However, the functional implications of [Ca2+]i in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca2+]i. Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca2+]i. We found that a free [Ca2+]i of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca2+]i of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca2+ concentrations, with docking efficiency being the most robust at 500 nM.Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca Before fusion with the plasma membrane (PM), secretory vesicles have to complete several steps, namely docking and priming. The least understood stage is docking due to a lack of suitable methods for measuring this activity In contrast to the static snapshots obtained with electron microscopy, TIRF-microscopy gives real-time access to dynamic processes that occur close to the PM. For example, LDCVs of neuroendocrine cells, such as chromaffin cells, can be observed in vivo before secretion. Their residence time and their mobility at the PM allow differentiation between docked and primed LDCVs 2+ plays a role in several stages of exocytosis, triggering fusion at micromolar concentrations and promoting priming at nanomolar concentrations 2+ in docking remains largely unknown. We assessed the effect of varying intracellular Ca2+ concentrations ([Ca2+]i) on both docking and priming in bovine chromaffin cells using TIRF-microscopy and simultaneous patch-clamp recordings 2+]i at the desired level, whereas TIRF-microscopy enabled the observation of fluorescently labeled LDCVs in the vicinity of the PM.Intracellular Ca2+]i from 100 nM to 700 nM resulted in an increased size of the immediately releasable pool (IRP) of vesicles and decreased lateral mobility of LDCVs, indicating enhanced priming. Maximal axial immobilization of LDCVs was observed when the [Ca2+]i was maintained at 500 nM. Additionally, at the same [Ca2+]i, the number of LDCVs retained for an intermediate time at the PM was reduced to a minimum. Both of these results indicate that increasing the [Ca2+]i to 500 nM induced optimal docking. These findings show for the first time that Ca2+ modulates docking and that this modulation occurs at a different [Ca2+]i than priming.Changing [Ca2+. The [Ca2+]i was measured with the ratiometric Ca2+ indicators Fura2 and FuraFF. We reduced Ca2+ influx with an extracellular medium containing 1 mM Ca2+ ([Ca2+]e) and 4 mM Mg2+ ([Mg2+]e). This approach allowed us to set a constant [Ca2+]i ranging from resting levels of approximately 100 nM up to the low micromolar range at which secretion is initiated. To examine the physiological significance of the various calcium concentrations, we loaded the cell for 2 min to establish constant [Ca2+]i and then imaged the fluorescently labeled LDCVs for additional 2 min. [Ca2+]i and membrane capacitance were measured throughout the experiment (2+]i) were performed simultaneously, the identical sets of cells were used to generate all the parameters analyzed in this work.LDCVs in bovine chromaffin cells were stained by over-expressing neuropeptide Y (NPY) fused to the red fluorescent protein mCherry, and their mobility near the PM was visualized by TIRF-microscopy. In order to control the intracellular medium we performed patch-clamp measurements in whole cell configuration and used different pipette solutions containing various amounts of free Caperiment . Finally2+]i. We pooled cells with similar [Ca2+]i and sorted them into five bins in which the average [Ca2+]i (±s.d.) of the cells was 73±31, 293±49, 492±39, 665±62, and 1206±336 nM  of vesicles is usually determined. However, in order to tightly control the [Ca2+]i, we reduced the [Ca2+]e and increased the [Mg2+]e, which substantially reduced the influx of Ca2+ and size of the Ca2+-microdomain during depolarization. Consequently, the apparent size of the RRP was also strongly diminished (data not shown). In contrast, vesicles in close proximity to the Ca2+ channels, such as vesicles belonging to the IRP, were less affected by the reduced size of the Ca2+-microdomain. Compared to the apparent size reduction of the RRP, the size of the IRP  had an average peak position at 30.5 nm, half-width of 0.97 and amplitude of 11.6%. The first Gaussian component (caged) had a peak position at 60.5 nm, half-width of 68.6 nm, and amplitude of 7.4%. The second Gaussian component (free) had a peak position at 186.8 nm, half-width of 83.0 nm, and amplitude of 0.2%. A log-normal fit is not described properly by peak position, amplitude, and width 2+]i calculating the integral of each fit component for every cell. We found that as [Ca2+]i was increased from 100 nM to 700 nM, the proportion of CDs reflecting immobility linearly increased with its maximum at 700 nM . Though no significant difference in the proportion of CDs representing caged mobility was found among all [Ca2+]i, the proportion of CDs reflecting caged mobility was the smallest at 700 nM free Ca2+. Finally, free mobility represented, at best, a quarter of all CDs and was also Ca2+-dependent. Overall, we observed that the maximum shift towards immobility was reached at 700 nM. Since immobile vesicles have been shown to be primed 2+]i.Through all [Cat 700 nM . Raising2+ . We selected cells (N = 19) in which the [Ca2+]i was constant at 590±68 nM (±s.d.), i.e. where priming was induced. Finally, we back-tracked LDCVs for up to 15 s from the time of secretion and performed the CD analysis . Nonetheless, the log-normal component, which corresponds to immobility, left-shifted for secreted vesicles compared to vesicles in resting cells. We calculated a median CD value of the log-normal fit of 36.8±1.4 nm for resting cells, independent of the [Ca2+]i, compared to a median value of 21.6 nm for secreted vesicles. Thus, our data show that, within 15 s of their fusion with the PM, primed LDCVs were highly immobile.The CD histogram was fitted by the same sum of one log-normal and two Gaussian distributions as above Eq. 8, . The int2+]i is transiently elevated after a train of depolarizations.In order to characterize the mobility state of LDCVs at the moment of fusion, we calculated the intersection points of each neighboring fit component of the secreted vesicles to outline borders in which the vesicle’s mobility state is defined . The int2+. This represents more than a one-third reduction compared to 100 nM free Ca2+  of each individual vesicle within the evanescent wave based on its fluorescence intensity. The relative changes in LDCV fluorescence over time were further checked with the CDz analysis  for which the most striking difference was observed. At 100 nM free Ca2+, roughly 10% of the LDCVs resided at the PM for the entire measurement period (2 min). These LDCVs might correspond to a population of LDCVs called dead-end LDCVs, which remain stationary at the PM and do not participate in secretion 2+]i of 300 nM. Finally, approximately two-thirds of the LDCVs remained at the PM for more than 0.5 s and less than 2 min  in comparison to resting conditions (i.e. [Ca2+]i<200 nM) 2+]i of 700 nM, supporting our results obtained by lateral LDCV mobility analysis.Patch-clamping the cells in whole cell configuration allowed us to maintain the chromaffin cells at a constant and defined [Ca2+. As shown by Voets et al. we cannot exclude that optimal priming conditions for LDCVs occur at higher concentrations 2+]i the cells start to secrete consequently docking and priming are not anymore in equilibrium conditions. As a result the pool size of primed LDCVs is decreased which is reflected in an increased LDCV’s mobility. This observation is in full agreement with previous findings Both, membrane capacitance measurements and lateral CD analysis, revealed that the pool size of primed LDCVs was maximal at 700 nM of free Ca2+]i was greater than 10 µM. At this [Ca2+]i LDCVs may undergo rapid docking and priming, which would result in one large movement just prior to fusion In recent years, the mobility of LDCVs prior to secretion has been a matter of debate. In a previous study, we showed that secreted vesicles are mainly immobile prior to secretion 2+]i did not affect this density, indicating that Ca2+ is not an obligatory docking factor. Instead, the [Ca2+]i might have a modulatory role in docking. In line with this argument, we showed that the total number of LDCVs visualized at the PM over 2 min was reduced at an [Ca2+]i of 500 nM, which could be explained by a reduced docking rate or increased undocking rate. Previously, the duration of LDCVs remaining close to the PM (i.e. in the evanescent field of TIRF-microscopy) has been used as a complementary method for assessing docking. Toonen et al. 2+]i from 100 to 500 nM led to a specific reduction in the pool of LDCVs visiting the PM for a short (0.5 to 5 s) period of time. One interpretation of these data is that high intracellular Ca2+ inhibits docking. Several lines of evidence argue against this hypothesis. First, if docking is impeded at higher [Ca2+]i, the fraction of LDCVs approaching the PM and staying less than 0.5 s in