These Ca2+ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC-induced phosphorylation in mouse eggs, alongside Ca2+ oscillations, using fluorescent C-kinase activity reporter (CKAR) probes. Ca2+ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr2+ all caused repetitive increases in PKC-induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca2+ transients. High frequency oscillations caused by PLCζ led to an integration of PKC-induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca2+ oscillations using an isoform-specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ-specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC-induced phosphorylation outlasts each Ca2+ increase in mouse eggs but that signal integration only occurs at a non-physiological, high Ca2+ oscillation frequency. The results also suggest that Ca2+-induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.Fertilization of mammalian eggs is characterized by a series of Ca Atypical PKCs (aPKC) ζ and ι/λ are neither regulated by Ca2+ nor by DAG. Isoforms from all three subfamilies have been found to be expressed in mammalian eggs  in response to phosphorylation. CKAR consists of a pseudo-substrate that is specific to PKC fused to a FHA2 domain that binds phosphothreonine. This fusion protein is in turn flanked by a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at either end. A change in FRET between the CFP and YFP is caused by changes in CKAR conformation when the PKC-specific substrate is phosphorylated and bound by the FHA2-binding domain , and δCKAR was a kind gift from Alexandra Newton.Phorbol esters (PMA), PKC inhibitors (Gö6976 and rottlerin), ionophores (thapsigargin and ionomycin), and other chemicals were purchased from Sigma–Aldrich . CKAR and MyrPalm-CKAR were obtained from Addgene  experiments, approximately 10 µl of sperm suspension was added to the dish containing the eggs.MF1 female mice were super-ovulated by intraperitoneal injection of 7.5 i.u. of PMSG  followed 48 h later by 10 i.u. of hCG  encoding CKAR, MyrPalm-CKAR . This potential for fluorescence signal “spill-over” can distort FRET ratios. In contrast, Rhod-dextran is a long-wavelength Ca2+ indicator with a fluorescence excitation and emission maxima of 530 and 576 nm, respectively, and it is the dextran-coupled version of Rhod2 that is retained in the cytoplasm. Fluorescence was captured using a 20 × 0.75 NA objective at 10-sec intervals with a cooled CCD camera  and In Vivo software. The excitation light source was a white LED lamp . Filters (10 nm bandwidth) were controlled using filter wheels . FRET signals were measured by taking the ratio of emission at 470 and 535 nm with excitation at 430 nm. Rhod-dextran was excited with 550 nm light and emission collected at 600 nm. A multi-band filter  was placed in the dichroic filter block housing to allow excitation and emission at the selected wavelengths. Image J and SigmaPlot software  were used for data analysis. Data are plotted as the ratio of cyan fluorescence to yellow emission and values normalized to the percentage change from the start of the experiment. Specific PKC inhibitors Gö6976 and rottlerin, were used to act upon conventional PKCα and PKCβ, and the novel PKCδ isoforms, respectively.Zona-free MII eggs were kept in HKSOM under mineral oil at 37°C on the heated stage chamber of an inverted microscope . For CaCKAR and MyrPalm-CKAR cRNA were expressed in eggs as described above, and δCKAR was co-injected with PLCζ and incubated for 2–8 h. Single snapshots were taken of the subcellular distribution corresponding to CKAR/MyrPalm-CKAR/δCKAR FRET probes using a confocal microscope , under a 20× (0.75 NA) lens, and an argon laser. FRET signal was determined using the same ratiometric settings as described above and data were analyzed using Image J.The % CKAR FRET changes of individual signal increases were calculated based on the mean of three random spikes taken from each oscillating egg trace and divided by the total number of eggs. The “n” refers to the total number of eggs examined for each experiment type.We found that CKAR was effectively expressed in mature mouse eggs, following microinjection of its cRNA, as indicated by fluorescence detected in the CFP and YFP channels. PKC activity, as reflected by the phosphorylation of CKAR, was monitored by measuring the ratio of the CFP to YFP signal intensity and plotted as the percentage change over the starting ratio versus time. These data are presented as the inverse of FRET efficiency, since there is an increase in this ratio with increased phosphorylation , with either thapsigargin  or ionomycin . Thus, it is unclear whether this oscillatory CKAR phosphorylation signal change occurring upon mouse fertilization involves the direct activation of cPKCs by each Ca2+ transient.PKC-induced phosphorylation was monitored during IVF, using both CKAR and MyrPalm-CKAR, and their FRET signal change measured every 10 sec, alongside the occurrence of cytosolic CaAR probe b. The osalm-CKAR . Hence azed eggs c. Moreov2+ transients. 2+ transients during IVF at a higher time resolution. The amplitude for the cytosolic CKAR  was relatively larger than that for MyrPalm-CKAR . However, for both CKAR and MyrPalm-CKAR, the peak of the FRET transient occurred 10–30 sec after the peak of the Ca2+ signal. In addition, the CKAR response displayed a slower decline, and did not return to baseline until ∼5 min after the Ca2+ transient had finished. However, it was notable that each CKAR signal increase had returned to near baseline value prior to initiation of the next Ca2+ spike, and hence there was no sign of progressive accumulation of the CKAR signal in A consistent feature of the CKAR-mediated oscillations at fertilization was that each of the FRET transients showed a different time-course relative to the Ca2+ oscillations in mouse eggs  compared to conventional CKAR . In contrast, 2+ oscillations and similar FRET oscillatory signal increases using either cytosolic CKAR (4.97 ± 0.33%) or δCKAR (4.47 ± 0.46%). These signal changes are comparable in amplitude and pattern to those seen at fertilization. These data suggest that PKCδ responds differently to Sr2+ compared with fertilization by sperm or with PLCζ.Since PKCδ can only be stimulated by DAG and not by Canal CKAR a. In add2+ oscillations in the egg that are critical for the physiological activation of embryo development  to measure these dynamic changes alongside Ca2+ oscillations. CKAR and its subcellular-targeted derivatives have shown to be specific for monitoring PKC-induced phosphorylation and are subject to dephosphorylation by cellular phosphatases  is within the plasma membrane (Violin et al., 2+ transients. In somatic cells, agonist stimulation can lead to DAG generation in the Golgi membranes as well as the plasma membrane (Gallegos et al., 2 specifically located in internal vesicles (Yu et al., 2 hydrolysis. Therefore, it is distinctly possible that the sperm-delivered PLCζ enables Ca2+-dependent DAG formation on intracellular PIP2-containing vesicles, facilitating repetitive PKC stimulation throughout the egg cytoplasm. Further experiments could address this possibility by the use of DAG-specific probes targeted to intracellular vesicles.One of the most remarkable results of the current study was that a PKC-induced response is detected with both the plasma membrane-targeted and cytoplasmic CKAR. Previous studies of PKC in live somatic cells have shown that agonists can lead to DAG production, although PKC oscillations only occur in the plasma membrane (Oancea and Meyer,


The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.Parameters influencing live imaging and imaging of fixed unstained and Carmine Alum stained whole mounts were evaluated. Artifacts induced by light scattering of GFP and Carmine Alum signals from epithelial cells were identified in live tissue as primarily due to fat cells and in whole mount tissue as due to dense Carmine Alum staining of epithelium. Carmine Alum autofluorescence was detected at excitation wavelengths from 750 to 950 nm with a peak of emission at 623 nm (~602-656 nm). Images of Carmine Alum fluorescence differed dramatically at emission wavelengths of 565–615 nm versus 650–710 nm. In the latter, a mostly epithelial (nuclear) visualization of Carmine Alum predominates. Autofluorescence with a peak emission of 495 nm was derived from the fixed and processed tissue itself as it was present in the unstained whole mount. Contribution of autofluorescence to the image decreases with increasing laser excitation wavelengths. SHG-B versus SHG-F signals revealed collagen fibers and could be found within single fibers, or in different fibers within the same layer. These differences presumably reflected different states of collagen fiber maturation. Loss of SHG signals from layer to layer could be ascribed to artifacts rendered by light scattering from the dense TEB structures, and unless bandpass emissions were selected, contained unfiltered non-SHG fluorescence and autofluorescent emissions. Flexibility in imaging can be increased using spectral emission imaging to optimize emission bandwidths and to separate SHG-B, GFP, and Carmine Alum signals, although conventional filters were also useful.Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing. Multiphigenesis ,3. Recenigenesis -8. Biophigenesis -16.Clinical modalities of imaging tissues non-invasively have been applied to animal models to explore mammary gland structures ,18. ThesConditions for optimal imaging of collagen fibrillar structure using SHG-B have been reported by Zoumi et al. . They foThe combination of SHG-B and immunocytochemistry has been used to demonstrate the association of collagen I fibers with terminal end buds in the developing mouse mammary gland and the promotion of collagen fibrillogenesis by macrophages . These sIn addition to the capacity to document collagen structure, SHG-B versus SHG-F potentially reveals information on the maturity of collagen fibers. Williams et al. have compared two day tendon with mature mouse tendon and conclude that fibrillar structure imaged in the SHG-F mode is more prominent compared with that imaged in the SHG-B, whereas in the mature tendon, the signals are identical . They suHowever, nearly all of these imaging techniques must be studied with live tissues in prospective studies and are limited by availability of animals and the proper timing required for lengthy imaging sessions. Various methods have been described for clearing fixed tissue for deep (millimeter) imaging in mouse organs such as brain tissue, and recently a method has been described that notably preserves fluorescence intensity ,25. HoweRecently, we found that multiphoton microscopy can be used to retroactively image Carmine Alum-stained whole mounts to explore the morphology of hyperplastic glands and the inner structures of TEBs and ducts in three dimensions, specifically to determine whether ducts are filled with cells, retain a central hollow lumen, or abnormally contain multiple chambers or densely arranged lateral buds . ImagingWild-type FVB mice, or mice carrying an enhanced Green Fluorescent Protein (GFP) transgene under the control of the chicken beta-actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer (FVB.Cg-Tg (ACTBEGFP) B5Nagy/J strain were obtained from The Jackson Laboratories, Bar Harbor, Maine). HAI-1 transgenic FVB mice (unpublished), that overexpress the Kunitz-type protease inhibitor HAI-1 in their mammary glands were obtained from an in-house breeding colony. Mice were maintained and bred within the Division of Comparative Medicine animal facilities with unrestricted access to food and drinking water. Animals were examined at least once daily. All protocols involving the animals have been approved by the Georgetown University Animal Care and Use Committee (GUACUC) and carried out under the protocol No. 09043. After euthanizing animals according to the approved protocol, mammary tissue (the #3 or #4 glands) was harvested from 6–8 week or six month old wild-type, GFP, or HAI-1 mice  and spread out on microscope slides or cover glasses  to mimic the arrangement of the tissue in the mouse as closely as possible. The glands were imaged immediately and then fixed and processed for whole mount preparation essentially as previously described . BrieflyConfocal, bright field, and multiphoton images were collected using a Zeiss510/META/NLO microscope  with Zeiss EC Plan-Neofluar 10x/0.30 M27, a Zeiss LD LCI Plan Apo 25x/0.8 lens (working distance 0.57 at 0.17 mm cover glass), or a Zeiss c-Apochromat 40X/ 1.2 NA W corr. UV–VIS-IR M27. For transmitted SHG signals, a Zeiss achromatic condenser 0.8 H D Ph DIC was used with the polarizer replaced by an IR filter to block excitation IR in the emission. In Figure An NDD located in the transmitted light path just beyond a Zeiss ACHR COND 0.8 HD PH DIC condenser and IR filter is a useful addition to image transmitted SHG and/or transmitted fluorescence. The major signal from the ChD transmittance NDD detector is the SHG-F originating from collagen fibers and Carmine Alum or GFP signals which are much weaker by comparison. For these studies, these signals were not separated, although addition of emission filters could be used to isolate the transmitted SHG signal if desired. Additional file All images are presented in raw form (no post processing) with the exception of the Z-image stacks which were processed for 3D reconstruction as described using Image J 1.44p , Metamorph Offline ver. 7.7.1.0 , or Zeiss 510 META (with Physiology Software ver. 3.5 and Multiple Time Series) Software ver. 3.5 . Contrast and brightness settings were changed identically for comparisons.Excised mammary glands from GFP-mice were first examined using conventional bright field with a stereofluorescence microscope Figure A a-c. NeMultiphoton imaging of mammary glands was performed to improve resolution and to achieve the benefits of three dimensional imaging (3D). First, emission from GFP and SHG signals were studied in live mouse mammary glands  in multiple animals are harvested for each treatment or time point. Carmine Alum stained whole mount preparations are typically prepared from these experiments so that the data can be analysed according to researcher and instrument availability without concern for sample deterioration. Previously, we had determined that Carmine Alum is fluorescent and can be imaged in 3D . For thoAs an example of the power of 3D imaging of Carmine Alum staining, mammary glands from HAI-1 mice were imaged in the same manner as Figure In a final experiment, details of the ductal side branches were explored in a mouse model of HAI-1 in which potential abnormalities had been identified previously at the bright field level . Imaging of SHG-B, Carmine Alum and SHG-F was performed and the resulting XY, 3D and orthogonal XY, XZ, and YZ images were compared Figure A-D. It iFigure The difference in SHG-B and SHG-F signals cannot be explained solely on light scattering properties of Carmine Alum-stained epithelial cells within the TEBs. Both SHG-B and SHG-F signals are seen in fibers within the same layer and in fibers associated in parallel orientations adjacent to TEBs Figure A-D as weAdditional file To improve upon the quality and to optimize and understand imaging of Carmine Alum fluorescence, we systematically explored excitation and emission conditions for detection of Carmine Alum. Lambda scans were obtained of a duct in cross section using excitation wavelengths 750, 800, 830, 860, 890, and 960 nm at 0.1% laser excitation power and adjusting laser gain to qualitatively compensate for the changes at different wavelengths Figure A. Since In all cases, at 750, 860 or 950 nm laser excitations, epithelial cell staining was isolated much more effectively and to greater Z-depths when emission bandwidths of 650–710 nm were used . Thuexample, ,22,31. Uexample, . They obexample, , the abiexample, . The imao so that the fibers are reoriented parallel to the laser beam.Zoumi et al. optimized SHG of 3D collagen raft cultures and found peak SHG-B at approximately 800 nm . We founin vivo. Thus, further studies are required to examine the utility of the SHG-F compared with SHG-B signals to reveal differences in collagen fiber properties and for quantitative comparison of SHG-detected fibers.As previously described , ECM fibImaging morphology in Carmine Alum-stained mammary gland whole mounts is limited in that the dense array of Carmine Alum-stained epithelial cells in terminal end buds scatters both the fluorescence and SHG signals depending upon imaging depth and the relative positions of the epithelial or collagen fiber structures with respect to the detectors. Study of unstained whole mounts suggests that the “shadowing” effect of TEBs upon SHG and Carmine Alum signals deeper within the tissue likely arises from the density of Carmine Alum staining itself. In any case, the investigator must pay careful attention to the surrounding structures before interpreting the structural data. Another potential limiting factor to acquiring deep tissue images in whole mounts is the working distance of the imaging lens. TEBs deeper into the whole mounts are accessible with long working distance lenses in the case that there are no intervening epithelial structures between the TEB chosen for imaging and the coverslip surface.For deepest imaging into the whole mount tissue, it is probably advisable to collect the Carmine Alum signal using the higher emission wavelengths above 623 nm to detect the epithelial cells and eliminate ECM/ stromal contribution from autofluorescence  and to the right (YZ). Dashed lines indicate planes of associated images; red for YZ, green for XZ, and blue for XY. Multiple SHG positive fibrillar layers are present (arrows). Scale bars = 50 μm.Click here for fileMultiphoton and SHG spectral emission scans using live GFP-mouse tissue. An isolated GFP mouse mammary gland was imaged in the lambda mode  using a Zeiss LSM510/META/NLO with MP excitation of 860 nm and a 25X/0.8 N.A. Zeiss lens. A. At left, the graph represents emission wavelength (X axis) plotted against average intensity (Y axis). At right, the profile of the mammary gland TEB is shown using the “lambda coded” setting. B. The graph at left indicates the emission wavelength profile for three ROIs shown in the lambda coded image at right. Higher magnification views of the ROI area are indicated by arrows and shown as insets. C. Extracted images with emission bandwidths of Em 404–446 nm, 446–478 nm, 500–532 nm, and 596–703 nm. Images of the SHG-B and GFP peaks at 431 nm and 506 nm, respectively, are in the first and third images. GFP positive cells are indicated by the arrow. Scale bars = 50 μm.Click