiovascular diseases are a leading cause of hospitalization and death for both men and women . As adva2+ homeostasis. As disruption of Ca2+ handling plays a key role in many cardiovascular diseases [2+ homeostasis may help explain its influence on cardiovascular health.Despite the popularity of testosterone supplementation in older adults, how testosterone affects the heart is not fully understood. The discovery of androgen receptors in individual heart cells (myocytes) ,11 suggediseases ,13, undeThe principal male sex hormone, testosterone, is an androgen steroid. Testosterone plays important roles in normal growth and development, and its levels decline with age in both men and women. The following discussion provides a broad overview of testosterone, and its receptors, including pathways involved in its biosynthesis, regulation, and metabolism.in utero, rises sharply in puberty, and then declines with age [Testosterone is produced primarily by the testes in men, although it also can be produced by the adrenal glands and other sites including adipose tissue and bone ,15. It iwith age ,18. Indewith age . Testostwith age ,15,20,21with age . Intereswith age ,23. Thiswith age ,25, alths, to activate the cAMP/protein kinase A (PKA) pathway. This promotes the transport of cholesterol into the mitochondria and increases transcriptional activation of gene-encoding enzymes involved in testosterone biosynthesis [Testosterone biosynthesis in men is controlled by the hypothalamic-pituitary-gonadal axis , as showynthesis . The levynthesis .Figure 14 and ∆5 pathways  [5 pathway, CYP17A1 converts pregnenolone (via a hydroxylation reaction) to 17α-hydroxypregnenolone followed by a lysis reaction to yield dehydroepiandrosterone (DHEA) [When serum testosterone levels are low, cholesterol in Leydig cells is transported to the inner mitochondrial membrane via a multi-protein complex in conjunction with the steroidogenic acute regulatory (StAR) protein , as shows Figure . In the  (HSD3β) ,31,32. Ce (DHEA) ,31,32. De (DHEA) ,31. Somee (DHEA) .Figure As discussed in the ‘Testosterone in men and women’ section, there is evidence that circulating testosterone decreases with age in both men and women ,18,19,23Biological effects of androgens are mediated by interactions with androgen receptors. The effects of androgens on androgen receptors associated with the male reproductive system are well documented . The knoMost of the biological actions of endogenous and exogenous androgens are genomic effects mediated by androgen receptors that are members of the nuclear receptor gene superfamily. In the absence of androgens, the androgen receptor forms a complex with heat-shock proteins in the cytosol . The bin2+ levels in osteoblasts, platelets, skeletal muscle cells, neurons, and, importantly, in cardiac myocytes [2+ oscillations and IP3-receptor-mediated Ca2+ release from internal stores in neonatal rat cardiomyocytes [While the classic genomic pathway mediates many of the biological effects of androgens, it is unlikely to be responsible for the rapid responses to androgens observed in some tissues, including the cardiovascular system. These transcription-independent effects are thought to be mediated by the activation of a nongenomic pathway, although the underlying molecular mechanisms are not well understood . For examyocytes . Indeed,myocytes . While amyocytes ,51,52. TClinical studies have shown that there are male-female differences in the ability of the heart to contract, even in the absence of cardiovascular disease. For example, women have a higher ejection fraction at rest than men , but menThe influence of androgens on myocardial contractility in humans has been investigated by examining the effects of anabolic-androgenic steroids on heart function. These steroids are synthetic derivatives of testosterone that are used therapeutically, in particular, to stimulate muscle growth . They alin vivo and in intact hearts. Some investigators have examined the influence of long-term supplementation with testosterone, or other androgens, on cardiac contractile function. However, most have inferred information about chronic testosterone effects from gonadectomy (GDX) experiments, where animals were subjected to bilateral removal of the testes (±testosterone replacement) for varying periods of time. As discussed in detail below, results of these investigations demonstrate that male sex hormones modulate cardiac contractile function in animal models.Various animal models have been used to investigate the influence of testosterone on myocardial function in vivo, although one study has investigated the effect of GDX on myocardial structure and function with M-mode echocardiography [Relatively little is known about the influence of testosterone on cardiac contractile function in animal models iography . These aiography . GDX alsiography . These ec.f. [c.f. [A number of studies have explored the effects of testosterone supplementation on myocardial contractility in intact hearts isolated from various animal models. These studies typically used various anabolic-androgenic steroids at high doses to mimic anabolic steroid abuse in exercise training. Most found that chronic administration (8–12 weeks) of 5–50 mg/kg/week of anabolic steroid  suppresses peak left ventricular contractile performance in young adult rats (-72 but cc.f. ). By conc.f. ,71,73 buf. [c.f.  treated f. [c.f. . These oc.f. [c.f. [2+ in individual cardiomyocytes, effects of testosterone on contractile function may arise from effects on mechanisms involved in intracellular Ca2+ handling, as discussed below.Other investigators have examined the influence of chronic testosterone withdrawal on cardiac contractility in GDX rodents, as summarized in Table c.f. ), there c.f. ,77,78 buf. [c.f. ,76). Conf. [c.f. . Interesf. [c.f. . Togethe2+ and contraction is known as excitation-contraction coupling . This small influx of Ca2+ triggers the release of a much larger amount of Ca2+ through Ca2+ release channels (ryanodine receptors (RyRs)) in the sarcoplasmic reticulum (SR) in a process known as Ca2+-induced Ca2+ release [2+ is released from the SR in the form of discrete, subcellular units called Ca2+ sparks that fuse to form the Ca2+ transient [2+ then binds to contractile proteins, known as myofilaments, which results in sarcomere shortening and cardiac contraction. Relaxation occurs when Ca2+ is taken back up into the SR by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), whose activity is regulated by the endogenous inhibitor, phospholamban (PLB). Some Ca2+ is also removed from the cell on a beat-to-beat basis, primarily by the bidirectional Na+/Ca2+ exchanger (NCX) that removes one Ca2+ in exchange for 3 Na+ [The process by which cardiac excitation triggers a rise in intracellular Caiewed by ,81). Thi release ,81. Ca2+ransient . Ca2+ thor 3 Na+ . The eff2+ release and contraction are initiated by the cardiac AP, some investigators have explored the influence of testosterone on AP properties (Table 50) but actually prolongs APD95 in rat papillary muscle [50 and APD90 [As SR Cay muscle . By contnd APD90 . One facnd APD90 ,36. Thernd APD90 . Consistnd APD90 , in partnd APD90 -87. Thusnd APD90  suggest 50 [90/95 , steady state K+ current (ISS) and inward rectifier K+ current (IK1), or on the expression of proteins or mRNA levels linked to these currents [+ current (IKur) and decreases the expression of the corresponding Kv1.5 protein  is the major repolarizing current [Ks also is slightly reduced by GDX [GDX may act to prolong the cardiac AP by modifying ionic currents to either