the evanescent field would be reduced by increasing [Ca2+]i2+]i, facilitating the access of LDCVs to the PM 2+ reduces docking, then LDCVs would only approach the PM and shortly thereafter move away, resulting in large axial mobility. We observed the opposite; the axial mobility was reduced to a minimum at an [Ca2+]i of 500 nM, showing the same Ca2+-dependency as the residence time of LDCVs at the PM. The alternative interpretation of our data is that Ca2+ modulates docking efficiency by reducing the undocking rate. If this conclusion is true, then in cells in which docking and priming are in equilibrium increasing the [Ca2+]i would stabilize the docked pool of LDCVs, thereby reducing the turnover rate of LDCVs at the PM. This is precisely what we observed in cells that did not secrete, i.e. cells maintained at [Ca2+]i up to 700 nM , the Ca2+ spread is different in lateral and axial directions due to the lateral overlap of Ca2+ microdomains. At a distance of 150 nm from the channels, the [Ca2+]i is >300 nM along the PM and approximately 240 nM perpendicular to the PM . Munc13 also contains an active calmodulin binding site 2+-sensor for priming. The most promising candidate for such a sensor is DOC2, a double C2 domain protein 2+-dependent manner (Kd ≈ 300 nM) and appears to facilitate priming 2+-sensor for docking is even more elusive, with only two identified docking factors. Recently de Wit et al. 2+-dependence. Whereas Toonen et al. 2+-binding site. Interestingly, the actin disruption, which appears to be a major step that enables docking. is not only mediated by Munc18, but also by scinderin 2+ binding site with a Kd of 585 nM, which would be compatible with our estimation of the Ca2+-sensitivity of docking.Overall our results point to intriguing implications about the molecular mechanism underlying this differential modulation of docking and priming. Several putative proteins have been suggested to mediate the Ca2+]i. This provides a framework in which the factors involved in these Ca2+-dependent reactions can now be identified.In conclusion, examining both the lateral and axial mobility of LDCVs in the evanescent wave enabled us to distinguish between docked and primed vesicles, which we showed to occur at different  and high [MgCl2] to reduce Ca2+-influx and obtain a constant [Ca2+]i over a long period of time (∼5 min). In the experiments in which we measured the mobility of LDCVs prior to fusion, we changed the [Ca2+] and [MgCl2] to 2.5 and 1 mM. All other components of the solution remained the same. The bath solutions were at pH 7.4, 315 mOsm. The three different intracellular solutions for patch-clamping experiments are shown 2+-buffer capacity in order to minimize perturbance of endogenous Ca2+-buffers and to enable exocytosis upon depolarization. Free EGTA and Ca2+-indicator concentrations were calculated using an IGOR macro . Osmolarity was adjusted with Cs-glutamic-acid to a value of 300 mOsm (pH 7.25). We used a combination of Fura2 (Kd(Ca2+) ∼140 nM) and FuraFF (Kd(Ca2+) ∼5.5 µM; both Invitrogen, Carlsbad, CA, USA) to measure the [Ca2+]i with high precision in a range of 0–10 µM. All chemicals were obtained from Sigma .The bath solutions contained (in mM): 145 NaCl, 2.4 KCl, 1 CaCl2+]i. Due to variations in the inherent Ca2+-buffer capacity of the cell, using only three different intracellular solutions allowed us to impose a wide range of [Ca2+]i: 70–220 nM with solution 1, 170–450 nM with solution 2, and 450–2000 nM with solution 3.An in vivo calibration curve was used to convert the ratio of fluorescent signals at both wavelengths (350 and 380 nm) into the i was <800 nM prior to stimulation fulfilled this condition. Cells that were maintained at higher [Ca2+]i did not fulfill this condition, and the calculation only approximates their IRP size.The upper limits of the IRPs were calculated using the formula from Voets et al. . CalculaEM, Belfast, Northern Ireland) and controlled, like the rest of the setup, by in-house software based on LabView . Final pixel size was 160 nm. The acquisition rate was 10 Hz with an exposure time of 75 ms. Additionally, a TILL Polychrome V monochromator  was mounted at the rear port of the microscope via a light guide to ratiometrically measure the [Ca2+]i .TIRF-microscopy was carried out as described by Becherer et al. 2+]i was determined every 2.5 s. Only cells with a constant [Ca2+]i for more than 30 s before the end of the loading procedure were used for experiments. After LDCV imaging (2 min at 10 Hz), the [Ca2+]i was determined again to verify that it was similar to the concentration measured at the end of the loading process. After an additional minute of recording, the cell was stimulated as described above to confirm the viability of the cell, its ability to secrete, and to measure the IRP size. In the experiments in which we measured secretion simultaneously with TIRF-microscopy and patch-clamping, the [Ca2+]i was continuously measured at 0.4 Hz throughout the experiment. The secretion was induced 10 s before the end of the 2-min imaging period. All measurements were performed at room temperature.In order to obtain stable and consistent measurements, cells were imaged after a loading period of 2 min without exciting the fluorescently labeled NPY. During this loading period, the i. The lateral CD histogram was best fitted by the sum of three components, namely one log-normal and two Gaussian functions (Eq. 8).nA is the amplitude of each peak, nx describes the peak position, and nw describes its half width (1–3 are the different parameters for the three components). 1w is the half width of the log-normal component, it has no unit, and is related to the variance σ by 1w =  σ·2 values. Because the CD analysis results in a large fraction of very small and only positive values, the CD distribution is skewed and best described by a log-normal curve We applied a weighted fit procedure using the inverse SD of the average data at each i .(TIF)Click here for additional data file.Figure S2Lateral CD frequency distribution with SEM. blue circles, 100 nM; green circles, 300 nM; red circles, 500 nM; orange circles, 700 nM; gray circles, >800 nM [Ca2+]i).(TIF)Click here for additional data file.Figure S3Axial CD frequency distribution with SEM. blue circles, 100 nM; green circles, 300 nM; red circles, 500 nM; orange circles, 700 nM; gray circles, >800 nM [Ca2+]i).(TIF)Click here for additional data file.Figure S4Lateral vs. axial CD correlation plot.  To analyze the correlation of lateral and axial mobility, the CD of each LDCV which was used for the analysis of the axial movements was plotted versus its lateral CD on a double-logarithmic scale: (A) 100 nM, (B) 500 nM and (C) 700 nM. A large number of points overlap. To visualize this, the density of the CDs is color encoded. The more points are present on one spot the darker becomes this spot. The plots were fitted by a linear curve (f(x) = a+bx) and the slope b is shown on each curve. Note that this slope was specifically reduced at 500 nM free Ca2+. This indicates that the mobility of LDCVs from and to the PM is decreased at lower [Ca2+]i than their mobility along the PM .(TIF)Click here for additional data file.Figure S5Comparison between lateral or axial CD histogram of secreted vesicles and fixed fluorescent beads. (A) Lateral CD histogram of secreted vesicles (solid line) and fixed beads (dashed line). (B) Axial CD histogram of secreted vesicles (solid line) and fixed beads (dashed line). Note that in contrast to the lateral histogram the shape of the axial CD histogram of secreted vesicles is similar to the shape of the axial histogram of fixed beads.(TIF)Click here for additional data file.Table S1Statistical analysis of the fluorescence intensity vs. residence time correlation plot. RT stands for residence time; NMFI stands for normalized median fluorescence intensity.(DOC)Click here for additional data file.Table S2Statistical analysis over lateral CD distribution. P value obtained through two way ANOVA on Rank using Holm-Sidak post-test.100 nM: N = 23, n = 719; 300 nM: N = 13, n = 403; 500 nM: N = 26, n = 695; 700 nM N = 14, n = 309; >800 nM: N = 16, n = 452.(DOC)Click here for additional data file.Table S3Statistical analysis over axial CD distribution. P value obtained through two way ANOVA on Rank using Holm-Sidak post-test. 100 nM: N = 23, n = 719; 300 nM: N = 13, n = 403; 500 nM: N = 26, n = 695; 700 nM N = 14, n = 309; >800 nM: N = 16, n = 452.(DOC)Click here for additional data file.Movie S1Bovine chromaffin cell perfused with [Ca2+] at 80 nM. Recording of the representative bovine chromaffin cells transfected with plasmid expressing NPY-mCherry kept at stable [Ca2+]i of ∼80 nM (see 0 nM see . LDCVs a(MOV)Click here for additional data file.Movie S2Bovine chromaffin cell perfused with [Ca2+] at 650 nM. Recording of the representative bovine chromaffin cells transfected with plasmid expressing NPY-mCherry kept at stable [Ca2+]i of ∼650 nM (see 0 nM see . LDCVs a(MOV)Click here for additional data file.