here for fileZ-stack movie of abnormal TEB from HAI-1 gland. The image stack of 1 μm sections totaling 74 μm was used to generate the 3D and orthogonal views shown in Figure Click here for fileScattering artifacts on SHG signals generated in thick specimens. Single XY and orthogonal images illustrate the variable ability of SHG signals to reach the detector. A. The Carmine Alum stained TEB overlying the fibers associated with several blood vessels prevent the transmitted SHG signal (green) reaching the ChD, NDD detector and illustrated in the diagram (compare SHG-B/SHG-F images with SHG-B/CA/SHG-F images). B. A single vessel detected by SHG-B (red) is closest to the coverslip. A TEB deeper into the tissue contains a gradient of blue Carmine Alum fluorescence to green (the latter color due to the background Carmine Alum staining included in the SHG-F detector). C. Loss of reflected signals occurs when the SHG-B source is deeper within the tissue relative to the TEB, and the TEB blocks the return of the reflected signal to the internal detector. Scale bars = 50 μm.Click here for fileo) compared with the view from the opposite direction (180o) demonstrates that the layer of SHG-F-detected fibers (green) lies deeper within the tissue and is most easily visualized from the 180o perspective. D. The image stack used to illustrate Figures Three dimensional (3D) representations of TEBs. Z-stack of images 178 μm deep was collected to include a TEB from a Carmine Alum whole mount. A. A 3D reconstruction was made using the Zeiss AIM software which illustrates a sheet of mostly parallel collagen fibrils visualized exclusively by SHG-F . The TEB is oriented so that the vector followed by the TEB is at a different angle than the vector along which the attached duct lies (arrows). The QuickTime movie of the Z-stack is shown in Additional file Click here for fileAssociation of SHG signals from fibers associated with TEBs. Movies 2–5 are QuickTime animations from Additional file Click here for fileEffects of emission bandwidth collection on Carmine Alum/ SHG-B/ SHG-F images. A. A lambda scan of a Carmine Alum stained TEB is illustrated together with a graph containing data from three ROIs . B. Images were extracted with defined wavelengths using the Zeiss Meta software. EM 628–703 nm identifies primarily the epithelial tissue whereas the Em 564–618 nm extracted image is striking in the appearance of the TEB-associated material, fat cell outlines, and the shadowing artifact on the TEB (arrow), C. 3D Carmine Alum images (blue) of the same TEB were obtained using Ch3 and fixed emission filters of Em 565–615 (at left) and Em 650–710 (at right) together with SHG-B (red) and SHG-F (green) 3D images. Scale bars = 100 μm.Click here for fileQuantification of data from Additional file Click here for fileQuantification of “Shadowing” in Carmine Alum Wholemounts. The TEB presented in Additional file Click here for fileComparison of Emission Spectra of GFP and non-GFP Unstained Whole Mount Tissue. A. GFP mice and non-GFP mice were prepared for whole mount without Carmine Alum staining and a lambda scan obtained from Em 361–704 nm using Ex 860 nm. ROI’s in a background area and one within the ductal epithelium were graphed as a function of normalized intensity (arrows indicate insets of ROIs at higher magnification). The intensity scale is normalized for comparison of ROIs. B. GFP mice were prepared for whole mount without Carmine Alum staining and lambda scans were obtained from Em 361–704 nm at Ex 890, 860, and 735 nm. ROI’s in a background area and one within the ductal epithelium were graphed as a function of relative intensity (arrows indicate insets of higher magnification). Average intensity of each ROI is plotted. Scale bars = 100 μm.Click here for fileComparison of excitation wavelengths for imaging unstained whole mount tissue. A. SHG-B and SHG-F (unfiltered) images were obtained of a single XY plane at a region rich in fibers (upper panels) from a GFP-mouse. At a deeper XY plane that included a median section through a TEB, the autofluorescent background signal was included in a three-color image (lower panels). Comparison of the images at Em 860 and 890 nm reveal little difference in the qualitative information present. B. SHG-B was detected within a focal plane including fibrillar structures and was compared for Ex 735, 800, and 890 nm. None was detected for Ex 735 nm (not shown). An ROI was selected for a region containing fibrils  and the average emission intensity plotted for each excitation wavelength. An extracted image was obtained for emission wavelengths containing the SHG maximum intensity: for Ex 800 nm the peak was 400 nm and for Ex 890 nm, the peak was 445 nm . C. The transmitted signal was detected for Ex 890 , but was not detected for Ex 800 or Ex 735 (not shown). A DIC image appears using Ex 800 in ChD, whereas SHG-F appears at Ex 890 (yellow asterisk). Scale bars = 50 μm.Click here for fileReduction in background fluorescence with narrowed SHG-B emission bandwidth. A. A lambda scan was obtained from Em 361–704 nm using Ex 860 nm. A subset of wavelengths is illustrated where the blue arrows indicate the SHG-B signal (420 and 431 nm). B. Bandwidth images were extracted using Zeiss Meta software; blue arrows indicate the SHG-B fibers seen in A. C. Linescans of pixel intensity performed indicated by dashed lines on the two images in B demonstrate the increased signal to noise detection of the fibers (blue arrows). The same TEB was imaged also in Additional file Click here for fileImaging unstained whole mounts containing muscle. A-D. A z-stack was obtained for unstained whole mount tissue at Ex 860 with Em 390–465 nm , Em 500–530 nm , ChD unfiltered , and brightfield (D). E. The average frame intensity of background subtracted z-stack images is graphed. The asterisk indicates the position of the SHG-B and SHG-F signal observed at 140 μm z-depth in A-C and E.Click here for file


Acute myeloid leukemia (AML) comprises a spectrum of myeloid malignancies which are often associated with distinct chromosomal abnormalities, and the analysis of such abnormalities provides us with important information for disease classification, treatment selection and prognosis. Some chromosomal abnormalities albeit recurrent are rare such as tetrasomy 8 or isochromosome 5p. In addition, erratic chromosomal rearrangements may occur in AML, sometimes unbalanced and also accompanied by other abnormalities. Knowledge on the contribution of rare abnormalities to AML disease, progression and prognosis is limited.NPM1.Here we report a unique case of acute monoblastic leukemia with gain of i(5)(p10), tetrasomy 8, an unbalanced translocation der(19)t and mutated NPM1. During the course of the disease we observed clonal evolution with gain of i(5)(p10), tetrasomy 8 and eventually duplication of der(19)t. By using the der(19)t as clonal marker, we found that i(5)(p10) and tetrasomy 8 were secondary genetic events and that tetrasomy 8 had clonally evolved from trisomy 8.Bone marrow cells were examined by conventional karyotyping, fluorescence in situ hybridization (FISH) and mutation analysis at diagnosis and follow-up. At diagnosis we detected trisomy 8, an unbalanced translocation der(19)t and mutated This case of acute monoblastic leukemia presents a combination of rare chromosomal abnormalities including the unbalanced translocation der(19)t, hitherto un-reported in AML. In addition, our case supports the hypothesis of a step-wise clonal evolution from trisomy 8 to tetrasomy 8 in AML. Reporting and collecting data of rare chromosomal abnormalities will add information to AML disease, progression and prognosis, and may eventually translate to improved patient management. Acute myeloid leukemia (AML) comprises a spectrum of hematologic malignancies with variable outcomes. A hallmark of AML is maturation arrest of myeloid cells, which accumulate in the peripheral blood and bone marrow. The un-differentiated myeloid cells show chromosomal abnormalities in about 55% of cases of adult AML . Some ofCytogenetics has become mandatory for the diagnosis of AML according to the 2008 revision of the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues .Trisomy 8 either as a sole or as an additional abnormality is the most common among numerical abnormalities associated with AML . In contGain of an isochromosome of the short arm of chromosome 5, i.e. i(5)p10), represents a very rare recurrent abnormality in AML ,12. To d0, represRecurrent translocations in AML are typically balanced and are often associated with a favorable prognosis. Erratic chromosomal rearrangements may occur in AML, sometimes unbalanced and also accompanied by other abnormalities. They are thought to contribute to disease progression and are usually associated with poorer prognosis .NPM1 mutation. By following the course of the disease we show that gain of i(5)(p10) and tetrasomy 8 represent secondary events in this case and that tetrasomy 8 has clonally evolved from trisomy 8.We present a unique case of AML with gain of i(5)(p10), tetrasomy 8, unbalanced translocation der(19)t and In June 2011 a 64 year old male patient was admitted to our hospital with recurrent epistaxis and ecchymosis. The diagnosis of AML, in this case acute monoblastic leukemia, was established. As the patient was not eligible for bone marrow transplantation due to