attenuate repolarization or prolong depolarization. Therefore, a number of investigators have examined the influence of GDX on sarcolemmal proteins and currents, as shown in Table currents ,74. By cprotein , as shown in Table Ca-L, at least in the rabbit model [2+ channels) is markedly reduced by GDX in hearts from male rodents, and this effect is reversed by testosterone replacement [2+ influx in the heart. In support of this, peak L- and T-type Ca2+ currents are enhanced in neonatal rat cardiomyocytes chronically exposed to testosterone (24–30 h in culture), an effect blocked by the nuclear androgen receptor antagonist, flutamide [Ca-L and increases the expression of Cav1.2 (the pore-forming subunit of the L-channel) in cultured human ventricular myocytes [2+ currents and that this effect is attenuated by GDX. Thus, Ca2+ influx may actually be inhibited by GDX, so enhanced Ca2+ influx does not account for the increase in APD observed in the GDX heart. On the other hand, Ca2+ influx via ICa-L is the primary trigger for SR Ca2+ release. Thus, effects of testosterone on ICa-L could have important effects on cardiac contractility as discussed in the next section.Prolongation of the AP by GDX also could arise through changes in ionic currents that prolong depolarization. Whether Nait model . By contlacement ,91. Thislutamide ,93. Chromyocytes . These f2+ levels is NCX. This exchanger primarily operates to remove one Ca2+ from the cell in exchange for three Na+ [c.f. [2+ from the cell, a reduction in NCX in GDX hearts could slow relaxation, as observed in perfused hearts from GDX animals . There a+ [c.f. ,91, as sin vivo . GDX also reduces peak contraction and slows relaxation in isolated perfused hearts, especially when hearts are working under high loads . Likewic.f. ,76) is a2+ sensitivity but reduces maximal myofilament responses to Ca2+ in skinned rat ventricular muscle [2+ could contribute to the reduction in contractility observed in GDX hearts. Another study has examined myosin heavy chain (MHC) composition in sham-operated and GDX rat hearts [Testosterone could modify cardiac contractile function by effects on the contractile proteins themselves. However, few studies have examined the impact of testosterone on myofilament proteins. One study has shown that testosterone deprivation has no effect on myofilament Car muscle . This det hearts . They fot hearts . Predomi2+ transients . Femaleild type . Furtherd by GDX , anotherd by GDX . As withmyocytes ,99. Thes2+ transients after GDX [2+ concentrations and pacing frequencies far below physiological for rats . By contrast, Curl et al. [2+ concentrations  and found that peak contractions and Ca2+ transients declined after GDX, especially at higher external Ca2+ concentrations  [The SR plays a crucial role in Cams Table . As withd by GDX , RyR-medat model . In supp+ sparks . Further females ,103. Tak(CaMKII) ,104,105 2+ content and SR Ca2+ reuptake mechanisms. Studies have shown that levels of the SR Ca2+-binding proteins calsequestrin and calreticulin are not affected by GDX [2+ content is reduced in myocytes from GDX animals when compared to sham-operated controls [2+ content in the setting of chronic testosterone withdrawal has been investigated. Table 2+ ATPase protein, SERCA2a, is not affected by GDX [2+ content in GDX hearts.Most investigations of the influence of testosterone on SR function have focussed on its effects on SR Cad by GDX . Howevercontrols . The mecd by GDX ,75,96. T17) is reduced by GDX [16) is reduced by GDX [c.f. [2+ may explain the slower Ca2+ transient decay [2+ content [Other investigators have examined the expression of the endogenous SERCA2a inhibitor, PLB, after chronic testosterone withdrawal Table . There id by GDX ,96. Therd by GDX , althougd by GDX , a reducDX [c.f. ). This rnt decay ,99 and r content  characte2+, which is reduced by GDX  effects of testosterone in cardiac muscle and isolated cardiomyocytes. It is well established that physiological testosterone levels fluctuate between 10 and 35 nM in adult men , and sim2+ current in neonatal rat cardiomyocytes. Furthermore, even lower concentrations of testosterone (100 nM) inhibit peak ICa-L in ventricular myocytes isolated from neonatal rats [K1 [Ks in guinea pig ventricular myocytes [in vivo is unclear because most studies used supra-physiological concentrations of drug.There is evidence that testosterone can acutely affect the cardiac AP, at least when higher concentrations of androgens are used. Acute application of testosterone has no effect on RMP but prolongs APD in guinea pig papillary muscle preparations when high concentrations are used  . On the tal rats  and adultal rats . Superfurats [K1  and applmyocytes . These s2+ handling directly. One group has shown that acute application of testosterone (100 nM) elicits voltage-dependent oscillations in intracellular Ca2+, along with IP3-receptor-mediated Ca2+ release from SR stores in neonatal rat ventricular myocytes [2+ transient [2+ homeostasis or contractions in cells from females. As most studies of the effects of testosterone on the heart have used cells from males, it is possible that there are male-female differences in responses to testosterone, and additional studies to address this issue are needed. These acute effects of androgens on the heart are seen only at high concentrations of steroid, so the physiological relevance of these findings is uncertain. Additional studies that explore the impact of more physiological concentrations on cardiac Ca2+-handling mechanisms are warranted.Others have examined effects of acute testosterone application on cardiomyocyte Camyocytes . By contmyocytes . This laransient . Whetherransient  study sh2+-handling mechanisms in ventricular myocytes, as illustrated in Figure + current, IKur, at least in rodent models. This reduction in IKur is secondary to a decrease in the expression of Kv1.5. Ca2+ transients also are smaller and slower in ventricular myocytes from GDX animals when compared to sham-operated controls, especially when cells are paced at physiological rates. The decrease in SR Ca2+ release arises as a consequence of changes in several components of the excitation-contraction coupling pathway. First, GDX reduces the density of L-type Ca2+ channels, so Ca2+ influx is reduced and there is less Ca2+ available to trigger SR Ca2+ release. Second, the amount of SR Ca2+ available for release is reduced by GDX, and the magnitude of Ca2+ sparks may decline. The decay of the Ca2+ transient is slowed as a consequence of a decrease in the rate of SR Ca2+ uptake along with prolongation of the APD. The decline in SR Ca2+ uptake arises through a reduction in phosphorylation of PLB by CaMKII and possibly also by PKA. Contractions are attenuated in GDX myocytes due to a decrease in the magnitude of the Ca2+ transient along with a reduction in the maximal myofilament responsiveness to Ca2+. Relaxation is slowed due to slower Ca2+ transient decay along with a shift from the fast α-MHC isoform to the slower β-MHC isoform. These findings demonstrate that GDX influences critical mechanisms involved in Ca2+ homeostasis and suggest that testosterone modulates myocardial function, at least in part, by effects on individual ventricular myocytes in rodent models. Additional experiments that explore the impact of testosterone on these mechanisms in cardiomyocytes from larger mammals including humans are needed and the key signaling pathways involved should be identified.The evidence reviewed here suggests that chronic testosterone withdrawal influences cardiac Ca2+ homeostasis at the level of the individual heart cell has a number of important clinical implications. For example, men have faster rates of repolarization than women [2+ handling and contraction linked to falling testosterone levels in older adults are likely to interact with diseases in the aging heart. For example, the observation that contractions and Ca2+ transients decline in low-testosterone states may promote heart failure with reduced ejection fraction [2+ dysregulation also is implicated in the pathogenesis of diseases such as myocardial ischemia and arrhythmias [The idea that testosterone regulates the cardiac action potential and Caan women , and casan women . This isan women ,117. Furfraction . Intracehythmias , where a