The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.5 genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶104–1∶105 and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶103–1∶104 and were protected from hepatitis, but two out of the five rabbits showed virus shedding.First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected. Hepatitis E virus (HEV) is a major cause of enterically transmitted hepatitis worldwide Previous studies have shown that rhesus monkeys and chimpanzees are susceptible to HEV of all known genotypes Recently, researchers have reported that HEV can be isolated from rabbits All of the animals in this study were handled in a manner approved by the Committee of Laboratory Animal Welfare and Ethics, Southeast University . The regulations issued by the review committee of laboratory animal welfare and ethics and the protocol for the review on laboratory animal welfare and ethics, Southeast University, were followed.Seven HEV strains isolated from humans and rabbits were used in the present study. One strain of genotype 1 HEV was collected from the stools of patients with epidemic hepatitis E in Xinjiang, China (GenBank No: JX857689). A genotype 3 strain was collected from the stools of a US organ transplant recipient with chronic hepatitis E (GenBank No: JN837481). Three genotype 4 HEV strains, H4-NJ153 (GenBank No: JX847414), H4-NJ112 (GenBank No: JX847413), and H4-NJ703 (GenBank No: AY789228), were collected from the stools of patients with acute HEV infection in Nanjing, China. Two strains of rabbit HEV, R-JS174 (GenBank No: JQ065065) and R-JS204 (GenBank No: JQ065068), were recovered from stool samples representing secondary rabbit transmission (rabbit to rabbit).The stool samples were diluted in phosphate-buffered saline  containing 1% bovine serum albumin (BSA) to make 10% (wt/vol) suspensions. These suspensions were further clarified by centrifugation at 5,000 rpm at 4°C for 20 min and filtered through 0.22 µm filters. The quantification of HEV RNA in the samples was determined using a real-time RT-PCR kit .Oryctolagus cuniculus European rabbits. Prior to inoculation, all rabbits were tested for anti-HEV IgG antibodies with enzyme-linked immunosorbent assay (ELISA) and confirmed to be seronegative for HEV infection as described previously Ninety specific-pathogen-free (SPF) female New Zealand white rabbits, weighing 1.5–2.0 kg and aged 4–5 months, were purchased from the animal farm of Nanjing Agricultural University, Nanjing, China. The rabbits used in the present study were E.coli-BL21(ED3)-pLysS strain  as described previously The candidate hepatitis E vaccine, HEV p179, used in this study was expressed in As shown in 5 genome equivalents of H4-NJ703.In the second experiment, 15 rabbits were randomly divided into three groups. Rabbits in group Vaccine-10 and group Vaccine-20 were vaccinated intramuscularly with 1 ml vaccine containing 10 µg and 20 µg HEV p179 vaccine, respectively, and five rabbits in another group received a placebo. Totally, three times of vaccination were administered by two weeks apart . Two weeks after the third vaccination, all animals were challenged intravenously with 3.3×10Serum and stool samples were collected per week post inoculation (wpi) and stored at −80°C. Liver, bile, duodenum, jejunum, and ileum samples were collected at the end of the experiment (10 wpi). For the sample preparation, 0.2 g of different tissue sample was homogenized in 2 ml sterile PBS buffer (10% w/v) and clarified by centrifugation at 4°C at 3,000 rpm for 15 min. These samples were also stored at −80°C for the detection of HEV RNA. The liver tissue samples used for histopathologic examination were fixed in 10% neutral buffered formalin immediately upon sampling.Anti-HEV IgG antibodies in rabbit sera were detected by an ELISA as described previously All rabbits were monitored weekly for 10 weeks after inoculation. The sera were separated from clotted blood by low-speed centrifugation for 15 min at 4°C and the activities of alanine aminotransferase (ALT) were measured on the day of collection using an automated bio-chemistry analyzer . ALT peak level exceeding two-fold of the base-line level was taken as biochemical evidence of hepatitis. This correlation was described in a previous study involving a non-human primate animal model CCG ACA GAA TTG ATT TCG TCG GC) and JM-5 . The internal primers were JM-3  and JM-4 . Reverse transcription and first-round PCR were performed using a One-Step RT-PCR Kit . Reverse transcription was performed at 50°C for 45 min and terminated by heating at 95°C for 15 min, after which PCR continued for 35 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 30 sec. This was followed by a final extension at 72°C for 10 min. Next, 3 µl of first-round PCR product was subjected to nested PCR using Taq DNA polymerase  consisting of 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec. This was followed by a final extension at 72°C for 10 min. The amplicons were separated and visualized by electrophoresis on a 1% (wt/vol) agarose gel. The expected final product of the universal nested RT-PCR was 236 bp in length.A universal RT-PCR assay was performed to detect HEV RNA in serum, stool, bile and liver tissue samples Liver tissue sections, 7 µm in thickness, were derived from HEV infected rabbits and normal rabbits and then placed on slides and examined for HEV antigens using indirect immunofluorescence staining as reported previously The liver tissues used for histologic examination were fixed in 10% neutral buffered formalin, routinely processed, sectioned at a thickness of 7 µm, and stained with hematoxylin and eosin. All sections were examined using an Olympus BH-2 microscope .The mean and standard deviation, or frequency and percentage, were determined for the continuous variables and categorical variables, respectively. In the first experiment, the comparison of positive rates of the IgG anti-HEV antibody in different groups was conducted by Fisher’s exact test. In order to evaluate the infective ability of different HEV strains to rabbits, the trends of anti-HEV IgG antibodies and feces shedding at different wpi were also conducted by Fisher’s exact test in the present study. In the second experiment, the analysis of the variance was conducted to test the differences of the titers of anti-HEV IgG (GMTs) and the serum ALT levels in the different vaccine groups. Statistical analyses as above were performed with Statistical Product and Service Solutions 13.0 .Rabbits from the control group did not show any clinical signs attributable to HEV infection and remained seronegative throughout the study . SimilarIn groups R-JS174 and R-JS204, 80% of the animals were seropositive and began to excrete virus one or two weeks after inoculation  and 3. HIn group H3, all five rabbits were positive for anti-HEV IgG antibodies at the end of the experiment . HoweverOne rabbit from group H4-NJ153 and two rabbits from group H4-NJ112 were antibody-positive but did not show any clinical signs of hepatitis. One rabbit from H4-NJ112 group was positive for anti-HEV-IgG and also positive for HEV RNA in a stool sample taken three weeks after inoculation. Viral shedding ceased one week later. The other animals remained negative for HEV RNA throughout the experiment.In group H4-NJ703, all rabbits were anti-HEV-IgG-positive at the end of the experiment. Three of the five rabbits showed symptoms of hepatitis such as anorexia, lethargy, and diarrhea. All five animals in group H4-NJ703 excreted virus in their stool. The virus shedding began one or two weeks after intravenous inoculation, and persisted for three to ten weeks post inoculation . Three rrd, 1st, 1st and 4th week, respectively). However, the values in groups H1, H4-NJ153, H4-NJ112 and the control group did not increased.The dynamic patterns of the mean S/CO values of anti-HEV antibodies of different groups were analyzed in 3 genome equivalents of H4-NJ703, no rabbits showed ALT elevation. Only two of five rabbits in this group showed fecal shedding and seroconversion. As shown in In the intravenous low dosage (iv-Low) group, in which the rabbits were inoculated with 3.3×104 genome equivalents of H4-NJ703, three animals shed virus in their feces and seroconverted to anti-HEV IgG antibodies. One of them also showed ALT elevation. Additionally, accumulations of inflammatory cells could be observed in liver section , all rabbits presented one or more signs of HEV infection. One rabbit had detectable viremia, two had elevated ALT , and all had fecal shedding and seroconvertion .However, in the three oral groups, only two rabbits from group or-High showed virus excretion and seroconversion. The remaining rabbits showed no virologic or serologic evidence of HEV infection. The animals in these groups showed a much lower rate of ALT elevation, fecal shedding, and seroconversion than those in the intravenous groups   .After viral challenge, three of the five rabbits in the unvaccinated control group showed a significant elevation in serum ALT, as indicated by a peak ALT level exceeding two-fold of the corresponding base-line level . All of the control animals excreted virus in their stools. This was first detectable one week after infection and continued for three to ten weeks. The vaccine inhibited ALT elevation when the animal in groups Vaccine-10 and Vaccine-20 were challenged by H4-NJ703. No animal in group Vaccine-20 showed HEV excretion in their feces but two of five rabbits in group Vaccine-10 did .Several types of animal models for HEV infection have been identified, characterized, and refined in the previous studies et al and Krawczynski, et al found that swine could be used as an animal model for HEV genotypes 3 and 4 but not for genotypes 1 or 2 In the present study, rabbits were successfully infected with H4-NJ703 (human HEV genotype 4), as indicated by viral shedding in the stools, characteristic histopathological changes, and elevation in the level of liver enzyme. However, the infectivity of H4-NJ153 and H4-NJ112 to rabbits was much lower than that of H4-NJ703 although all these three strains are in the same genotype. In this way, these results indicated that although different HEV strains had very similar genomic organization, they could vary significantly in their ability to infect rabbits. Such results were also found in previous studies 5 genome equivalents was insufficient to induce a high infection rate in three oral groups because only two out of the five inoculated rabbits showed some measurable infection (Although the fecal-oral route is the most common route of HEV transmission, almost all experimental studies on HEV have used intravenous inoculation as the route of infection because the former is inefficient nfection .5 genome equivalents of H4-NJ703 showed fecal virus shedding and biochemical evidence of hepatitis. Two doses of candidate HEV p179 vaccine, at either 10 µg or 20 µg per dose, were highly effective in preventing ALT elevation following the challenge with HEV. However, although no animal in group Vaccine-20 showed HEV RNA excretion in the feces, two of five rabbits in group Vaccine-10 did. This was consistent with previous reports In this study, nearly all of the placebo-treated rabbits intravenously challenged with 3.3×10The major implication of this work is that rabbits can serve as an alternative animal model for the study of HEV. The present study also has several limitations. First, although the rabbits in group H3 became positive to anti-HEV antibodies, no fecal shedding of HEV RNA and no elevations of serum ALT were found in these animals. In order to assess the infectivity of genotype 3 HEV in rabbit, more different genotype 3 HEV strains should be studied. Second, in order to compare the ability of the infectivity of different HEV strains in rabbits, methods to titrate the active viral particles in the inoculum must be developed. Third, the present data indicated significant differences in the duration of viral excretion among different animals in the same group. In this way, the interactions between the virus and rabbits, including the mechanisms underlying HEV infection in rabbits should be further studied.In summary, the present study demonstrates that rabbits may serve as a non-primate small animal model for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.