comorbidities, induction therapy (7+3) with daunorubicin and cytarabine followed by three cycles of consolidation treatment with high-dose cytarabine was administered and led to complete hematologic and cytogenetic remission. A first relapse occurred 14 months thereafter, which was treated with induction therapy as before and, again, resulted to complete remission. At 19 months a second relapse occurred with additional infiltration of inguinal lymph nodes and the skin. In January 2013 induction treatment with cisplatin, gemcitabine and dexamethasone was administered, which resulted in hematologic and partial cytogenetic remission at four month thereafter. In July 2013 the patient vastly relapsed and passed away despite of continued consolidation therapy.At diagnosis conventional cytogenetics revealed the following karyotype: 47,XY,+8,add19)(p13)[5]/46,XY[15]. The initial karyotype was defined more precisely with multicolor fluorescence in situ hybridization 24X-FISH) analysis, which yielded to the following karyotype: 47,XY,+8,der(19)t[6]/46,XY[19]. At first relapse a marked clonal evolution had occurred with gain of isochromosome 5p and tetrasomy 8. The evolved karyotype was determined as: 49,XY,+i(5)(p10),+8,+8,der(19)t[7]/46,XY[14] [5]4X-FISH aE2A specific break-apart probe (D19S883) was used. Fusion signals were observed on both normal and derivative chromosomes 19, which indicated that the breakpoint on chromosome 19 ought to be located distal from the E2A gene locus , Hgb 14.1 g/dl, and platelets 56 G/L. The immunophenotyping and the cytological analysis were consistent with acute monoblastic leukemia.NPM1 mutation. To the best of our knowledge such a case has not been reported previously.We described a case of acute monoblastic leukemia with a unique combination of rare aberrations, i.e. gain of i(5)(p10), tetrasomy 8, der(19)t and In myeloid malignancies the occurrence of i(5)p10) is very rare and to date only 23 cases have been reported in the literature (p10) could have been underestimated by conventional G-banding as i(5)(p10) might have been misinterpreted as 5q-, which is frequent in patients with MDS or AML. By using locus specific probes towards 5p and 5q, FISH analysis allowed clear discrimination between 5q- and i(5)(p10) both on metaphases and on interphase nuclei Figure . This apTetrasomy 8 has been reported in about 120 cases to date . It was E2A, have been reported in approximately 400 cases in AML and in the context of various fusion partners such as MLL on 11q23 and others [E2A (TCF3) specific break-apart probe for chromosome-band 19p13 and obtained fusion signals only. This indicated absence of a chromosomal break within this gene locus , PTBP1 and STK11, and there is only one report of involvement of FSTL3 with CCND1 in a case with B-CLL [Apart from trisomy 8 our case presented with an unbalanced translocation der(19)t at diagnosis. Although both breakpoint regions  are intermittently involved in various translocations and other aberrations in hematologic malignancies, a literature search (PubMed and Mitelman database) on t in AML did not return a single hit, which suggests that the observed derivative chromosome 19 ought be erratic in AML ,32. Therth B-CLL .miR-21, miR-301, miR-142s, miR-142as) located and involvement in neoplastic diseases has been suggested [The band 17q23 has been identified as a “fragile site” in cancer with higher incidence of chromosomal breakage . Neverthuggested ,37. The NPM1 harbour chromosomal abnormalities in only about 15% of cases and the most frequent abnormalities are +8, –4 or –Y [NPM1 is retained during clonal evolution, it is thought that NPM1 mutations represent a founder mutation, and additional mutations or chromosomal abnormalities are thought to be secondary. In our case the NPM1 mutation did not change during clonal evolution. However, as we observed trisomy 8 and der(19)t at diagnosis, it is not clear which of the abnormalities conferred the founder.AMLs with mutated –4 or –Y ,39. As mIn vitro, a proliferative advantage of tetrasomy 8 over trisomy 8 has been observed in AML [By using the International Prognostic Scoring System in AML our case initially classified as “Intermediate-II”, as trisomy 8 is not a favourable prognostic factor, but does not score as an adverse prognostic factor either ,3. Howevd in AML . Cases wd in AML ,13,25. TNPM1 mutation. During the follow-up of the disease we observed that gain of i(5)(p10) and tetrasomy 8 represented secondary genetic events in this case. By using the der(19)t as clonal marker we formally demonstrated that tetrasomy 8 has evolved from trisomy 8. This case is only the third, illustrating a step-wise clonal evolution from trisomy 8 to tetrasomy 8 in AML.We described the first case of acute monoblastic leukemia with gain of i(5)(p10), tetrasomy 8, an unbalanced translocation der(19)t and Reporting and collecting data on rare chromosomal abnormalities of AML will add information to pathogenesis, disease progression and prognosis, and may eventually translate to improved patient management.Bone marrow aspirates were cultured for 24 h and 48 h in bone marrow culture medium . Metaphase chromosomes were prepared and GTG-banded by standard techniques. A minimum of 20 metaphases were karyotyped according to the International System for Human Cytogenetic Nomenclature (ISCN) 2013 .E2A (TCF3) gene locus on chromosome 19 was analyzed with the XL E2A break-apart probe . Metaphase chromosomes were also analyzed with the 24XCyte multicolor FISH probe set (Metasystems). All hybridizations were performed on a ThermoBrite instrument (Abbott Molecular) with denaturation at 78°C for 2 minutes followed by over-night incubation at 37°C and subsequent standard washing procedures, which were completed by counterstaining with DAPI . For interphase FISH cells from 24 h cultures were used, and more than 200 nuclei were analyzed. All of the FISH analyses were performed with a Zeiss Axio-Imager 1 M fluorescence microscope in combination with the Metafer 4 software (Metasystems).Polysomy 8 and i(5)(p10) were simultaneously analyzed by using a probe mixture of the Vysis CEP 8 probe  and the Vysis LSI 5p/5q Dual Color probe . The NPM1 and FLT3 gene mutation analysis as described [DNA and RNA were prepared from bone marrow aspirates by standard procedures and subjected to escribed ,43.Peripheral blood cells were examined by an automated hematologic analyzer . Bone marrow smears were stained with Wright-Giemsa and analyzed according to routine clinical laboratory procedures. Immunophenotyping of bone marrow cells was performed on an 8-color Navios™ Flow Cytometer  according to the manufacturer’s instructions.The patient gave written informed consent for the publication of this case including accompanying images prior to his passing away. A copy of this statement is available for review by the Editor-in-Chief of this journal.The authors declared that they have no competing interests.CP and GH performed the cytogenetic studies, DV and MF collected and compiled clinical data, JB and CP designed the study, HS participated in the design of the study and its coordination, JB and CP drafted the manuscript and all authors read and approved the final manuscript.


The action potential triggers Ca2+ influx through Ca2+ channels which causes local changes of [Ca2+]i for vesicle release. However, any direct role of extracellular Ca2+ (besides Ca2+ influx) on Ca2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.Neurotransmitter release is elicited by an elevation of 2+ and caffeine-induced release of stored Ca2+, we found that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2–3 µM). The IC50 for extracellular Ca2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca2+ concentration ([Ca2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca2+]o. The calcimimetics Mg2+, Cd2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.Using photolysis of caged Ca2+ hypothesis of synaptic release, physiological levels of extracellular Ca2+ play dual roles in evoked exocytosis by providing a source of Ca2+ influx, and by directly regulating quantal size and release probability in neuronal cells.As an extension of the classic Ca Presynaptic Ca2+ current is thus decreased by 30%, which corresponds to the more than 33% [Ca2+]o drop in a glutamatergic synapse 2+]o decreases even down to 0.1–0.3 mM from 1.2 mM 2+ modulates ion channels in neurons; for example, lowering [Ca2+]o shifts the voltage dependence of sodium channels to more negative potentials, via a biophysical surface charge effect 2+]o may have a biochemical effect on cell function by regulating non-selective cation channels and TRPM7 channel Early studies using ion-sensitive electrodes showed that a local electrical stimulus decreases [Ca2+]i rise directly triggers vesicle release 2+ also plays a direct role in vesicle exocytosis from outside cells. To investigate this possibility, we triggered vesicle release using photolysis of caged Ca2+ or caffeine-induced release of stored Ca2+ in the absence of membrane depolarization (without Ca2+ influx through voltage-dependent Ca2+ channels) at various levels of [Ca2+]o. We found that extracellular Ca2+ directly regulates [Ca2+]i-dependent vesicle exocytosis in the somata of sensory dorsal root ganglion (DRG) neurons and neuroendocrine chromaffin cells. Interestingly, physiological levels of [Ca2+]o inhibited [Ca2+]i-dependent exocytosis, partially by altering the quantal size and frequency of single vesicle release.For neurotransmitter release, it is well established that an AP-induced i and UV flashes in DRG cells, we used an IX-71 inverted microscope  equipped with a monochromator-based system . NP-EGTA is very selective for Ca2+ and intracellular dialysis of NP-EGTA generated a small loading transient, which did not affect membrane capacitance (data not shown) 2+ indicator fura-6F was excited at 1 Hz with 5-ms light pulses at 340 and 380 nm for the measurement of [Ca2+]i in the presence of NP-EGTA 2+ experiments was similar to that reported 2+]i to ∼300 nM after the loading transient.For measurements of [Ca2+]i in cells in slices, we used the Uniblitz shutter-based fluorescence system , a DVC1412 CCD camera , and a BX-51 upright microscope (Olympus). The images were acquired with TILLvisION software . The adrenal slice was loaded with fluo-4 AM or fura-2 AM by local puffer for ∼120 s via a patch pipette tip of ∼5 µm diameter. Individual cells were imaged by DIC optics using a 40× water-immersion lens (NA = 0.8). For fluo-4, Ca2+ transients were recorded by measuring the emission at 525 nm, resulting from excitation at 488 nm 2+ transients were recorded by measuring the ratio of emission at 510 nm, resulting from excitation at 340 and 380 nm For measurements of i through photolysis of caged Ca2+2+ directly regulates the [Ca2+]i-dependent exocytosis. We did not use APs, because AP-induced Ca2+ influx causes microdomain changes in [Ca2+]i which cannot be controlled under various levels of [Ca2+]o2+ compound nitrophenyl-EGTA. We elicited a [Ca2+]i rise using photolysis to a level near the threshold of evoked exocytosis. A train of low-energy UV flashes was applied to produce a [Ca2+]i plateau of ∼2 µM, resulting in a large Cm increase of ∼2 pF in a Ca2+-free bath  fully inhibited the [Ca2+]i-dependent ΔCm. In a second cell, the UV-flash evoked a similar level of [Ca2+]i rise and ΔCm in 2.5 mM [Ca2+]o was inhibited again, albeit by 40% compared to the Ca2+-free bath . Compared to 0 mM [Ca2+]o, the evoked [Ca2+]i rise was similar (106±8%), but ΔCm was reduced by 82±8% in 2.5 mM [Ca2+]o , which is a well-known feature of Ca2+-dependent exocytosis for synaptic transmission in hippocampal neurons and somatic release in chromaffin cells . Taken together, extracellular Ca2+ inhibited Ca2+-regulated vesicle exocytosis.To confirm the ECIE with a non-capacitance assay and investigate its role in transmitter release, we used a neuroendocrine cell which is a widely used model for exocytosis to 59±9% . The pho2+]i (2–3 µM). These data are not consistent with other studies using photolysis of caged Ca2+ to much higher [Ca2+]i levels (6–25 µM) 2+]i by using high-energy UV flashes and 10 mM nitrophenyl-EGTA. Extracellular Ca2+ still reversibly inhibited exocytosis when the UV flash-induced [Ca2+]i rise was increased to ∼4 µM  and tested its effect on evoked exocytosis. The caffeine-induced exocytosis was 35% greater in 1.65 mM than in 2.5 mM [Ca2+]o, while the evoked [Ca2+]i rise was similar , Mg2+ (1.26 mM), and Ca2+ (1.38 mM) . Neither calhex (1 µM) nor calindol (1 µM) affected the caffeine-induced [Ca2+]i rise in DRG neurons , which inhibits secretion in parathyroid cells affected A. In th1.38 mM) B. Secon [Ca2+]i . However [Ca2+]o C, D, or neurons . Third, re found  as reporFinally, we undertook a RNAi knockdown approach to test whether the CaSR was responsible for ECIE. Two short hairpin RNAs (shRNAs) for rat CaSR were designed and tested in HEK 293 cells. Both shRNAs efficiently knocked down over-expressed rat CaSR . However2+ and other divalent ions, as well as their potency order  placed in an adrenal slice 2+ and Mg2+ both inhibited exocytosis  by ∼27% (2+]o (5–90 mM)  To investigate the extracellular Ca by ∼27% D, table by ∼27% C. In ad by ∼27% D, table2+ compound or caffeine to elevate [Ca2+]i without Ca2+ influx. This permitted us to determine whether extracellular Ca2+ directly regulated vesicle release. We found that ECIE occurred in the somata of DRG neurons and adrenal chromaffin cells.In this work we used photolysis of a caged Ca2+]i induced by UV flash photolysis, the [Ca2+]i-induced Cm increase was enhanced by removing external Ca2+ in DRG neurons  and release probability (by 60%), and modulated the release kinetics of single vesicle exocytosis (2+]o (∼34%) significantly increased exocytosis in DRG neurons  for a given rise in o. A 34% change (from 2.5 to 1.65 mM) 2+]o significantly increased total exocytosis  the opening of a fusion pore is contributed to by both an intracellular force (Fin by synaptotagmins out of ECIE); (2) the total threshold force to open the fusion pore is Ftotal, such that Fin+Fout≥Ftotal is the condition to open fusion pore/exocytosis. This hypothesis explains that increasing Fin by higher [Ca2+]i reduces the effect of [Ca2+]o on ECIE.ECIE was induced by physiological changes in i. As an additional effect of [Ca2+]o, ECIE had never been examined previously under proper conditions. Most previous work used membrane depolarization/APs to trigger vesicle release 2+ channels are also clustered. AP-triggered vesicle release is induced by Ca2+ microdomains near the Ca2+ channel  2+]i (2∼3 µM) was triggered (2+]i (∼6 µM) greatly attenuated this inhibition  inhibited exocytosis, we failed to link the CaSR to ECIE (2+-binding protein at the extracellular side of the plasma membrane may be responsible for ECIE. (2) Generally Ca2+-dependent secretion is SNARE-dependent, although did not determine whether ECIE is SNARE dependent, because neurotoxins against somatic secretion in DRG neurons are not available. (3) Conformational changes of the Ca2+ channel induced by membrane depolarization and Ca2+ binding are reported to facilitate vesicle exocytosis in PC12 cells and chromaffin cells 2+ influx. However, this effect is different from ECIE since there is no membrane depolarization to open Ca2+ channel for the Ca2+ entry. (4) It is known that extracellular Ca2+ could shifts the voltage-dependent gating of Na+ and Ca2+ channels through a surface charge effect Regarding mechanisms of ECIE, (1) we investigated whether ECIE is mediated  to ECIE , S8. An 2+ directly regulated exocytosis. It is known that under pathological conditions, such as brain ischemia, extracellular Ca2+ and Mg2+ decrease greatly 2+]i. Thus, ECIE may affect neurotransmitter release more in neural somata than in synapses, where synchronized exocytosis is triggered by Ca2+ microdomains (10∼100 µM) following an AP 2+ hypothesis of exocytotic transmitter release In summary, our study demonstrated that extracellular CaFigure S1Facilitation of photolysis-induced exocytosis by cAMP elevation with forskolin in DRG neurons. (A) ΔCm and [Ca2+]i signals in response to Ca2+ release by trains of UV flashes (4 flashes at 0.5 Hz). The neuron was perfused with standard bath solutions containing 0 mM Ca2+, 0 mM Ca2++100 µM forskolin, 2.5 mM Ca2+, and 2.5 mM Ca2++100 µM forskolin, respectively, at 120 s intervals. UV flashes produced similar [Ca2+]i changes, while the corresponding exocytosis was facilitated by forskolin both in the presence and absence of external Ca2+. The initial value of Cm is noted. [Ca2+]i was monitored by Fura-6F measurements. (B) Comparison of averaged ΔCm traces with forskolin in 0 Ca2+  and 2.5 mM Ca2+ . (C–D) Average results from 4 cells showing that forskolin enhanced exocytosis in both 0 and 2.5 mM Ca2+. Exocytosis in 0 and 2.5 mM Ca2+ in the absence of forskolin was 70±8% and 22±9% of that with forskolin treatment. The corresponding [Ca2+]i values were similar in the presence and absence of forskolin. Compared to the [Ca2+]i rise with forskolin application, the [Ca2+]i rise was 88±6% in 0 [Ca2+]o and 103±5% in 2.5 mM [Ca2+]o solutions.(DOC)Click here for additional data file.Figure S2ECIE measured by combined membrane capacitance and amperometry in cultured rat adrenal chromaffin cells.(A) Representative ΔCm and amperometric current (Iamp) responses to [Ca2+]i rises induced by UV flashes in the presence (2.5 mM) or absence (0 mM) of external Ca2+. The arrowheads indicate the time points of UV flashes. A [Ca2+]i increases and secretion signals  were first induced by a train of UV flashes in 2.5 mM extracellular Ca2+ solution (2.5 Ca2+) (left). Subsequently, another similar [Ca2+]i increase induced by the second UV train produced much larger secretion signals in Ca2+-free solution (0 Ca2+) (middle). Finally, a similar [Ca2+]i rise induced by the third UV train triggered secretion signals similar to that by the first UV train when extracellular solution was changed back to 2.5 mM (right). (B) Statistics. Following UV flashes, the secretion signals were significantly smaller in 0 vs 2.5 mM Ca2+ , while the [Ca2+]i values were similar (107±4% of control).(DOC)Click here for additional data file.Figure S32+ did not inhibit high [Ca2+]i-induced exocytosis.Extracellular Ca Increase of [Ca2+]i to ∼8 µM almost abolished the extracellular Ca2+ inhibition of photolysis-induced exocytosis in DRG neurons.(DOC)Click here for additional data file.Figure S4Positive control of specific reagents  against CaSR. (A) Typical trace of [Ca2+]i measurement in CaSR-transfected HEK 293 cells. Cells initially bathed in 10 µM [Ca2+]o solution. [Ca2+]o at 1 mM induced a [Ca2+]i rise, which was smaller in the presence of calhex (1 µM) (n = 4). [Ca2+]i was monitored using Fura-2 AM. (B) Cells bathed in 0.25 mM [Ca2+]o solution. Calindol (1 µM) induced a [Ca2+]i rise (n = 5).