Upwardly-oriented capsules dehisce at the apex, whereas pendent ones dehisce at the base, in both cases ensuring that seeds are released from an upright position; 3) in manipulation experiments on Silene chungtienensis, upward fruits  exhibit much greater dispersal distances and more dispersive pattern than pendent ones, and have a more even distribution of dispersal direction than horizontal ones. Our results suggest that fruit orientation may have important function in seed dispersal, which may be the reason why the phenomenon that stalk erection after flowering occurs widely.That stalks reorient after flowering to face upwards is a common phenomenon in many flowering plants, indicating the potential importance of fruit orientation on seed dispersal. But this idea has not been subject to an empirical test. We examined this hypothesis by analysing the evolutionary correlation between fruit orientation and other characters and by investigating the effects of fruit orientation on seed dispersal. We found that 1) in a sub-alpine plant community, upward fruit orientation strongly correlates with fruits that act as seed containers, which are often of dry type and are dispersed by non-animal vectors; 2) as exemplified by the Campanulaceae  The fruit is a characteristic structure in flowering plants, and its character affect the way of seed dispersal. It is well known that specific morphological features may enhance dispersal by the relevant agents24567Anisodus luridus (Solanaceae), Wang et al.111A striking feature of fruits that act as seed containers is their upward orientation. Many plants whose flowers are not upwardly orientated erect their stalks after flowering, thus holding the fruits in an upward orientationTo test this hypothesis, the first question we asked was: do some taxa exhibit upward fruit or stalk erection more frequently than others? An upward fruit orientation is likely to be more important for plants that produce containers, which require a suitable position from which to release seeds. We would therefore expect to find a correlation between fruit orientation and the function of the fruit, i.e., whether it acts as a diaspore or as a seed container. Furthermore, fruit orientation may also correlates to other fruit characters, such as fruit type and dispersal mechanisms. Erecting the stalks after flowering had been more frequently reported in capsule- and follicle-bearing taxa1s. str. are dominated by capsules, but the orientation of the fruits varies among them remarkably, being either pendent or upward. Furthermore, the position of dehiscence in the capsule also varies in this familySecondly, we asked why there are a number of plants that keep their containers pendent e.g.,  and whatFinally, we asked how fruit orientation could affect seed dispersal. We anticipate that an upward fruit may have better control over the release of seed relative to other fruit orientations, thus promoting dispersal. These effects may be achieved through dispersal distance, dispersal direction and/or the general pattern of seed distribution. All of these dispersal parameters may have potential influence on plant fitness through their effects on the fate of seedsSilene chungtienensis (Caryophyllaceae) as a model system.To answer these questions, we first investigated the distribution pattern of fruit orientation and its correlation with other characters in a natural plant community, and then used a model system, the bluebell family, to investigate the evolutionary pattern of fruit orientation and dehiscence position. Finally, we examined the effects of fruit orientation on seed dispersal by conducting phenotypic manipulation experiments in the field, using In the community we studied in SW China, 80 species from 66 genera and 34 families were included in the analysis. The distribution of the fruit characters is shown in Fruit orientation and all three other characters  evolves in a correlated way, given that the dependent evolutionary model fit the data better , causing collinearity. When testing each predictor separately, all of them had a significant influence .When the three predictors  were considered together in the Bayesian mixed model MCMCglmm and onlyAsyneuma , many members of Campanula and all members of Adenophora and Hanabusaya produce pendent rather than upward capsules. We further investigated why pendent capsules occur in these plants, and a strong evolutionary correlation between fruit orientation and dehiscence position was discovered . Specifically, almost all species with upward capsules have an apical dehiscence position, whereas species with pendent capsules have a basal dehiscence position. Likelihood . We noticed that Merciera tenuifolia (with indehiscent fruit) does not obey the correlation we mentioned above. Interestingly, however, this species produces few large seeds rather than numerous small ones, and is thus quite different from other members of the family in the dispersal mechanism.The Bayesian Inference tree based on four plastid markers with posterior probabilities is shown in kelihood  and parskelihood  analysisF2,43 = 89.789, P < 0.001). The distance in the pendent group was much shorter than that in the other two groups. There was no significant difference in mean dispersal distance between upward and horizontal groups  samples of the pendent group showed a strongly clustered pattern of seed distribution. In contrast, only five samples (out of 15) from the upward group and nine samples (out of 16) from the horizontal group showed a clustered pattern; other samples showed a random pattern of distribution. When seed positions from samples within each group were pooled, all three groups showed a clustered distribution; however, the intensity of clustering differed among them. The pendent group showed the strongest clustering pattern  that are dispersed by wind  and also have pores at the top of erect capsules and at the base of hanging ones, preventing the seeds from simply falling out”. This correlation between fruit orientation and dehiscence position is the very one that we found in the Campanulaceae s. str. in this study. The dehiscence position is always in the upper part in upward capsules, whereas it is at the base in pendent ones, in both cases ensuring that seeds are released from the upright position. Our analysis of character evolution showed that the ancestral condition was likely to be an upward capsule accompanied by apical dehiscence, and that the pendent capsule combined with basal dehiscence independently evolved several times in this family.Seeds from capsules and follicles are usually small and have no features to facilitate dispersal by specific vectors. These seeds could be indirectly dispersed by windCampanulaCampanula, we found that closely related species may have different dehiscence positions. This suggest that dehiscence position, which strongly