The role of Ca2+ was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [Ca2+] was increased. This effect was inhibited by dipyridamole, intracellular Ca2+ chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high K+ saline. Ca2+ levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with Ca2+ entry through the deoxygenation-induced pathway (Psickle), activating the Gardos channel. [Ca2+] required for phosphatidylserine scrambling are in the range achievable in vivo.Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates Ca Patients with sickle cell disease (SCD) display a range of symptoms which include chronic anemia together with ischemic pain and organ damage . The und2+-dependent scramblase moves APs rapidly in both directions thus disrupting phospholipid asymmetry , RBCs at 1% Hct were exposed to the calcium ionophore bromo-A23187 (1–6 μM), vanadate (1 mM), EDTA (2 mM), and different [Ca2+]os for 30 min to achieve the requisite final [Ca2+]o [r2([H+]i/[H+]o)2 = 2.05 [2+]i. After 30 min, RBCs were treated with Co2+  after which they were processed for annexin-labelling, as above. Annexin was used to label PS because it is important to compare findings with extensive reports in the literature using this PS marker  was used because it does not fluoresce. These experiments were carried out in LK or HK saline, composition as above except for the addition of 0.15 mM MgCl2 to keep intracellular [Mg2+] at physiological levels. Finally, to show Ca2+-loading of RBCs, cells were loaded with fluo-4-AM  with fluo-4 fluorescence also then measured in the FITC channel by FACS.Salines and HbS cell suspensions were first equilibrated with humidified air (oxygenated) or N [Ca2+]o . This wa2 = 2.05 , 36, to r e.g., , 37–39).–39.2 .Unless otherwise stated, data are presented as means ± S.E.M. for blood samples from n = 36). The effect of different [Ca2+]os  on the percentage of HbS cells showing PS exposure was then investigated. In oxygenated (20% O2) HbS cells, PS exposure was lower and although the extent of exposure was augmented when RBCs were incubated at higher [Ca2+]os, the effect was small and not significant (2), PS exposure was always higher than that observed in oxygenated HbS cells. There was also a marked increase in PS at the higher [Ca2+]os . This inhibitory effect did not persist over an 18 hour incubation, probably because the available cytoplasmic MAPTA becomes saturated with Ca2+.PS exposure in HbS cell samples taken from SCD patients and immediately labelled with FITC-annexin ranged from 0.4 to 16.0% with a mean of 2.3 ± 0.5%  reduced PS exposure in deoxygenated HbS cells (P < .01), consistent with Ca2+ entry via Psickle stimulating exposure. DIDS, although better known as a band 3 inhibitor, is also a partial Psickle inhibitor  directly [2+ pump and also the flippase. Following 30 min incubation with bromo-A23187 to alter [Ca2+]i, Co2+ (0.4 mM) was then added to block Ca2+ permeability via A23187 thereby keeping intracellular [Ca2+] constant during annexin labelling (for which 2.5 mM extracellular [Ca2+] is required). Results are shown in 2+]i increased above about 600 nM. A sigmoidal dependence of PS exposure with [Ca2+] was then apparent with an EC50 of 1.31 ± 0.84 μM (n = 6). Peak exposures varied from 16–46%, mean 28 ± 5 (n = 6) with a plateau reached at about 10 μM and without further change at higher [Ca2+]is ([Ca2+]s up to 600 μM were tested).Treatment of RBCs with the divalent cation ionophore bromo-A23187 was used to alter intracellular . LK saline was found to increase the percentage of positive cells (μM) which, for example, reduced percentage of positive cells from 44% to 28% at 10 μM Ca2+. Finally, the effect of the calmodulin inhibitor W-7 was tested . This important finding suggests coordination of these eryptotic events. Results also implicate a role for RBC shrinkage and shape change.Whilst it is well known that RBCs from SCD patients show elevated levels of PS exposure and that these are increased upon deoxygenation, the mechanism is not clear. The present results explore more fully that the role of Ca2+ levels had little effect on PS exposure in oxygenated HbS cells. Under deoxygenated conditions, however, PS exposure increased with [Ca2+]o. This effect was partially inhibited by dipyridamole i which then promotes PS exposure by Gardos channel activation, loss of intracellular solutes, and red cell shrinkage. Importantly, high K+ salines were effective over all incubation times (up to 18 hours). Shrinkage has been shown previously to stimulate PS exposure in both normal RBCs and HbS cells s using bromo-A23187. RBCs were first treated with vanadate (to inhibit both the plasma membrane Ca2+ pump and the flippase) and subsequently with Co2+ . Results showed that PS exposure was stimulated by micromolar Ca2+ concentrations with an EC50 of about 1.2 μM. This concentration is similar, though slightly higher, compared with that required for half-maximal activation of the Gardos channel activation   , and whic events  and faci


The physiological outcome of injury-induced Ca2+ inflow is, however, unknown.Nitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+-sensitive fluorescent dye, Fura-2/AM.In this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response.We demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular CaThe data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization. Endothelial injury is regarded as the early event that leads to the onset and progression of severe vascular disorders, such as thrombosis, hypertension, and atherosclerosis . EndotheAging is accompanied by a decline in the healthy function of multiple organ systems, leading to increased incidence of mortality from many diseases. Oxidative stress and elevated ROS (Reactive oxygen species) has been implicated in the mechanism of senescence and aging; they are also involved in cancer, diabetes, neurodegenerative, cardiovascular and other diseases,17 Overp2 i in ECs nearby the lesion site i within lesioned endothelium, there is no report of injury-induce NO release in blood vessels. Understanding the signal transduction machinery leading to endothelial activation and NO synthesis might be clinically relevant to design alternative strategies to restore endothelial integrity and prevent ISR.Nitric oxide (NO), a gasotransmitter that may be synthesized and released by ECs ,19, mighlum (ER) ,22. Thislum (ER) . Cholestlum (ER) . That NOlum (ER) . Moreoveion site ,27. The ll types -31. Cxs ll types ,33. Desp2+-sensitive fluorochrome, and DAF-FM diacetate, a NO-sensitive fluorescent dye. We provided, for the first time, the evidence that mechanical injury induces a long lasting NO production (NOP) in cells nearby the lesion site which requires Ca2+ entry through uncoupled Cx37Hcs, Cx40Hcs, and Cx43Hc. These results might aid in developing novel pharmacological treatments devoted to restore endothelial integrity upon invasive medical interventions.In the present investigation, we sought to determine whether NO synthesis occurs in ECs facing the injury site of excised rat aorta. We further aimed at elucidating the signal transduction pathway responsible for NO release by lesioned endothelium. These goals were accomplished by loading ECs with Fura-2/AM, a CaWistar rats aged 2-3 months were sacrificed with an overdose of diethyl ether. The thoracic and abdominal aorta were dissected out and perfused with physiological salt solution (PSS). The vessel was cleaned of the surrounding connective tissue, cut in ~5 mm long rings, stored in PSS at room temperature (22-24 °C) and used within 5 hours. All the animal protocols were approved by the East Tennessee State University's Animal Care and Use Committee. All experiments conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The animal handling was under the continuous control of the Veterinary Surgeon of the University of Pavia.2, 1 MgCl2, 10 Glucose, 10 Hepes. In Ca2+-free solution (0 Ca2+), Ca2+ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. Aortic rings were bathed in 0 Ca2+ for no longer than 90 sec before inducing the injury. Control experiments have demonstrated that such a short pre-incubation period is not able deplete intracellular Ca2+ stores i and NO measurements[Ca2+]i in intact endothelium has been previously described i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed "ratio"). An increase in [Ca2+]i causes an increase in the ratio. Ratio measurements were performed and plotted on-line every 5 s. The experiments were performed at room temperature (21-23 °C).The technique used to evaluate changes in phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) was purchased from Calbiochem . All other chemicals were purchased from Sigma. Cx-mimetic peptides, including ectively ,19. Thei2+]i occurring within the ECs adjacent to the lesion site and which have lost their contact with the scraped cells. The Ca2+ signal consisted in an initial transient peak, mainly due to InsP3-dependent Ca2+ release, followed by a prolonged decay phase caused by Ca2+ entry from the extracellular milieu i elevation or the rate of its decay phase to the baseline. This result concurs with previous evidence about the efficacy of gap junction blockers as selective uncoupling drugs in rat aortic rings i induced by the injury and does not spread farther away  affected the accompanying increase in DAF-FM fluorescence strongly hint at the endothelial origin of the NO signal. Nevertheless, the possibility that NO synthesis also takes place within the tunica media of damaged rat aortic rings cannot be ruled out.The changes in DAF-FM fluorescence measured from the endothelial layer could also reflect some NOP occurring within the underlying SMCs, which generate a Cal injury . This Caund edge . However+ influx . As a co2+-dependent eNOS activation at the wound edge of rat aortic endothelium. However, the decay phase of the Ca2+ response to injury is not significantly affected by 1 hour pre-treatment with Cx-mimetic peptides. This feature concurs with the highly localized, sub-membranal nature of the Ca2+ signal engaging eNOS in vascular ECs i that features the decay phase of the Ca2+ response to injury, therefore, suggests that Cx isoforms other than Cx 37, 40, and 43 are expressed in the native endothelium of rat aorta. This hypothesis is strongly supported by the recent discovery of Cx32 mRNA and protein in both cultured and blood vessel ECs [2+ influx when transfected in HeLa cells [2+ entry across the plasmalemma are Cx26 [3+ and heptanol, which block CxHcs and impair injury-induced Ca2+ entry in the intact endothelium of rat aorta [2+ response to mechanical injury in rat aortic endothelium. It is worth of noting that Cx-mimetic peptides, either separately or in combination, caused an increase in the InsP3-dependent initial Ca2+ peak. A recent study reported that CxHcs may mediate InsP3 efflux into the extracellular space [3 produced at the wound edge from moving out of the cytosol, thus increasing the extent of Ca2+ release in the first raw of cells.The results presented in the current investigation support a role for Cx37Hcs, Cx40Hcs, and Cx43Hcs in driving the Caular ECs ,58, whiclsewhere , the phassel ECs . Cx32HcsLa cells  and in MLa cells . Additioare Cx26  and Cx45are Cx26 . That Cxare Cx26  and by Sare Cx26 . Both grare Cx26 . Howeverare Cx26 . Moreoveare Cx26 , Px hemiat aorta . Accordiar space . It is, 2+ entry via Cx37Hcs, Cx40Hcs, and Cx43Hcs. Our data indicate that the membranal compartmentalization of CxHcs and eNOS depends on is enabled by caveolae. Experiments are under way in our laboratory to assess the role played by CxHcs-induced NO synthesis in the regeneration of rat aortic endothelium. These results outline the importance of designing novel prosthetic devices able to release NO after vascular interventions, such as arterial bypass grafting and intravascular stent deployment [2+ influx at the lesion site [In conclusion, this study provides the first clear-cut evidence that arterial damage activates surviving ECs to produce NO by inducing Caployment . Indeed,ployment . Alternaion site . These sion site ,86. In pion site .The authors declare that they have no competing interests.RBR: conceived the study, carried out the experiments, analyzed and interpreted the data, drafted the manuscript. JEAC: conceived the study, carried out the experiments, analyzed and interpreted the data. AR: conceived the study, carried out the experiments, analyzed and interpreted the data. ADC: critically revised the manuscript. MPC: critically revised the manuscript. SM: critically revised the manuscript. GG: conceived the study and critically revised the manuscript. FM: conceived the study, analyzed and interpreted the data and drafted the manuscript. FT: conceived the study, analyzed and interpreted the data, critically revised the drafted manuscript. All authors read and approved the final manuscript.RBR: Professor of Physiology at Benemérita Universidad Autónoma de Puebla. JEAC: Researcher at University of Pavia. AR: Postdoctoral Fellow at University of Calgary. ADC: Assistant Professor of Cardiac Surgery at Second University of Naples. MPC: Assistant Professor of Anatomy at University of Naples "Federico II". SM: Full Professor of Anatomy at University of Naples "Federico II". GG: Assistant Professor of Anatomy at University of Molise. FM: Assistant Professor of Physiology at University of Pavia. FT: Associate Professor of Physiology at University of Pavia.


FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs) of acute myeloid leukemia-M5 (AML-M5), R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene,  Acute monocytic leukemia, also named acute myeloid leukemia-M5 (AML-M5), is one of the most common subtypes of AML defined by the French-American-British (FAB), which is comprised by more than 80% of monoblasts (AML-M5a) or 30–80% monoblasts with (pro)monocytic differentiation (AML-M5b). Responses to chemotherapy and prognosis in AML-M5 patients are varied. The recently proposed World Health Organization (WHO) classification attempts to associate the prognostic variance with cytogenetic abnormalities It is necessary to evaluate the chromosomal changes in a whole genomic level. However, banding analysis, currently as the first choice to evaluate the genomic abnormalities of AML, is frequently to get inconclusive results due to chromosome condensation, imperfect banding, and the presence of few metaphases. FISH is a more sensitive technique, but is limited by the genomic coverage properties. Array CGH provides some distinct advantages including the potential for comprehensive genomic profiling and the ability to delimit the boundaries of specific genomic aberrations. Recently, it is considered as a powerful method to analyze genomic imbalances in hematopoietic malignancies, especially to determine cryptic and recurrent anomalies not observed by routine techniques The primary aim of present study is to assess, by means of array CGH assay, the possible existence of unbalanced chromosomal abnormalities, and delineate the copy number alterations (CNAs) in 24 cases of adult AML-M5 patients. Besides this, recurrent CNAs harboring cancer genes are also analyzed. The novel candidate oncogenes and tumor suppressor genes aberrations which might be implicated in the leukemogenesis of AML-M5 were revealed in this study.Karyotypes were available for 16 AML-M5 cases . 11 of tMLL and 5q35.3 carrying AGXT2L2 and COL23A1 were detected in case #8 and case #16, respectively. 12 recurrent CNAs were mapped in A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb were recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y . The cryBy array CGH, 3q26.2-qter was identified as a recurrent chromosomal anomaly. To investigate whether the gain of 3q26.2-qter, was closely associated with AML-M5 or other subtypes of AML, FISH with EVI (3q26.2) break-apart probe was hybridized in bone marrow cells of 69 cases of de novo AMLs . Normal FOXN3 gene at 14q were observed in two cases (case #13 and #24) (FOXN3 in a cohort of acute leukemia samples including 78 AMLs and 19 ALLs (FOXN3 was found in the AML group (0. 90±0.12) (p<0.05) (FOXN3 was detected in the ALL group (1.16±0.65), but the expression was not significantly different from the controls nor the AML groups (p>0.05)  . We subs 19 ALLs . In comp(p<0.05) . No sign(p<0.05) . A low t(p>0.05) . Gain ofcase #18 . Consist9/L, hemoglobin concentration <80 g/L, platelet count <50×109/L, higher percent of blast cells in bone marrow (comparing to the average blast ratio in bone marrow) and responses to treatment, such as number of CR. By statistic analysis, no significant difference was observed between the two groups  count >10×10o groups .In the last few years, the classification of AML has developed from a single morphological level to a MICM basis. As one of the diagnostic methods, genetic abnormality examination plays an important role in further sub-classification of AML and gives more clues to therapy selection and prognosis RPN1 gene at the breakpoint of 3q21.3 juxtaposing to EVI1 gene at the breakpoint of 3q26.2 is the key mechanism of this type of AML. However, the significance of 3q26.2-qter amplication is not well known in AML although it is considered as a recurrent chromosomal abnormality in several solid tumors EVI gene by array CGH. However, neither its recurrence in AML nor its specific association with AML-M5 was confirmed by FISH. There's limitation on the confirmatory FISH analysis since it only focus on EVI gene. Recent study by Hussenet and his colleagues reported that SOX2 is an oncogene activated by recurrent 3q26.3 amplifications in human lung squamous cell carcinomas Array CGH improves the detectable rate of chromosomal abnormalities, which make it easier to map the recurrent chromosomal aberrations of AML-M5. As a subtype of AML, it seems that AML-M5 carries both the common recurrent CNAs which have been reported in the previous AML studies and novel recurrent CNAs which are noted as candidate AML-M5 associated CNAs. However, whether these novel recurrent CNAs are specially associated with AML-M5 are needed to be clarified. Gain of 3q26.2-qter is the sole large recurrent chromosomal change in the present study. There is an overlap between 3q26.2-qter in case 12 and 3q21.1-qter in case 18. It is known that 3q21q26 anomaly is the most common karyotype in acute myeloid patients with 3q abnormalities FOXN3 deletion is observed in leukemia. The FOXN3 acts as a transcriptional repressor and has been implicated in DNA damage-inducible cell cycle arrests in saccharomyces cerevisiae FOXN3 in mamals are not known. Reduced expression of FOXN3 has been reported in several cancers, such as renal cell carcinoma and oral squamous cell carcinoma et al. found that FOXN3 recruited SKIP, which played a role in cell growth and differentiation through the TGF-β pathway and repressed genes important for tumorigenesis FOXN3 gene is recurrent in this study, suggesting that FOXN3 possibly serves as a novel TSG and contributes to the leukemogenesis. However, subsequent expression study suggested that reduced expression of FOXN3 was not only presented in AML-M5 but also other subtypes of AML, indicating that the FOXN3 encodes a myeloid marker rather than a specific leukemogenic protein of AML-M5. The MLL gene is reported to be highly associated with AML with complex karyotypes MLL frequently co-exists with the deletion of CDKN2A. CDKN2A encode cyclin-dependent kinase inhibitor, which have emerged as an important effecter in aging as well as a potent tumor suppressor. The main genetic alterations involving CDKN2A are deletions by bi- or monoallele or hypermethylation of 5′ GpG island in its promotor CDKN2A has been reported, but deletion of CDKN2A is rare MLL amplication and CDKN2A deletions in AML-M5 and the cooperation of these genes rearrangements in leukemogenesis of AML-M5 needs to be further clarified.By the advantages of array CGH, the candidate genes in the cryptic recurrent CNAs have been singled out. It is the first time that Due to the limitation of case number, no statistical difference was shown between the number of CNAs and clinico-biological parameters in our analysis. More cases are necessary to evaluate the clinical significance of the number of CNAs.In summary, we genetically defined AML-M5 by karyotype, FISH and array CGH. Array CGH is necessary to complement the traditional cytogenetic analysis in revealing the cryptic genomic alterations in AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs coexisting in AML and novel CNAs in AML-M5. The recurrent CNAs harbor oncogenes or tumor suppressor genes. Further studies are needed to understand the role of these genes in the leukemogenic network of AML.This research project was approved by both the Ethical Committee of the First Affiliated Hospital of China Medical University and the Institutional Review Board (IRB) at the University of Oklahoma Health Sciences Center (OUHSC) (IRB #13100). Before data collection a written informed consent from was obtained from each participant. Bone marrow samples were collected from 24 adults with AML-M5  who referred to the First Affiliated Hospital of China Medical University between May 2009 and December 2010. The patients aged <14 years old were excluded in this study. The diagnosis was made according to the French-American-British (FAB) Cooperative Group criteria Twenty two patients were newly diagnosed and received a single course of daunorubicin plus cytarabine (DA) or idarubicin plus cytarabine (IA) regimen for induction treatment. For the patients who failed to achieve complete remission (CR) or relapsed from CR, salvage therapy was used, including low-dose cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) (CAG) or fludarabine, cytarabine and G-CSF (FLAG) regimen. Intermediated dose cytarabine (IDAra-C) ± antharcycline, mitoxantrone plus cytarabine (MA) and DA were chosen as postremission therapy for patients achieved CR and it lasted for 6–8 courses. No further therapy was applied to patients remaining in remission after postremission therapy. The details of each regimen were listed in 9/L, platelet count ≥100×109/L, no myeloid blasts in the peripheral blood and no extramedullary disease. Patients who did not fulfill the above criteria were defined as unremission. Relapse was defined as the reappearance of more than 5% blasts in bone marrow aspirates or extramedullary leukemia in patients with a previously documented CR.For response assessment, bone marrow aspiration was performed between days 21 and 28 after induction therapy. CR was defined as a bone marrow with normal hematopoiesis, blasts in bone marrow less than 5%, granulocyte count ≥1.0×10Bone marrow mononuclear cells (MNCs) were isolated by Ficoll-paque gradient centrifugation , aliquoted into fetal calf