(DOC)Click here for additional data file.Figure S52+]i rise induced by caffeine.Calhex and calindol had no effect on [Ca (A) [Ca2+]i rise induced by caffeine was monitored by Fura-2 measurements. Left traces show the [Ca2+]i rise induced by 20 mM caffeine in the presence of 2.5 mM [Ca2+]o and 1 µM calhex. Right traces show the [Ca2+]i rise induced by caffeine in the presence of 0 mM [Ca2+]o and 1 µM calindol. [Ca2+]i was monitored by Fura-2 measurements. There was a 90-s interval between continuous recordings. (B) Statistics of the caffeine-induced [Ca2+]i rise. Compared to signals in 0 mM [Ca2+]o and 2.5 mM [Ca2+]o, the [Ca2+]i rise was similar in calindol  or calhex .(DOC)Click here for additional data file.Figure S6NSCCs did not exist on DRG neurons. DRG neurons were on-cell patched. The upper four traces show consecutive current recordings at 60-s intervals in different perfusion solutions . The lowest trace shows the stimulation pulse. DRG neurons were stimulated with a step depolarization from −40 mV to +150 mV, followed by a hyperpolarization to −100 mV. Voltage-induced membrane currents in different external solutions were the same and this excludes the existence of NSCCs in the somata of DRG neurons (DOC)Click here for additional data file.Figure S7CaSR is not linked to SNARE complex. (A) Immunoblot of exocytosis-related proteins in DRG extracts with antibodies against syntaxin 1, synaptotagmin 1, SNAP 25, and complexin 1 and 2. GAPDH was used as a control. For the complexin doublet, the upper band is complexin 2 and the lower band is complexin 1. (B) Immunoprecipitation using a polyclonal antibody against aa 45–81 in complexin 2 with or without 3.5 mM Ca2+. Bound proteins were detected with monoclonal antibodies to syntaxin 1, SNAP 25, and CaSR. The upper strip shows that complexin antibody immunoprecipitated syntaxin 1 and SNAP 25. The lower strip shows no CaSR in the complexin immunocomplex.(DOC)Click here for additional data file.Figure S8CaSR knockdown had no effect on ECIE. (A) Western blot of CaSR and β-actin in shRNA 1 (sh 1), shRNA 2 (sh 2) and control shRNA-treated HEK 293 cells. Note that the lower band (CaSR) is missing in sh 1 and sh 2-treated cells. The upper band labeled with an asterisk (*) is a non-specific band recognized by the antibody. (B) Electrophysiology of ECIE in control shRNA, sh 1 and sh 2-treated DRG neurons. Experiments were done 4–5 days after transfection. (C) Statistics of normalized exocytosis and [Ca2+]i rise. The caffeine (20 mM)-triggered exocytosis in 2.5 mM Ca2+ was reduced to 41±8% (n = 6), 40±5% (n = 6) and 50±6% (n = 6) in control shRNA, sh 1 and sh 2, respectively. Extracellular Ca2+ inhibition was unaffected in sh 1 and sh 2-treated DRG neurons compared to that of control. [Ca2+]i was monitored by Fura-2 measurements. Compared to the [Ca2+]i rise in 0 mM Ca2+, the caffeine-induced Ca2+ rise was greater in 2.5 mM Ca2+ in the control cells . In sh 1 and sh 2-transfected neurons. the caffeine-induced [Ca2+]i rise was similar to that in 0 mM Ca2+ and 2.5 mM Ca2+ .(DOC)Click here for additional data file.Figure S92+ decrease modulated exocytosis in chromaffin cells.Physiological levels of extracellular Ca (A) Left, amperometric recording from an adrenal slice. A 34% reduction of [Ca2+]o increased the 20 mM caffeine-induced amperometric signal. Upper traces show amperometric recordings, lower traces show the corresponding ∫Iampdt signals. Right, the caffeine-induced [Ca2+]i rise was similar when changing [Ca2+]o in another rat adrenal slice. [Ca2+]i was measured using Fura-2 AM. (B) Statistics of normalized exocytosis and [Ca2+]i rise signals. A 34% [Ca2+]o reduction increased caffeine-induced exocytosis signals by 68±12% in chromaffin cells . The caffeine (20 mM)-induced [Ca2+]i rise was similar at [Ca2+]o of 1.65 mM and 2.5 mM in chromaffin cells .(DOC)Click here for additional data file.


In the heart, it is essential to activate cellular contraction in each heartbeat. Nevertheless Ca2+ is not only a key element in excitation-contraction coupling (EC coupling), but it is also a pivotal second messenger in cardiac signal transduction, being able to control processes such as excitability, metabolism, and transcriptional regulation. Regarding the latter, Ca2+ activates Ca2+-dependent transcription factors by a process called excitation-transcription coupling (ET coupling). ET coupling is an integrated process by which the common signaling pathways that regulate EC coupling activate transcription factors. Although ET coupling has been extensively studied in neurons and other cell types, less is known in cardiac muscle. Some hints have been found in studies on the development of cardiac hypertrophy, where two Ca2+-dependent enzymes are key actors: Ca2+/Calmodulin kinase II (CaMKII) and phosphatase calcineurin, both of which are activated by the complex Ca2+/Calmodulin. The question now is how ET coupling occurs in cardiomyocytes, where intracellular Ca2+ is continuously oscillating. In this focused review, we will draw attention to location of Ca2+ signaling: intranuclear ([Ca2+]n) or cytoplasmic ([Ca2+]c), and the specific ionic channels involved in the activation of cardiac ET coupling. Specifically, we will highlight the role of the 1,4,5 inositol triphosphate receptors (IP3Rs) in the elevation of [Ca2+]n levels, which are important to locally activate CaMKII, and the role of transient receptor potential channels canonical (TRPCs) in [Ca2+]c, needed to activate calcineurin (Cn).Ca Ca2+ entry activates intracellular Ca2+ release channels, named ryanodine receptors (RyRs), located in the membrane of the sarcoplasmic reticulum (SR). RyRs amplify the initial Ca2+ signal, providing enough Ca2+ to activate contractile myofibrils. Relaxation occurs when cytosolic Ca2+ concentration ([Ca2+]c) returns to diastolic values, due mainly to Ca2+ pumped back into the SR by the Ca2+-ATPase (SERCA) and extrusion from the cell via the Na+/Ca2+ exchanger  are being elucidated  , which is imported into the nucleus where it activates the transcription factor GATA . The location, RyR in the SR and IP3R in the nuclear envelope (NE) and perinuclear area, preferentially affects cytosolic and [Ca2+]n, respectively. When Ca2+ enters through the sarcolemma, the specific channel involved may also help to differentiate contractile vs. transcriptional Ca2+. LTCCs are mainly involved in contraction, while other less known Ca2+ permeating channels in the cardiomyocyte, such as TRPCs, play an important role in hypertrophy development  the location of Ca2+ release channels is different in SR and NE; (2) some molecules preferentially affect [Ca2+]n; and (3) [Ca2+]n signal decay is slower, due mainly to the lack of SERCA in the inner membrane of the NE , is crucial for EC coupling in ventricular myocytes. Other Ca2+]n more than [Ca2+]c. For instance, endothelin, which activates Gq and PLC producing IP3, increases [Ca2+]n in both atrial  induces cardiac hypertrophy and heart failure (HF). It is not known whether the cell is stimulated by an increase in the time-average [Ca2+]i or if, because [Ca2+]n dynamics are slower than cytoplasmic ones, there is an accumulation of Ca2+ in the nucleoplasm at higher frequencies.Oscillating CaThe question of how ET coupling can co-exist in cardiac myocytes in which i and activation of Cn  results in decreased myosin and ribosomal RNA expression  divided into 2 general subfamilies based on structural and functional similarities: TRPC1/4/5 and TRPC3/6/7. TRPC2 is not expressed in humans  versus receptor-operated Ca2+ entry (ROCE)  Figure . TRPC1/42+ sensor in the endoplasmic reticulum/SR, which, when is Ca2+ depleted, clusters proximal to the plasma membrane to activate Orai1, the pore-forming subunit of the Ca2+ release-activated channel  and Orai1 as mediators of SOCE. STIM1 serves as a Ca2+]i, besides its major role in EC coupling, is an important messenger in signal transduction regulating cardiac hypertrophy by activation of Ca2+-dependent transcription factors. Here we have attempted to present some of the pathways by which cardiac Ca2+ signaling is involved in ET coupling, notably during cardiac hypertrophy development. Although the profound influence of Ca2+ signaling on gene expression has been recognized mainly in neurons  but is initiated in many cases by an elevation in [Ca2+]i. New discoveries are expected in the near future on cardiac Ca2+ regulation to further enrich our understanding in this fascinating research field.In conclusion, [CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although successful identification and implementation of new drugs for children with AML remain a formidable challenge, enthusiasm for novel molecular therapeutic approaches is great given the potential for significant clinical benefit for children who do not have other curative options.Approximately two-thirds of children with acute myeloid leukemia (AML) are cured with intensive multi-agent chemotherapy. However, refractory and relapsed AML remains a significant source of childhood cancer mortality, highlighting the need for new therapies. Further therapy intensification with traditional cytotoxic chemotherapy in pediatric AML is not feasible given the risks of both