co-varies with fruit orientation, is not a conservative enough feature to be used as a key character for classification at the subgenus-level , promoting greater dispersal distances. Furthermore, after fruits swing in one direction the stalk usually rebounds in the opposite direction . This is a natural botanical garden, located in the biodiversity hotspot region in SW China, which was established to protect natural vegetation from grazing. We recorded the common plant species in meadow and shrub habitats, and then constructed a phylogenetic tree by grafting the genera present in the study area onto a backbone phylogenetic hypothesis using the online program PhylomaticViola delavayi have elaiosomes that facilitate ant dispersal, they are autochores in the primary dispersal process.For each species, we assigned states for the following four characters: fruit function (diaspore or seed container), fruit orientation (upward or non-upward), fruit type (dry or fleshy) and expected dispersal vector . We also noted whether stalk erection took place after flowering (present or absent), characterized by a non-upward-oriented flower/inflorescence followed by an upward fruit/infructescences. The information involved were obtained either from direct observation or from the literature  to reconstruct the relationships within the family, building on previous phylogenetic studies in the family level carried out by Haberle et al.et al.393738et al.Campanula species  that showed variation in dehiscence position and fruit orientation were newly generated in the current study (Because the fruit orientation (upward or pendent) and the dehiscence position (at the apex or the base) varies among taxa within the Campanulaceae nt study , while ant study . We infeAnalysis of character evolution was based on the topology of Bayesian inference tree with branch lengths. To make the results more reliable, we performed our reconstruction using both the maximum parsimony and maximum likelihood method conducted in Mesquite 4.2To collect the character information, we consulted descriptions and botanical illustrations in publications , and checked specimens in the herbaria . For several species that detailed information  cannot be obtained by these ways, we searched for photos or illustrations on the internet using Google Image and then checked the identification reliability according to specimens or descriptions. Only those images with reliable identification were used in analysis. Direct observations in the field were also an important source of data, especially for taxa from China. The character information we used and their sources were listed in s. str., using BayesTriats (V2)χ2 distribution with four degrees of freedom  as a model. This plant has pendulous flowers which are pollinated by nocturnal moths, and its stalks become erect after flowering, holding the capsules upward  under natural conditions, ensuring adequate seeds for experimental study. Natural fruits with stems were harvested just before dehiscence, retaining only the top fruit by cutting the others off. These fruits were then assigned into three groups (2) plate daubed with Vaseline , modified from Weiblen & Thomsonet al.To investigate the effects of fruit orientation on seed dispersal, we conducted phenotypic manipulation experiments in Shangri-La Alpine Botanical Garden, between Aug. 10 and Sep. 2 in 2014. We used s upward . When mae groups : 1) the G function (R package spatstatG(r)and the nearest neighbour distances r, representing a general distribution pattern of points . We then used the envelope function (in the spatstat package) to test whether the results differed significantly from complete spatial randomness (CSR). We also estimated the general seed distribution pattern for each group, by pooling the seed coordinates for all samples in that group. Curves from the three groups were plotted on one graph; a steeper (more rapid increase) curve indicates a higher degree of clustering.Most of the seeds dispersed after five to seven days, and we then captured images of the distribution of seeds on plates using a high resolution digital camera . These images were analysed using PointPickerHow to cite this article: Niu, Y. et al. Post-floral Erection of Stalks Provides Insight into the Evolution of Fruit Orientation and Its Effects on Seed Dispersal. Sci. Rep.6, 20146; doi: 10.1038/srep20146 (2016).


A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC50) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC50 = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC50 = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆Gsolv) were also considered.The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide  contains supplementary material, which is available to authorized users. In spite of extensive investigations and clinical efforts made over more than two decades on the extinction of HIV, AIDS is still a substantial threat to global health -7. HIV t1) diols  for each of the diastereomeric peptides (see Tables a and b) and PCU-lactam-CO-EAIS(a and b). Due to the current lack of synthetic procedures for the required lactam/lactone starting structures, the synthesis of these peptides are not feasible.In order to obtain a better theoretical understanding about the difference characteristics between the lactam/lactone-peptides, two pairs of theoretical cage peptides were studied, namely PCU-lactone-NH-EAIS(bind) between a target protein and a set of ligands over the trajectory of molecular dynamics (MD) simulation. In this method  are defined in equations In order to calculate the theoretical binding energies, we used our MD simulations at 10 ps intervals, as suggested by Stoica and co-workers . The frebind) than the corresponding lactone-peptide diastereomers. The theoretical results [both ΔGBind(PBSA) and ΔGBind(GBSA)] for Lactam-NH-EAIS(a) peptide showed better binding affinity against CSA-HIV PR in comparison to PCU-lactone-CO-EAIS(a) peptide inhibitors. On the other hand, the case for (b) has slightly higher binding energy with ΔGBind(PBSA) for the lactam peptide, while with ΔGBind(GBSA) the same pattern as before was observed (better/lower binding energy for the lactam peptide). These results largely support the lower experimental IC50 values for PCU-lactam-NH-EAIS inhibitor (IC50 = 0.076 μm) than the PCU-lactone-CO-EAIS compound (IC50 = 0.850 μm).The first important observation is that for all cases, the PCU-lactam-peptides diastereomers showed better binding energies  give better binding energies than the corresponding lactone. It therefore appears likely that the source of better binding energy for the cage peptides originates from the PCU skeleton.50 values for the peptides were measured for a diastereomeric mixture [resulting from the two cage enantiomers attached to the (S)-peptide] it makes sense to look at the average of the calculated binding free energies of the PCU model compounds as well. In the case of both PB and GB solvation models the same order for binding free energy values was observed for the PCU models (top four models in Tables i.e. PCU-lactam models (either a or b) exhibit stronger ΔGbind in comparison to the PCU-lactone). It is important to note that for both cases (PCU-model and peptide) that the PCU-lactone consistently gave the weakest binding energies.Since the experimental ICbind. This is the case for both MM-PBSA and MM-GBSA methods. However, the mentioned difference was more evident in MM-GBSA data. Two cases for the electronic energy contributions are worth pointing out. Lower ΔEEle value  was reported for the PCU-lactone-CO-EAIS(a) in