serum (FCS) with 10% DMSO, and cryopreserved in the liquid phase of a liquid nitrogen tank.5 to 2×106 blasts with Trizol , and resuspended in 50 μl DEPC-treated water. Complementary DNA was made from ∼20 ng of RNA using the SuperScript III first strand synthesis kit and oligo-dT priming .Liquid nitrogen-cryopreserved (liquid phase) bone marrow mononuclear cells were washed and recovered by centrifugation. Following that, DNA was extracted with phenol-chloroform, and precipitated with ammonium acetate, ethanol and glycogen. RNA was prepared from approximately 2×10Short-term cultures of unstimulated bone marrow samples were prepared and fixed according to our standard laboratory protocols. Karyotype analysis was performed by R banding technique. Chromosome study could not been performed on six bone marrow samples due to the limitation of cell number. The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature.Array CGH analysis was performed on all DNA samples obtained from 24 patients with AML-M5 following the manufacturer's protocol with minor modifications, . The array consisted of approximately 720,000–mer oligonucleotide probes that spanned both coding and noncoding sequences with average genome coverage of approximately 2 kilobases. A commercially available pooled normal control DNA was used  for reference. The patients and the reference DNA were labeled with either Cyanine 3 (Cy-3) or Cyanine 5 (Cy-5) by random priming  and then hybridized to the chip via incubating in the MAUI hybridization system . After 40-hours hybridization at 42°C, the slides were washed and scanned using a GenePix 4000B . NimbleScan version 2.4 and the SignalMap version 1.9 were applied for data analysis . The genomic locations were retrieved from National Center for Biotechnology Information (NCBI) build 36 (hg 18). Frequently affected regions recently detected as copy number polymorphisms were excluded from data analysis according to the Chromosome Number Variation (CNV) database in our lab and genomic variants in human genome (Build 36: Mar. 2006). The genomic data of array CGH are available in NCBI Gene Expression Omnibus (GEO) under accession number GSE53429.Confirmatory FISH analysis was performed using different probes. (1) vysis LSI MLL dual color break apart rearrangement probe and vysis LSI CDKN2A (9p21)/CEP9 probe  on case #18. (2) EVI t inv(3) Break, Dual-Color Probe  on 69 de novo AML patients' bone marrow cells. The FAB subtypes and karyotypes of the patients were showed in FOXN3 on bone marrow MNCs of 97 patients with acute leukemia and 16 normal controls who had normal myelograms . Hybridization with TEL/AML1 probe demonstrated an extra red signal in interphase (E) and translocation of chromosome 21 in metaphase (F). (G) Arrow indicated the derivative inv(3) showed by R-banding. FISH with EVI(3q26) probe showed two fusion signals plus an extra red signal (TEL'EVI1) in interphase (H) and inv(3) with a variant breakpoint of EVI gene in metaphase (I). Figure S2, The comparisons of FOXN3 Levels in health control, ALL and AML. The significantly reduced FOXN3 level was observed between AML and health controls. *: p<0.05. The FOXN3 level was lower in ALL than health control whereas no significantly difference was observed. Figure S3, The comparisons of FOXN3 Levels in health control, M5 and non-M5. No significantly difference was observed on the FOXN3 levels between AML-M5 and non-AML-M5 (p>0.05). Table S1, The results of karyotype and array CGH in 24 AML-M5. Table S2, FAB subtypes, karyotypes and FISH with EVI (3q26) probe analysis on 69 de novo AML patients. Table S3, Clinical information and FOXN3 expression levels of 97 acute leukemia samples and 16 normal controls. Table S4, The clinical characterizations of 24 cases of AML-M5. Table S5, Drug dose and duration of chemotherapy. Table S6, Primers in qRT-PCR for FOXN3.Figure S1, 3q anomaly identified by karyotype and FISH in two of the 69 de novo AMLs. (A)–(F) showed t in case with ID 37 and (G)–(I) showed inv(3) in case with ID 65. Arrows indicated the der(3)t (A) and der(21)t (B) observed by R-banding. FISH with EVI(3q26) probe showed two fusion signals plus an extra red signal (TEL'EVI1) in interphase (C) and translocation of chromosome 3 with a variant breakpoint of (7Z)Click here for additional data file.


Fish, unlike terrestrial vertebrates, obtain Ca2+ from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca2+ uptake functions to cope with fluctuating Ca2+ levels in aquatic environments. Cortisol was previously found to regulate Ca2+ uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca2+ fresh water stimulated Ca2+ influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca2+ influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca2+ transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca2+ uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca2+ uptake, the involvement of vitamin D3 was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D3 in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca2+ uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D3 signaling to stimulate Ca2+ uptake in zebrafish.Cortisol was reported to downregulate body-fluid Ca CSs, which consist of glucocorticoids (GCs) and mineralocorticoids (MCs), are vital hormones for mammals, and are involved in regulating the osmolality and ion levels, body fluids, energy metabolism, respiration, and immune reactions CS is synthesized in the adrenal cortex of mammals, but in the interrenal tissue of the head kidneys in teleosts. Physiological functions of CSs in teleosts are similar to those in mammals, and CS signaling is also mediated by the GC receptor (GR) and MC receptor (MR), which are ligand-activated transcription factors 3 was a vital calcitrophic endocrine to regulate Ca2+ homeostasis in vertebrates. Liver vitamin D-25hydroxylase (CYP27A1) converts vitamin D3 precursor to 25-hydroxyvitamin D3 (25(OH)D3), which is then converted to 1,25-dihydroxytamin D3 2D3), the active form of the vitamin D3, by renal 1α-OHase (CYP27B1) 3 spreads its function through its receptor, vitamin D3 receptor (VDR). VDR is a ligand-active transcription factor, and duodenal trpv6 (ecac) is the target of the vitamin D3-VDR complex in mammals trpv6 expression by vitamin D3 is one of important pathways to enhance Ca2+ uptake in mammals 3 had been demonstrated to elevate serum Ca2+ level 3 was also proposed to be associated with Ca2+ transport in the gills based on the vitamin D3 deficiency-induced impairment of growth and mineralization in the fish skeleton 2D3 or mRNA expression of gill/kidney VDR have been observed in salmon undergoing smoltification and migrating from freshwater (low Ca2+ concentrations) to seawater (high Ca2+ concentrations), suggesting that synthesis of the sterol and its receptor might be regulated depending upon ambient Ca2+ concentrations 3 metabolism although the actions varied depending on species 3 and Ca2+ homeostasis in mammals; however, it is unknown whether this connection is also developed in fish.Vitamin D2+ transport 2+ mainly via absorption from the environment rather than by dietary means. In adult fish, the predominant route of Ca2+ entry from the environment is across the gill epithelium while in larvae, the body skin is the major route of Ca2+ uptake before full development of the gills occurs 2+ uptake function is well regulated for maintaining the internal Ca2+ homeostasis during acclimation to aquatic environments with fluctuating Ca2+ levels (<0.01 mM in soft fresh water to >10 mM in seawater) 2+ transport is carried out through the operation of apical epithelial Ca2+ channels , and basolateral plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) 2+ transport 2+ uptake in teleosts; however, it is still unclear if cortisol controls only ECaCs or other Ca2+ transporters (NCX and PMCA), and if this control pathway is mediated by the GR, MR, or both. Elucidation of these issues would enhance our understanding of the molecular physiological mechanisms of cortisol's control of epithelial Ca2+ transport, an essential component associated with bone structure and formation in vertebrates.In teleosts, cortisol is well demonstrated to regulate the mechanisms of ionic and osmotic balance, but only few studies investigated the role of cortisol in controlling transepithelial Ca2+ uptake function 2+ uptake mechanisms. The present study attempted to address 3 specific questions: (I) Does cortisol control zebrafish Ca2+ uptake function by regulating the ecac, ncx1b, and/or pmca2? (II) Does cortisol control zebrafish Ca2+ uptake function through mediation by the GR, MR, or both receptors? (III) Does cortisol control zebrafish Ca2+ uptake function by regulating vital calcitrophic endocrine suchlike vitamin D3? The effects of environmental Ca2+ levels and exogenous cortisol on Ca2+ contents and influx, the mRNA expressions of Ca2+ transporters , the steroidogenesis enzymes 11β-hydroxylase and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), CS receptors (gr and mr) and vitamin D3-related genes  were investigated. Moreover, effects of knockdown of the GR or MR on Ca2+ contents and influx, and the expression of Ca2+ transporters and vitamin D3-related genes in zebrafish embryos were examined.Recently, zebrafish have become a model for research on ion regulation and related endocrine controls due to the well-developed genetic database and applicability of various molecular physiological approaches Danio rerio) were kept in local tap water ([Ca2+] = 0.2 mM) at 28.5°C under a 14∶10-h light-dark photoperiod at the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee .Zebrafish (2+ (0.02 mM) levels were prepared with double-deionized water  supplemented with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. Ca2+ concentrations  of the high- and low-Ca2+ media were 2 and 0.02 mM, respectively, but the other ion concentrations of the 3 media were the same  as those in local tap water. Variations in ion concentrations were maintained within 10% of the predicted values. Fertilized zebrafish eggs were transferred to high- and low- Ca2+ media, respectively, and incubated thereafter until sampling at 3 d post-fertilization (dpf). The sampling time in this study was based previously Artificial fresh waters with high- (2 mM) and low-CaFor cortisol incubation experiments, we based cortisol dosage from previous study 3 (13.1 N) was added to samples for digestion at 60°C overnight. Digested solutions were diluted with double-deionized water, and the total Ca2+ content was measured with a Z-8000 atomic absorption spectrophotometer . Standard solutions  were used to make the standard curves.Zebrafish embryos were anesthetized with MS-222 (Sigma) and then briefly rinsed in deionized water. 