short-term and long-term organ dysfunction. Substantial emphasis has been placed upon the development of molecularly targeted therapeutic approaches for adults and children with high-risk subtypes of AML with the goal of improving remission induction and minimizing relapse. Several promising agents are currently in clinical testing or late preclinical development for AML, including monoclonal antibodies against leukemia cell surface proteins, kinase inhibitors, proteasome inhibitors, epigenetic agents, and chimeric antigen receptor engineered T cell immunotherapies. Many of these therapies have been specifically tested in children with relapsed/refractory AML in Phase 1 and 2 trials with a smaller number of new agents under Phase 3 evaluation for children with  Acute myeloid leukemia (AML) is a group of genetically heterogeneous diseases that account for approximately 20% of pediatric leukemias with approximately 800 newly diagnosed children and adolescents annually in the United States (U.S.) , 2. CurrGiven the higher incidence of AML in adult patients and the limited success of conventional chemotherapy in maintaining long-term remission in this population, various new agents are under active investigation in early-phase clinical trials conducted within predominantly adult cooperative groups. Many of these novel agents aim to target genetic and molecular alterations hypothesized to be involved in leukemogenesis and/or therapy resistance. However, progress in the development and successful implementation of new therapies has been limited by the underlying biologic heterogeneity of AML, the ability of older patients to tolerate intensive therapy, patients’ associated medical co-morbidities, and rapid disease progression at time of relapse.There is burgeoning evidence that the inherent biology of AML differs between children and adults, as suggested by the discordant incidences of leukemia-associated genetic alterations, patterns of epigenetic changes, and rates of remission induction –8. A comApproximately 80% of childhood AML cases express CD33, a glycosylated sialic acid-binding transmembrane receptor protein of the lectin family. High CD33 surface expression is associated with worse outcomes in children and adults with AML –12. The Gemtuzumab ozogamicin has been specifically studied in pediatric AML via the Children’s Oncology Group (COG) and other consortia. Initial testing of GO monotherapy in the relapsed/refractory setting demonstrated similar pharmacokinetics to those in adult patients and morphologic remission rates of 28–54%, allowing many children to undergo successful subsequent HSCT –21. Studde novo AML. In the COG pilot trial AAML03P1, CR rates >80% were achieved in children treated with GO and chemotherapy in the induction and post-induction setting  in comparison to children treated with chemotherapy alone (p = 0.09). This increase in TRM was associated with a larger fraction of GO patients with prolonged time to neutrophil recovery in intensification II. Furthermore, this increase in TRM offset gains from the improved relapse rate, and thus no difference in OS was observed between the treatment arms in analyses of all patients. On subgroup analysis, high-risk patients had improved OS and decreased relapse risk   antibodies, which bind autologous T cells and redirect them specifically against tumor cell antigens. Such approaches have proven successful in early-phase testing for children and adults with leukemia, including the CD19/CD3 BiTE antibody blinatumomab (MT103) for B-precursor ALL , 37. Pretoxicity –40. BiTEOther antibody-based approaches for AML in early-phase clinical testing include targeting of surface proteins CD30, CD45, CD98, CD123, CTLA-4, or EphA3  . Some ofFLT3-ITD) with a high mutant:wild-type allelic ratio (>0.4) or point mutations in the FLT3 activation loop of the tyrosine kinase domain (FLT3-TKD) occur in 15–20% of pediatric AML patients  that target the FLT3 receptor. In the clinic, TKIs targeting mutant FLT3 have been studied primarily in early-phase trials in adult patients. Modest efficacy of first-generation FLT3 inhibitors  in adults with relapsed AML has been reported, both as monotherapy and in conjunction with cytotoxic chemotherapy  (de novo MLL-rearranged B-precursor acute lymphoblastic leukemias (ALLs), which overexpress the wild-type FLT3 receptor  treated on a Phase 1 trial at SJCRH with sorafenib, clofarabine, and cytarabine achieved CR or CR with incomplete blood count recovery (CRi)  .de novo FLT3-ITD AML treated with sorafenib and chemotherapy, although OS did not differ between the chemotherapy and chemotherapy/sorafenib arms  (FLT3-ITD AML are non-randomly assigned to treatment with sorafenib in combination with standard chemotherapy for assessment of feasibility (stage 1) and efficacy (stage 2), then taken to HSCT with the best available donor. Interim analyses of AAML1031 data have demonstrated the feasibility and safety of sorafenib with chemotherapy in stage 1, and stage 2 efficacy studies are now underway. A 1-year sorafenib maintenance phase for patients post-HSCT (or post-chemotherapy completion for non-transplanted patients) is underway in AAML1031 based in part upon safety and outcome data from a multi-institutional pilot study  . In thisot study , 79. Simot study . Althougctor AML .FLT3-ITD AML. Recent Phase 1 and 2 studies of quizartinib in adults with relapsed/refractory AML demonstrated acceptable toxicity profiles and preferential responses of FLT3-ITD patients , have garnered considerable attention for treatment of adults with patients , 81. A fpatients , 82. TheFLT3 point mutations that emerge during TKI therapy, including FLT3 D835 point mutations. Limited preclinical evaluation of crenolanib incubated with primary leukemia specimens from children with TKI-resistant FLT3-ITD/FLT3 D835 AML demonstrated moderate in vitro anti-leukemic activity  mutations , 83, a Pactivity . Most reactivity . CrenolaFLT3-ITD alterations   . Althoug1869114) , such rein vitro and in vivo with the Aurora kinase A inhibitor alisertib (MLN8237)  . ClinicaMLN8237) . Early-pMLN8237) .de novo settings, where it has been reasonably well-tolerated and has demonstrated efficacy in combination with other agents  (Bortezomib was first studied in children with relapsed/refractory solid tumors and leukemia in the COG and TACL consortia –102. The1371981) .DNMT3A, IDH1 and IDH2, TET2) are commonly mutated in adult AML and myelodysplastic syndromes (MDS), although the frequency of these mutations appears much lower in pediatric AML   –115. A fZ004777) .de novo AML or MDS  family of proteins recognize acetylated lysine residues of histone proteins and modulate gene expression via recruitment of transcriptional regulators . Inhibitn of MYC , 124. Con of MYC , 126. Thpoptosis . A Phasepoptosis .in vitro activity against human AML cell lines harboring various genetic alterations and in vivo efficacy in AML cell line xenotransplantation models, likely via G1 cell cycle arrest and induction of apoptosis , have been evaluated in the preclinical setting for various hematologic malignancies, including AML , 129. Tepoptosis , 130. A poptosis .Rapid progress has been made recently in the development of cancer immunotherapy using human T cells genetically engineered with synthetic chimeric antigen receptors (CARs) to target tumor antigens . Currentin vitro cytotoxicity against human AML cell lines and primary specimens , decrease acquired drug resistance, and increase potential for additive or synergistic cytotoxicity. At a genomics level, significant collaborative efforts are ongoing via the SJCRH–Washington University Pediatric Cancer Genome Project, the National Cancer Institute’s therapeutically applicable research to generate effective treatments (TARGET) AML Project, and other consortia to delineate more precisely various genetic subgroups of pediatric AML and to identify “actionable” lesions for molecularly targeted therapies , 143.One important consideration will be the degree to which results should be extrapolated from adult clinical trial data for children with AML given the inherent, but incompletely understood, differences in biology and in therapeutic responses. Results from early-phase clinical trials of new agents and the number of novel drugs in preclinical development are encouraging, but significant challenges persist in drug development for pediatric oncology. Evaluation of new agents in children with relapsed leukemia in early-phase clinical trials remains particularly challenging given the limited availability of novel drugs for pediatric studies. Moreover, study accrual can be challenging due to rapid disease progression in this population that hinders enrollment. Evaluation of meaningful responses in this setting is also difficult given the use of stringent disease response definitions that limit the number of cycles of therapy administered despite drug mechanisms that may require multiple courses of treatment to achieve optimal response. Given the relative rarity of childhood AML, its biologic heterogeneity, and the prospect of tailored therapeutics for ever-smaller genetic subgroups, collaborative trials across pediatric oncology trial consortia may be a more efficient means by which to identify new molecularly targeted agents for children with specific biologic subtypes of AML. Nonetheless, investigation of promising new therapies is essential to decrease relapse and improve cure for children with AML, and enthusiasm remains high for the development of molecularly targeted approaches in this emerging era of precision medicine.