comparison to the PCU-lactam-NH-EAIS(a) peptide . Lower ΔEEle value of this compound is attributed to the potential electronic interactions . We have previously demonstrated [3H, see Figure 3H) and Asp revealed a weaker interaction for the PCU-lactam-NH-EAIS (average of about 13 Å) than for the corresponding PCU-lactone peptide .Amongst the component of binding free energies, the van der Waals (VDW) interactions between the ligands and the HIV protease showed the largest contribution to the ΔGnstrated ,13 that We calculated the entropic contribution using normal mode analysis for the PCU-lactam-NH-EAIS(a) and PCU-lactone-NH-EAIS(a) Table .The binding free energy has slightly improved, but the same trend was observed.A more detailed study will be reported later to address the potential segment of the PCU-based compounds causing this change in their binding affinities.1H NMR signals in various solvents suggested the lactim–lactam tautomeric equilibrium exists [3)2SO the major tautomer is the lactam form, while for dioxane the lactim is dominant. It is therefore most likely that the lactam form is the active form in aqueous solution . The conformation for the amide side chain with the lowest energy was determined. In both cases this conformation involved intramolecular hydrogen bonding (O5 - - HN1 for the lactam and N4H - - O1 for the lactone).In this section, we considered the PCU-lactam, lactim and lactone models shown in Figure m exists ,20. In  of PCU-lactam model in comparison to the corresponding atoms in the lactone case. The less negative atomic charge of O5 for PCU-lactam (−0.63987 a.u) than the corresponding value for the PCU-lactone (−0.65063 a.u) can be explained due to the intramolecular hydrogen bonding interaction in the lactam compound.According to the reported data in Table Analysis of the charge properties mapped onto the optimized structure will assist with the characterization of how the electronic properties are related to chemical activity . In addi2 and O5 atoms)  appeared less electron rich for O2 and electron difficient for N4H. With the C = O2 group of the PCU-lactam model, clearly more polarized than the corresponding carbonyl of the lactone. To recognize reactive domains on the compound’s surface in solution, the calculation of ESP charges was also performed in water. According to the obtained ESP values  for PCU-lactam inhibitors in comparison to the lactone series.The lactam structure which is supposed to interact with the two aspartic acid residues in the HIV PR  and quadrupole coupling constants (χ) generally known as NQR parameters. The calculated parameters for the The departure of the EFG tensor from the axial symmetry is characterized by the parameter η. The larger the value, the smaller the deviation from axial symmetry. This deviation can be caused by electronic influences such as by hydrogen bonding or packing forces in crystals.2) than that of the lactam carbonyl oxygen atom (0.1). It is notable that larger values for η were observed in more polar solvents, as expected.A higher value of the asymmetric parameter (η), around 0.9 was obtained for the oxygen atoms of the lactim hydroxyl group (O50 for the synthesized peptides with the obtained ΔGBind(PB/GB-SA) values it was argued that the source of stronger binding affinity for the cage peptides originates from the PCU skeleton. In order to verify our assumption, it serves best to focus on the quadrupole coupling constant data (χ) of the cage (PCU) part of the molecule, which is also expected to interact with the aspartase segments of the PR  were smaller for the PCU-lactam than for the lactone model. Smaller χ values implied that the quadrupole charge on these atoms were more delocalised. This will enable a stronger hydrogen bond interaction of these atoms (O2 and O3) with the Asp25/Asp25′ groups of the PR. The same observation holded for O3 of the lactim compound. As was the case with the asymmetry parameter, the quadrupole constant of O2 atom for the lactim also reflects the change from carbonyl oxygen to the oxygen atom in hydroxyl group.From the comparison of the measured ICR Figure . Our prehibitors ,13. Acco5) involved in the lactam side chain carbonyl exhibited less charge density  versus the corresponding lactone oxygen atom. Since the lactam side chain carbonyl (O5) is in fact involved in intramolecular hydrogen bonding with its NH group (N1H), it should be more delocalized resulting in a smaller χ value. This observation is in good agreement with the reported natural atomic charges on the oxygen nuclei of PCU-lactam and lactim , polarizability and dipole moments of PCU-lactam, lactim and lactone models were calculated next. The HOMO and LUMO frontier molecular orbitals are demonstrated in Figure i.e. hydrogen bond) interaction between the substrate and the Asp25/Asp25′ groups. The LUMO around lactim functional group appeared to be most prominent of the three compounds.It is evident that HOMO orbital of the lactam functional group is larger in comparison to corresponding orbitals located around the lactone and lactim cases. The size of the HOMO orbitals (which is an indication of the electron density) of the PCU-lactam and lactone models can be rationalized in terms of the corresponding experimental binding affinities. The lactam model exhibits larger electron density, which can potentially result into a better electrostatic (ind) to the field E which induces it. The dipole moment is the first derivative of the energy with respect to an applied electric field. It is used as a measure of the asymmetry in the molecular charge distribution. Much effort has been made to look for a correlation between the electric dipole moment of drug-like compounds and their biological activity [Polarizability of a molecule is defined as the ease of distortion of the electron cloud of a molecular entity by an electric field (such as when in close proximity of a charged reagent). It is experimentally measured as the ratio of induced dipole moment  with the AIS side chain. Comparison of the MM-PB (GB) SA binding free energy data of this compound in lactone series with the one of the most active PCU-lactam-NH-EAIS peptide (IC50 = 0.076 μM) that was synthesized before in our laboratory, reflect a higher binding affinity of the PCU-lactam peptide peptides against South African HIV-protease than the lactone series. This result supports the experimentally observed trend for HIV-PR IC50 values of the PCU-lactam-NH-EAIS inhibitor (IC50 = 0.076 μm) versus the lactone-peptides (IC50 = 0.850 μm).A series of three novel PCU-lactone-CO-EAIS peptides were synthesized and tested for HIV-protease activity. Two of them exhibited significant activities (~1 μM). The most active inhibitor amongst these synthesized PCU-lactone peptides was the PCU-lactone peptide 2 and O3) of the PCU-lactam, which can potentially react better with the active catalytic aspartic acid residues of HIV PR. It appears that the higher charge density, polarizability and the dipole moment due to the hetero-atoms of cage-lactam plays an essential role in their higher experimental activity and binding affinity than the corresponding cage-lactone peptides.From an electronic structure standpoint, a relatively more negative atomic charge was observed on the oxygen and nitrogen atoms of cage lactam model in comparison with cage lactone. The NQR results revealed higher delocalisation of charge distribution around oxygen atoms (ONMR spectra and HRMS data of all compounds are provided with the supplementary material. The 3D structures of all calculated compounds are also provided.