30 individuals were pooled as 1 sample. HNO45Ca2+ -containing medium for a subsequent 4-h incubation. After incubation, embryos were washed several times in isotope-free water medium. Six embryos were pooled into 1 vial, anesthetized with MS-222, and digested with tissue solubilizer  at 60°C for 8 h. The digested solutions were supplemented with counting solution , and the radioactivities of the solutions were counted with a liquid scintillation beta counter . The Ca2+ influx was calculated using the following formula: Jin = Qembryo Xout−1t−1 W−1; where Jin is the influx (pmol·mg−1·h−1), Qembryo is the radioactivity of the embryo  at the end of incubation, Xout is the specific activity of the incubation medium (cpm/pmol), t is the incubation time (h), and W is the average body wet weight of different-stage embryos (mg).By following previously described methods g for 30 min, the supernatants were obtained. The samples were then mixed with an equal volume of isopropanol. Pellets were precipitated by centrifugation at 4°C and 12,000×g for 30 min, washed with 70% alcohol, and stored at −20°C until use.After anesthetized with 0.03% MS222, appropriate amounts of zebrafish tissues or embryos were collected and homogenized in 1 ml Trizol reagent , then mixed with 0.2 ml chloroform, and thoroughly shaken. After centrifugation at 4°C and 12,000×20, 250 ng random primers, 5 mM dithiothreitol, 40 units RNase inhibitor, and 200 units Superscript RT (Invitrogen) for 1 h at 50°C followed a 70°C incubation for 15 min. For PCR amplification, 2 µl cDNA was used as template in a 50-µL final reaction volume containing 0.25 mM dNTPs, 2.5 units Taq polymerase , and 0.2 µM of each primer (For complementary (c)DNA synthesis, 1∼5 µg of total RNA was reverse-transcribed in a final volume 20 µl containing 0.5 mM dNTPs, 2.5 µM oligo (dT)h primer . 30 cyclqPCR was performed with a LightCycler real-time PCR system  in a final volume of 10 µl, containing 5 µl 2× SYBR Green I Master (Roche Applied System), 300 nM of the primer pairs, and 20∼30 ng cDNA. The standard curve for each gene was checked in a linear range with β-actin as an internal control. The primer sets for the qPCR are shown in ecac or gr Fragments were obtained by PCR and inserted into the pGEM-T easy vector . The inserted fragments were amplified with the T7 and SP6 primers by PCR, and the products as templates were used for the in vitro transcription with T7 and SP6 RNA polymerase (Roche) in the presence of digoxigenin (DIG)-UTP (Roche) to, respectively, synthesize sense and anti-sense probes. Zebrafish embryos were anesthetized on ice and fixed with 4% paraformaldehyde (PFA) in a phosphate-buffered saline  solution at 4°C overnight. To do in situ hybrizization, we followed previously 2 each) on the yolk sac surface of an embryo were chosen for counting.Zebrafish 2 and 5% CO2.Adult fish were anesthetized with 0.03% MS222 and then gills were dissected and directly transferred to the pre-incubation DMEM medium (Invitrogen) containing 50 mg/ml of penicillin (Invitrogen), 50 µg/ml of streptomycin (Invitrogen) and 20% Fetal Bovine Serum (Invitrogen). Individual gill arches were carefully separated from the whole gill structure. Each gill arch was cut lengthwise, and the cut filament was designated as one sample to be incubated in a well (96-well). The cut gill filaments were incubated with the freshly prepared pre-incubation medium (control group) and the DMEM with supplementary 20 mg/l cortisol, respectively. The media were freshly prepared and replaced twice per day. Organ culture was carried out at 28°C for 1 d in 96-well culture plates in a humidified chamber supplied with 95% O5′-GTATCTTTTAGTCTCCAT-3′) and GR MO (5′-TCCAGTCCTCCTTGATCCAT-3′) were prepared with 1× Danieau solution 2, and 5.0 mM HEPES; pH 7.6). A standard control MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as the control. To confirm MO specificity, fragments of the GR and MR containing the MO-targeted sequences were PCR-amplified with gene-specific primers  were injected into embryos at the 1∼2 cell stage, and embryos were sampled at 3 dpf.To rescue the defects caused by the MO, a full-length GR was PCR-amplified with a specific primer  and clonThirty embryos were pooled as one sample and homogenized. Protein of 50 mg/well was loaded to a 10% SDS-PAGE at 100 V for 2 h. After separation, proteins were transferred onto polyvinylidene difluoride membrane (Millipore) at 100 V for 2 h. After being blocked for 1.5 h in 5% nonfat milk, the blots were incubated with GR (Santa Cruz Biotechnology) or MR polyclonal antibody (Abcam) overnight 4°C, diluted 1∶500 and with an alkaline-phos-phatase-conjugated goat anti-rabbit IgG  for another 2 h. The blots were developed with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium.Fresh zebrafish gills were fixed with 4% PFA at 4°C for 3 h and then immersed serially in PBS containing 5, 10, and 20% sucrose for 15 min at room temperature. Finally, gills were soaked in a mixed PBS solution (OCT compound: 20% sucrose at 1∶2) overnight and then embedded with OCT compound embedding medium  at 20°C. Cryosections at 6 µm were made with a cryostat , and these were placed onto poly-L-lysine-coated slides .Prepared slides were rinsed in PBS and blocked with 3% BSA for 30 min. Afterward, the slides were first incubated with an α5 monoclonal antibody against the α-subunit of the avian Na,K-ATPase (NKA)  overnight at 4°C. The slides were washed twice with PBS and incubated with an Alexa Fluor 568 goat anti-mouse IgG antibody  for 2 h at room temperature. Images were acquired with a Leica TCS-NT confocal laser scanning microscope (Leica) or an Axioplan 2 imaging microscope.ecac genomic sequence was obtained from a zebrafish genome database (http://www.ncbi.nlm.nih.gov/projects/genome/guide/zebrafish/). Potential regulatory elements upstream of the ecac gene were predicted by Genomatix MatInspector (http://www.genomatix.de).The zebrafish t-test.Data are presented as the mean±SD and were analyzed by one-way ANOVA and Student's mr and gr mRNAs were universally expressed in all tissues studied (gr was first detected at 1 h post-fertilization (hpf) and throughout development; however, mr mRNA only began to be expressed at 12 hpf , cortisol-treated groups (20 and 40 mg/l) showed significant increases in Ca2+ content and influx  and gr and stimulated hsd11b2, but did not affect that of the mr  revealed signals of GFP translation , confirm2+ content and influx in 3-dpf zebrafish embryos, but the MR MO had no effects  after injections with the MOs. Compared to the control group (control MO injection without cortisol), both groups of the control MO with cortisol and the MR MO with cortisol exhibited a significantly higher Ca did not . Similarcortisol .2+ uptake mechanism, the zebrafish GR-SB MO and/or GR cRNA were co-injected into zebrafish 1∼2-cell-stage embryos. Compared to the control MO-injected group, GR-SB MO, similar to GR MO, also caused significant decreases in both Ca2+ influx and ecac mRNA expression in 3-dpf zebrafish embryos  or 0.02 mM (low) Ca2+ medium. Compared to the control MO in low Ca2+ medium, the GR morphants in low Ca2+ medium were significantly lower in the ECaC mRNA expression at 3 dpf, but the MR morphants in low Ca2+ medium were similar to the control group  was used to treat zebrafish embryos. To trace the effect of exogenous cortisol, 1- and 3-dpf embryos were sampled. Cortisol caused differential effects on mRNA expressions of ecac and the vitamin D3-related genes in 1-dpf or 3-dpf zebrafish embryos. According to the qPCR, cortisol significantly stimulated ecac and cyp27b1 mRNA expression in 1-and 3-dpf zebrafish embryos; however, cortisol only significantly stimulated the mRNA expressions of 3 receptor avitamin D (vdra), cyp27a1l and cyp27a1in 1- or 3-dpf embryos  expression of zebrafish 11β-hydroxylase was stimulated by low-Ca2+ environment; (II) exogenous cortisol increased zebrafish Ca2+ influx and content through upregulating the