Cellular Ca2+ signaling is an important regulator of many physiological processes, and it has been widely reported to participate in the regulation of apoptotic cell death in cancer cells and tissues. However, in MPM the role of cellular Ca2+ has been poorly investigated. Therefore, we examined whether Ca2+ is involved in MPM. We found that mesothelioma cell lines and short-term cultures obtained from MPM-affected patients exhibited a critical dysregulation in Ca2+ signaling. We determined that this characteristic was associated with resistance to apoptotic stimuli and that correction of intracellular Ca2+ signaling resulted in the rescue of efficient apoptotic responses. In addition, we discovered that mitochondrial Ca2+-uptake plays a pivotal role as an inducer of apoptosis in MPM. Altogether, these findings suggest the identification of new MPM markers, which in turn could be potential targets for new therapeutic approaches.The failure of apoptosis may contribute to the formation of cancer and to its resistance to therapy. Malignant pleural mesothelioma (MPM) is an aggressive tumor that responds poorly to standard chemo- and radio-therapies. Several studies have demonstrated that a plethora of oncogenes and tumor suppressors contribute to MPM onset/progression. Importantly, most of these genes are involved in the regulation of calcium (Ca Human malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm that leads to patient death within a few months or years –3. Prese2+ distribution within intracellular compartments m) uptake have been observed [2+]m overload is a pro-apoptotic mechanism that induces mitochondrial swelling, which perturbs or ruptures the outer mitochondrial membrane, inducing the release of mitochondrial apoptotic factors into the cytosol m cytosol , 17.2+ in the onset or progression of cancer, highlighting Ca2+ handling as a potential target for the pharmacological treatment of tumors [2+ homeostasis and alterations in the principal pumps and channels responsible for appropriate Ca2+ handling have been found in cancers of different histotypes [2+ ([Ca2+]ER) release m uptake in response to stimulation of the cells with BK when compared with cell cultures derived from biopsies of healthy individuals (HM). In parallel, we found the same features in MPM commercial cell lines . In MPM cells, the [Ca2+]c increases were significantly smaller than those in control cells . The ER also participates in the subsequent decrease in [Ca2+]c by removing Ca2+ from the cytoplasm and recovering the internal Ca2+ stores through the action of sarco- and endoplasmic reticulum Ca2+-ATPases (ATP2A2). It is clear that ATP2A2 pumps are the principal regulator for the maintenance of [Ca2+]ER. One of the most common compounds used to induce intracellular Ca2+ accumulation, the sesquiterpene thapsigargin (TG), is a specific and potent inhibitor of ATP2A2. Taking advantage of this feature, we decided to evaluate the native store filling of the ER compartment in normal and mesothelioma cells. Cells were loaded in Ca2+-free medium with the Ca2+-indicator Fura-2-acetoxymethylester (FURA-2/AM) for 30 min, and the levels of the thapsigargin-releasable Ca2+ were assessed. We found that in MPM cells, the thapsigargin-dependent intracellular Ca2+ elevation was significantly lower when compared with those observed in HMC cells ; this is often due to store-operated Ca2+ entry (SOCE) that is no longer functional and/or suppressed by Ca2+ efflux across the plasma membrane (PM) i was markedly lower in the MPM cells compared with the control cells  . To inves Figure .2+ across the PM, specifically Ca2+ channels and pumps such as TRP-channels (TRPCs) and PM Ca2+-ATPase pumps (ATP2Bs) i should increase mitochondrial Ca2+ uptake, thereby activating the apoptotic machinery. MPP89 cells were pretreated with a high external [Ca2+] (10 mM) or with sodium orthovanadate , a general inhibitor of P-type ATPases, in particular ATP2Bs [To evaluate if the disruption of Car ATP2Bs .2+] or NaV, the growth capacity of the cells was affected . Silencing ATP2B4 in MPM cells expressing a mtAEQ restores the [Ca2+]m uptake  complex m uptake in normal and MPM cells. HMCs were transfected using a specific shRNA to silence MCU expression. Compared to the shRNA control, cells transfected with shRNA-MCU (shMCU) showed a significant decrease in the MCU protein levels  or with 1 μM NaV for 5 days, and the growth capacity of the cells was assessed. We found that similar to the mesothelioma cell lines, the primary mesothelioma short-term cultures were less proliferative in the presence of high external [Ca2+] or NaV after 5 days of incubation  apoptotic pathway, and although many of these proteins are involved in Ca2+-handling, the direct relationship between Ca2+ and the regulation of apoptosis has yet to be addressed.Few research studies have examined the importance of Ca2+-dependent pathway of apoptosis m handling. For example, MCU silencing attenuates [Ca2+]m [2+]m levels suppress cell death in a variety of cancerous cell lines, including those derived from the cervix, central nervous system and lymph tissues m , 10, and tissues , 50, 51. tissues . Neverth2+]m levels and survival as a possible fundamental aspect of the pathology of MPM. The results were surprising. We detected a remarkable reduction in cell growth accompanied by an increase in the apoptotic response and a restored sensitivity to proapoptotic agents.Based on these findings, we evaluated the involvement of the MCU in regulating the m uptake. In addition, a marked increase in the levels of MCU protein was found following miR-25 inhibition (data not shown).Interestingly, one of the most recent advances in cancer research is the demonstration that microRNAs (miRs) cause a variety of human disorders, particularly cancer , 53, and stimuli . One int stimuli . TherefoNevertheless it may be possible to consider miR-25 as a possible diagnostic marker and a novel therapeutic target, it is well known that a single miR can target multiple downstream transcripts and that many transcripts can be regulated by multiple miRs. Thus, further investigations are ongoing to discern the exact relationship between MPM, MCU and miR-25.2+ signaling plays an important role in MPM; ii) the downregulation of Ca2+ handling exerts an adverse effect on the execution of the apoptotic process and iii) on the sensitivity to apoptotic stimuli; iv) the recovery of Ca2+ homeostasis enhances Ca2+ uptake by the mitochondria, thereby promoting apoptotic activity; and v) MCU is a key mediator of MPM, and vi) its pharmacological modulation restores the rate of apoptosis and the sensitivity to apoptotic stimuli.In conclusion, i) intracellular CaHuman normal mesothelial cells (HMs) were obtained from biopsies collected from young non-oncologic patients (age 29–35 ys) affected by pneumothorax at the Surgical Clinic of the University/Hospital, Ferrara.2+]i measurements, cells were seeded on 24-mm glass coverslips. For aequorin measurements, the cells were seeded before transfection onto 13-mm glass coverslips and allowed to grow to 50–60% confluence. For the proliferation assay, cells were seeded on 12-well plates. For immunoblotting analysis, cells were seeded on 6-well plates with the same conditions for growth.MPM cell lines of commercial origin, such as MPP89, were from our cell line collections , and pri2 flasks containing RPMI 1640 medium supplemented with 10% FBS (CAMBREX), 2 mM L-glutamine (CAMBREX), 25 U/ml of penicillin (CAMBREX) and 25 mg/ml of streptomycin (CAMBREX) and added with 200U/ml of collagenase type 2 (Wortington). The samples were incubated at 37°C in a 5% CO2-humidified atmosphere over night to separate the cells from the tissue. The next day, the samples were washed with PBS 1X, mixed by pipetting, centrifuged for 10 minutes at 1000 r.p.m. and plated in new 25-cm2 flasks with 5 ml of complete medium. The cultures were maintained at 37°C in a 5% CO2-humidified atmosphere. The medium was changed twice a week, and the cultures were split according to their growth rateHMC and MPM cells were derived from surgical pleural samples of patients. Informed written consent was obtained from the patients to established MPM cell lines. This study was approved by the County Ethical Committee, Ferrara, Italy. Samples were obtained in sterile conditions and put in RPMI medium supplied with 50 U/ml of penicillin (CAMBREX) and 50 mg/ml of streptomycin (CAMBREX). The biopsy samples were cut in small pieces and transferred to 25-cmDifferent morphological and proliferative parameters of HMC and MPM cultures were studied using immunofluorescence techniques. A panel of antibodies panel containing mouse anti-human MSLN HBME-1 (DAKO), goat anti-human CALB2 (SANTA CRUZ), mouse anti-human KRT 8–18 (SANTA CRUZ), and mouse anti-human VIM (SIGMA-ALDRICH) was used to characterized the cell lines. Immunofluorescence was carried out by covering fixed cells with 75 μl of the primary antibody diluted in PBS 1X as follows: mouse anti-human MSLN HBME-1 1:50, goat anti-human CALB2 1:50, mouse anti-human KRT 8-18 1:100, mouse anti-human VIM 1:200. The cells were incubated for 30 min at 37°C and subsequently washed three times in PBS 1X for 10 min. The slides were then dried on blotting paper and covered with 75 μl of secondary antibodies (anti-mouse or anti-goat) diluted in PBS 1X 1:50. The cells were incubated for another 30 min at 37°C and then washed three times in PBS 1X for 10 min. Finally, the samples were assembled on a chamber slide with 2 ml of glycerol-PBS 1X 7:3 supplemented with 0.5 mg/ml DAPI.2+ response was evaluated using the fluorescent Ca2+ indicator Fura-2/AM (Life Technologies). Cells were grown on 24-mm coverslips and incubated at 37°C for 30 min in 1 mM Ca2+/KRB supplemented with 2.5 mM Fura-2/AM, 0.02% Pluronic F-68 (Sigma), 0.1 mM Sulfinpyrazone (Sigma). The cells were then washed and supplied with 1 mM Ca2+/KRB. To determine the Ca2+ cytosolic response, the cells were placed in an open Leyden chamber on a 37°C thermo-controlled stage and exposed to 340/380 wavelength light using the Olympus Xcellence multiple wavelength high-resolution fluorescence microscopy system equipped with a UAPON 40 × 0340-2 objective, numerical aperture 1.35 (Olympus). The fluorescence data were expressed as emission ratios. Fluorescence was measured every 100 ms, and the [Ca2+]i was calculated by the ratio method using the equation: [Ca2+]c 1/4 Kd (R–Rmin)/(R–Rmax) Sf2/Sf1.The cytosolic Ca2+ levels, cells grown on 24-mm coverslips were incubated for 30 min in 1 mM Ca2+-free medium/KRB supplemented with 2.5 mM Fura-2/AM, 0.02% Pluronic F-68 (Sigma), 0.1 mM Sulfinpyrazone (Sigma) and EGTA 100 μM. The fluorescence was measured every 100 ms. After 2 min., thapsigargin 100 nM was added to the cells, and the amount of Ca2+ released was estimated by normalization of the 340/380 ratio.To measure the thapsigargin-releasable Ca2+ depletion by incubation for 1 h at 4°C in KRB supplemented with 100 mM EGTA and 40 mM tBHQ  (Sigma). The cells were then washed with KRB supplemented with 2% BSA and 1 mM EGTA. Cells that were transfected with cytAEQ and mtAEQ were reconstituted with coelenterazine  for 2 h in KRB supplemented with 1 mM CaCl2. All aequorin measurements were carried out in 1 mM Ca2+/KRB (cytAEQ and mtAEQ) or 100 mM EGTA/KRB (erAEQ). ER refilling was trigger by perfusing with 1 mM Ca2+/KRB until a steady state was reached. An agonist was added to the same medium, as specified in the figures. The experiments were terminated by lysing the cells with 100 mM digitonin in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O). The output of the discriminator was captured by a Thorn EMI photon-counting board and stored in an IBM-compatible computer for further analyses. The aequorin luminescence data were calibrated offline into [Ca2+] values using a computer algorithm based on the Ca2+ response curve of wild-type and mutant aequorins.All aequorin measurements were performed as previously described . The cel2O2 (SIGMA-ALDRICH), C2-ceramide , thapsigargin .For immunoblot and immunostaining analysis, the following antibodies were used: anti-TUBB , anti-GAPDH , anti-MCU , anti-CASP3 , anti-PARP1 , anti-TRPC-1 , anti-TRPC-4 , anti-ATP2Bs , anti-HSPD1 , anti-ATP2B4 , anti-PML , anti-AKT , anti-pTHR308 AKT , anti-BCL2 , anti- BCL2L1 , anti-Cytochrome C , anti-ITPR3 , anti-pSER/THR AKT Substrate , anti-ATP2A2 , anti-ACTN , anti-CANX . Other chemicals used are the following: Sodium orthovanadate , menadione (SIGMA-ALDRICH), HFor immunoblotting, cells were scraped into ice-cold phosphate-buffered saline and lysed in a modified 10 mM Tris buffer pH 7.4 containing 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM EDTA and protease inhibitor cocktail. After 30 min of incubation on ice, the lysates were cleared via centrifugation at 12,000 g at 4°C for 10 min. Protein concentrations were determined using the Bio-Rad procedure. Protein extracts, 15 or 25 μg, were separated on 4–12% bis-tris acrylamide and 4–20% tris-glycine gels  and electro-transferred to PVDF or nitrocellulose membrane according to standard procedures. Non-specific binding sites were saturated by incubating the membranes with TBS-Tween 20 (0.05%) supplemented with 5% nonfat powdered milk for 1 h. Next, the membranes were incubated overnight with primary antibodies, and the binding was assessed using appropriate HRP-labeled secondary antibodies  plus a chemiluminescent substrate . The chemiluminescence signals were detected using ImageQuant LAS 4010 . The immunoblots shown are representative of at least four independent experiments.3VO4, 2 mM NaF, 1 mM PMSF and protease inhibitor cocktail). The lysates were clarified by centrifugation for 10 minutes at 13200 rpm before use. The total protein content of the cell lysates was measured with the Lowry assay. The extracted proteins (1000 μg) were incubated overnight with an ITPR3 antibody at 4°C. The next day, beads were added to the lysates. Precipitation of the immune complexes was carried out for 3 h at 4°C. Afterwards, the beads were washed with PBS supplemented with phosphatase inhibitors and PMSF. The samples were proceed by SDS-PAGE and analyzed using a standard western blotting technique.Immunoprecipitations were carried out using protein G-coated sepharose beads  following the manufacturer's instructions. For whole-cell extracts, cells were incubated on ice for 20 minutes in the following lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 and supplemented with protease and phosphatase inhibitors . For each condition, at least 25 independent visual fields were counted. Mesothelioma cells were washed with PBS, fixed in 4% formaldehyde for 10 min and washed with PBS. Then, the cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS and blocked in PBS containing 2% BSA and 0.05% Triton X-100 for 1 h. The cells were then incubated with primary antibody (anti-cytochrome c and anti-HSPD1) for 3 h at room temperature and washed three times with PBS. The appropriate isotype-matched, AlexaFluor-conjugated secondary antibodies  were used. Images were obtained with a Nikon Swept Field confocal equipped with a CFI Plan Apo VC60XH objective (n.a. 1.4)  and an Andor DU885 EM-CCD camera . Statistical evaluation was performed using the colocalization counter JACOP, available in the Fiji software.® transfection reagent according to the manufacturer's instructions.The shRNA targeting MCU (TRCN0000133861) was purchased from Sigma-Aldrich. The siRNA targeting ATP2B4 (GS493) was purchased from Qiagen. shRNA and siRNA were transfected using the HiPerfectm was measured by loading cells with 20 nM tetramethyl rhodamine methyl ester  for 30 min at 37°C. The TMRM excitation was performed at 560 nm and emission was collected through a 590 to 650 nm band-pass filter. Images were taken every 5 s with a fixed 20-ms exposure time using a Nikon Swept Field confocal equipped with CFI Plan Apo VC60XH objective (n.a. 1.4)  and an Andor DU885 EM-CCD camera . FCCP , an uncoupler of oxidative phosphorylation, was added after 12 acquisitions to completely eliminate the electrical gradient established by the respiratory chain. Where indicated, cells were pre-treated with menadione. The values are expressed in a.f.u. (arbitrary fluorescence units).Mitochondrial Ψm levels, the ratio between the residual TMRM fluorescence (obtained after FCCP addition) and the initial TMRM fluorescence was calculated. The values obtained represent the amount of the potential-indicating probe incorporated in the different experimental conditions.To establish the mitochondrial Ψ2 and humidity, and then 51-plane z-stacks were acquired with a voxel dimension of 133 × 133 × 200 nm (X Y × Z). The mitochondrial network was described in number and volume using the 3D object counter available in software Fiji .Mesothelioma cells were seeded onto 24-mm glass coverslips, allowed to grow to 50–60% confluence and then transfected with 1.5 μg of plasmid DNA mtGFP. After 36 h of expression, the cells were treated as described and then imaged with a Nikon Swept Field confocal microscope equipped with a CFI Plan Apo VC60XH objective (n.a. 1.4)  and an Andor DU885 EM-CCD camera . When indicated, the cells were treated with the reported compound. The coverslip were placed in an incubation chamber with controlled temperature, COThe cells were seeded onto 24-mm glass coverslips and allowed to grow to 60–70% confluence. The cells were then incubated with MitoSOX-Red indicator (Life Technologies Ltd) for 30 min and washed. When indicated, cells were treated with the pro-apoptotic compound menadione. After washing, the amount of mitochondrial superoxide was determined using a Tali™ image-based cytometer (Life Technologies). The data are presented as a histogram showing the % of the mean intensity of MitoSOX fluorescence.595 was measured.Cell growth curves were generated by crystal violet staining. Cells were seeded with a low cell density (5.000 per well) in 12-well plates in triplicate and allowed to grow for 5 days. At 1, 2, 3, 4 and 5 days, the cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Crystal violet was dissolved with 1 mol/l acetic acid, and the An refers to the number of independent experiments. The probability of significant differences between experimental groups was determined by Student's t test.The results are expressed as the mean ± SEM, and the