expression of ecac but not those of ncx1b or pmca2; (III) exogenous cortisol probably through a feedback pathway modulated the mRNA expression of 11β-hydroxylase, hsd11b2 and gr but not that of mr in zebrafish; and (IV) translational knockdown of GR but not MR caused defects in the binding of cortisol or mRNA expression of vitamin D3-related genes, and thus impaired the ecac expression and Ca2+ uptake function of zebrafish ECaC via the GR, a regulatory pathway that could be mediated by vitamin D3.Glucocorticoid (cortisol) showed different impact in Ca2+ from the aquatic environment with fluctuating Ca2+ levels, and therefore the internal Ca2+ homeostasis is impacted by environmental Ca2+ levels. Fish (at least teleost) bone is acellular, and thus the bone does not provide a pool for Ca2+ as it does in terrestrial vertebrates 2+ homeostasis and bone formation , fish have to regulate the function of Ca2+ uptake to cope with a fluctuating environment. Like other teleosts, zebrafish can enhance Ca2+ uptake function by stimulating ECaC expression during acclimation to low-Ca2+ fresh water 2+ fresh water caused an increase in serum cortisol levels in rainbow trout. The present study further explored the mechanism behind this phenomenon. Low-Ca2+ fresh water stimulated the mRNA expression of 11β-hydroxylase, the enzyme in the final step of cortisol synthesis, which suggests that environmental conditions affect steroidogenesis and thus cortisol levels in zebrafish. All these results imply a possible role of cortisol in the control of Ca2+ uptake. To test this hypothesis, we treated zebrafish with exogenous cortisol. Similar to trout and eel ecac and Ca2+ influx, and these functional enhancements resulted in increased Ca2+ contents in the whole body (the present study). The present comprehensive data from molecular to the physiological level demonstrated the calciotropic effects of cortisol, and these effects showed a dose-dependent pattern and were of physiological significance.Fish mainly obtain Ca2+ uptake function through the operations of the 3 transporters in zebrafish 2+ transporters (NCX and PMCA). The present study first reports that exogenous cortisol affected ecac only but not those of ncx1b or pmca2; similar result found in cultured gills with exogenous cortisol  and simultaneously stimulated that of hsd11b2 (increasing the cortisone level), reflecting a feedback mechanism in controlling levels of corticoids. In mammals, HSD11B2 converts cortisol to cortisone in MR-specific tissues, and this prevents cortisol binding to the MR in these tissues and thus allows aldosterone to bind to the MR hsd11b2 by exogenous cortisol treatment may be a feedback to control cortisol levels in zebrafish. This notion was further supported by the data of 11β-hydroxylase and the gr; gene expressions of zebrafish 11β-hydroxylase and the gr were inhibited by exogenous cortisol treatment. In mammals, dexamethasone treatment suppressed the secretion of the adrenocorticotrophic hormone, resulting in a decline in serum corticosterone levels gr expressions were significantly downregulated by dexamethasone treatment gr in gills and liver 2+ mechanism through sophisticated feedback pathways, in which the expressions of 11β-hydroxylase, hsd11b2, and CRs are differentially modulated.In the present study, exogenous cortisol suppressed the mRNA expression of 2+ uptake in teleosts 2+ uptake function through the GR but not the MR. In addition to experiments of exogenous cortisol treatment, we used a gene-specific MO to abolish endogenous cortisol signaling in zebrafish and directly explored the effect of cortisol-signaling defects on the Ca2+ uptake function. Translational knockdown of the MR did not affect Ca2+ uptake in zebrafish, but GR knockdown evidently impaired the Ca2+ uptake mechanism by decreasing both Ca2+ influx and content. Moreover, these defects in the Ca2+ uptake mechanism were due to suppression of ecac expression but not the expressions of ncx1b or pmca2. GR defect was also demonstrated to downregulate ecac -expressing cell density in skin of zebrafish. These results indicated that the target of the GR is the ecac. Interestingly, the Ca2+ influx, ecac mRNA and ecac -expressing cell density in MR MO-injected zebrafish morphants could still be stimulated (compared to the control) by exogenous cortisol, suggesting that the effects of exogenous cortisol on Ca2+ uptake function is not through the MR. On the other hand, exogenous cortisol did not cause further stimulation (compared to the control) in Ca2+ influx or ecac mRNA in GR morphants, but successfully rescued the Ca2+ uptake functional defects caused by the GR MO. Similar effect was also found in GR MO-injected zebrafish with low Ca2+ freshwater. GR MO morphant can abolish stimulation of low Ca2+ freshwater on ecac mRNA expression, but MR MO morphant can not. Reinforcing these results, overexpression of the GR by injection with GR cRNA rescued the Ca2+ uptake mechanism that was impaired in GR MO morphants. This evidence strongly suggests that endogenous cortisol stimulates Ca2+ uptake through the GR, but not the MR, in zebrafish.Physiological functions of cortisol signaling are mediated by the GR and MR, which are ligand-activated transcription factors. In many previous studies, exogenous cortisol treatment was found to stimulate Ca+/Cl− vs. Ca2+) and species , and clarification of this point requires further studies. On the other hand, it was noted that zebrafish MR expression was not regulated by exogenous cortisol in the present study. Teleosts might not synthesize aldosterone, and DOC was proposed to play a similar role as aldosterone Some previous in vitro studies indicated that the trout MR and GR could bind cortisol and stimulate transcriptional activity in the mammalian cell lines transfected with a reporter plasmid 2+ absorption in chickens 2+ uptake and trpv6 (ecac) gene expression in mice trpv6, and suggested that dexamethasone might not directly regulate trpv6 transcription, but instead, downregulates the Ca2+ uptake function through other hormones. Based on a bioinformatics analysis, there are some putative GREs in the promoter region of the zebrafish ecac  in the promoter region of ecac3 was also reported to elevate the serum Ca2+ level ecac promoter region 2D3 treatment ecac mRNA expression by1,25(OH)2D3 in zebrafish embryos. Taken together, vitamin D3-VDR control of ecac expression and function may also exist in fish. However, the associations between cortisol, vitamin D3 and Ca2+ handling in fish were not clear until the present study. CYP27A1 is an enzyme to synthesize 25(OH)D3, the vitamin D3 precusor, and subsequently 25(OH)D3 is converted to 1,25(OH)2 D3 (ctive vitamin D3) by CYP27B1 cyp27b1, cyp27a1 and/or cyp27a1 like (cyp27a1l) at 1- and 3 dpf, and also affected vdra mRNA expression at 1 dpf. These implied that cortisol starts affecting the vitamin D3-VDR signaling from early stage. Cortisol could not only stimulate synthesis of vitamin D3 precursors but also accelerate the synthesis of active vitamin D3. Moreover, our subsequent knockdown experiment further reinforced this notion. Knockdown of GR, but not MR, was found to suppress the expressions of cyp27b1, cyp27a1, cyp27a1l and vdra at 1- and/or 3 dpf. According to these results, we suggested that cortisol probably regulates Ca2+ handling through vitamin D3-VDR system, besides directly regulating ecac expression.In addition to direct regulation of 2+ uptake in zebrafish. Cortisol appears to cause different effects on Ca2+ uptake between teleosts and higher vertebrates. Sources of Ca2+ fundamentally differ among vertebrates. For terrestrial vertebrates, food is the major source of Ca2+, and the intestines serve as a primary site for Ca2+ uptake. However, fish are constantly facing aquatic environments with variable Ca2+ concentrations (of as low as <0.01 mM in soft fresh water), and gills or the skin serve as the primary site for Ca2+ uptake 2+ uptake, which is a challenging issue to be explored from an evolutionary point of view.In this study, cortisol was suggested to directly or indirectly stimulate CaTable S1Primers for the RT-PCR analysis.(DOC)Click here for additional data file.Table S2Primers for the qPCR analysis.(DOC)Click here for additional data file.Table S3Primers for cRNA expression cloning.(DOC)Click here for additional data file.Figure S1Effect of exogenous cortisol on mRNA expression of ecac, pmca2 and ncx1b in cultured gills.ecac mRNA expression was analyzed by qPCR and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 5).(TIF)Click here for additional data file.Figure S2Upstream regulatory region of the zebrafish ecac gene. The transcription initiation sites are marked by +1, and the start codon (ATG) is marked by a square. The putative upstream regulatory elements are underlined. The core sequence of each element is shown in bold font. GRE, glucocorticoid-responsive element; VDRE, vitamin D3-responsive element; ARE, androgen-responsive element.(TIF)Click here for additional data file.Figure S3Co-localization of gr mRNA by in situ hybridization with anti-NKA using immunocytochemical analysis of zebrafish gill cryosections. (A) in situ hybridization of gr mRNA; (B) immunocytochemical staining of NKA. Arrow indicated gr mRNA and NKA protein signals at similar area. Scale bar 5 µm.(TIF)Click here for additional data file.