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants  on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted. The MutS homologue hMSH5 is a member of the DNA mismatch repair (MMR) family of proteins Undoubtedly, accurate and timely repair of DSBs is essential for cell survival and development in vivo reporter system. Our data indicates that hMSH5 promotes recombinational DSB repair, in which DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal region to the DSB. Given the importance of appropriate DSB repair, subtle changes inherited to the polymorphisms of DNA repair genes can affect the capacity of DSB repair and thereby increasing the risk of cancer predisposition Recent studies support a role for hMSH5 in the repair of cisplatin-induced DSBs, in which hMSH5 deficiency has been shown to elevate cisplatin-induced G2 arrest and increase cisplatin-triggered γ-H2AX foci formation L85F, 293T/f-hMSH5Y202C, 293T/f-hMSH5V206F, 293T/f-hMSH5R351G, 293T/f-hMSH5L377F and 293T/f-hMSH5P786S were generated by a similar procedure that has been previously described for 293T/f-hMSH5 The generation of the reporter cell line 293TLα/pMMR-IR3 was described previously 3VO4, and 1×protease inhibitor cocktail (Thermo Scientific). Soluble fractions were obtained after a 30 min centrifugation at 14,000 rpm.Harvested cells were rinsed with PBS before lysate preparation. Cells were lysed in CelLytic-M Mammalian Cell Lysis Reagent  supplemented with 10 mM NaF, 1 mM NaoC for 2 hours. Immunoprecipitates were captured by the use of 40 µl BSA-saturated 50% slurry of Protein A/rG Agarose beads.Stable cell lines harboring the wild-type hMSH5 and the series of hMSH5 variants were transiently transfected with pcDNA6/Myc-hMSH4 by the calcium phosphate approach. Cells were collected 40 hours after transfection for lysate preparation. Cell lysates of each preparation was incubated with 5 µl mouse α-Myc antibody  or mouse α-hMSH5 antiserum Cell lysates or immunoprecipitated proteins were separated by 4–20% gradient SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with indicated antibodies. Mouse α-hMSH5 antibody SceI)3xNLS construct Reporter cells were transiently transfected with the pCMV(I-5′-AGGGGTTCTTGGAGAAGAA) 5′-AAGGAGAGTGC GGCGCTTC) 5′-CAGGTGGTCTCTGCATCCA).The generation of pmH1P-based RNAi constructs was carried out as previously described L85F, 293T/f-hMSH5Y202C, 293T/f-hMSH5V206F, 293T/f-hMSH5R351G, 293T/f-hMSH5L377F and 293T/f-hMSH5P786S were used to perform clonogenic survival analysis in triplicate 6-cm plates (500 cells were seeded in each plate). Sixteen hours after seeding, cells were treated with indicated doses of cisplatin, bleomycin, doxorubicin, or camptothecin for one hour. Treated cells were maintained in culture for approximately 10 days to allow colony formation, and colonies containing more than 50 cells were counted. Cisplatin, bleomycin, doxorubicin, and camptothecin were purchased from Sigma.Stable cell lines 293T/f-hMSH5, 293T/f-hMSH5SceI) was transfected into the reporter cell line 293TLα/pMMR-IR3 by using an Amaxa Nucleofector , in which individual hMSH5 variant was co-expressed with I-SceI. Approximately 75% of the transfected cells were harvested for Western blotting analysis at 24 hours post transfection. Recombination frequency (percent of RFP positive cells) was determined by FACS analysis of 100,000 cells at 6 days post transfection .To generate a defined DSB, the plasmid pCBA-. The results of the analysis indicate that hMSH5 transient expression increases the rate of recombination by at least 50%  (SceI transfection at which the expression of I-SceI was readily detectable (i.e. −303/−57 or 57/338)  . The DSBtectable . In resp 57/338) . However 57/338) . This ob 57/338) . DSB-ind 57/338) . In addi 57/338) . These dTo determine the regulation of DSB-induced hMSH5 chromatin loading and to reveal the interrelationship among the hMSH5 interacting proteins, ChIP analysis was performed in conjunction with RNAi-mediated gene silencing at the proximal and distal loci (−303/−57 and 814/1037) . The effLikewise, RNAi-mediated hRad51 depletion led to the reduction of hMSH5 and hMSH4 loading similarly to that elicited by hMRE11 silencing; whereas the loading of hRad51 could only be affected by hMRE11 . This obY742F , bleomycin, doxorubicin, and camptothecin. In comparison to that of the hMSH5 wild-type controls, hMSH5 to CDDP . In respensitive . Among cresponse . Finallytothecin . These oR351G, also promoted recombinational DSB repair . This observation tends to suggest that the loading of hRad51 and hMSH5 can also be regulated by the availability of the donor template. Development of a procedure allowing instantaneous DSB induction at a defined locus, for monitoring precise time-dependent loading patterns of various repair proteins, would be important to address how the existence of a homology donor influences the choice of DSB repair pathway.Y202C is unique in several aspects. It stimulates recombination and renders cells resistant to CDDP, bleomycin and doxorubicin, and this variant is also predicated to be deleterious by the SIFT analysis (sift.bii.a-star.edu.sg; e.g. NHEJ-mediated DSB repair and the induction of apoptosis. Obviously, further studies will be needed to decipher how hMSH5 variants impact cellular responses to anticancer therapy. Taken together, our current study has demonstrated a role for hMSH5 in recombinational DSB repair. The outcomes of this study should make it possible to explore the detailed molecular mechanisms involved with hMSH5 as well as the effects of various hMSH5 variants on cancer predisposition and cellular resistance to chemotherapeutics.Our examination of six non-synonymous hMSH5 variants has indicated that these polymorphic variants could exert distinct effects on cellular responses to different anticancer agents. Although DSB is commonly generated in cells treated with CDDP, bleomycin, doxorubicin, or camptothecin, the process of DSB induction in each case is mechanistically different and may also be cell cycle dependent. For instance, CDDP creates a variety of DNA lesions, of which the interstrand crosslinks are probably responsible for creating DSBs at replication forks


Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca2+-channels in Ca2+ signalling pathways.Fluorescent microscopy was used to detect changes in Ca3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca2+ and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca2+ oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca2+→ NO→cGMP→cADPR→Ca2+, which determines periodic or steady operation of a short PFL based on Ca2+-induced Ca2+ release via RyR by generating cADPR, a coagonist of Ca2+ at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca2+-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca2+-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G.ACh activating M2+-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.This study presents a kinetic model of Ca Moreover, in adipocytes activation of Mq proteins (Gq)→Phospholipase C (PLC)→ diacylglycerol (DAG)/inositol-3-phosphate (IP3) → protein kinase C (PKC)/IP3receptor (IP3R)→Ca2+ (A).GOn the contrary hepatic 2+ release from intracellular stores by IP3 and ACh 2+-oscillations in various nonexcitable cells involving the PLC/IP3/Ca2+ dependent pathway (A) 2+-oscillations implicating either the classical PLC/IP3R signalling pathway (A) Since the discovery of Ca2+, the functional relevance of oscillatory regimes and the mechanisms of autoregulation of Ca2+-signalling machinery are not yet completely understood The metabolic effects of Ca2+-signalling pathways with multiple feedback and feedforward loops are implicated in the metabolic control of electrically nonexitable cells and may bring about complex of nonlinear dynamic behavior characterized by the set of temporal and spatial patterns usually registered as: Ca2+ elementary events (sparks), Ca2+-spikes, oscillations, spatial waves Without doubt, different Ca2+ oscillations was presented by Endo and coworkers in 1970 2+ induced Ca2+ release (CICR) in skinned muscle fibers. Soon after, Ca2+ oscillations were registered in Physarym P. by Ridgway and Durham 2+ oscillations in non-excitable cells using single hepatocytes. Since that time various modes of Ca2+ sparks, oscillations and waves and switching phenomena have been registered in hepatocytes The first example of Ca3R, with or without a modulatory role of the Gq→PLC→IP3R→Ca2+ axis 2+ and involving the long PFL Ca2+→PLC→IP3→IP3R→Ca2+2+-sensing receptors or G-proteins by PKC This dynamic versatility is generally believed to involve oscillatory mechanisms based on: a short positive feedback loop (PFL) in the form of CICR via IP3R and RyR 2+ oscillations and waves in response to ACh or CCK Some authors have also considered tandem oscillatory mechanisms involving both types of CICR via IP3R has been mirrored in mathematical modeling. Most models describe various feedback mechanisms related to IP3R and PLC This focus of interest in IP2+ waves in colonic interstitial and smooth muscle cells was proposed in 1993 2+→NO→Ca2+ positive feedback, which was based on known dependence of NO synthesis by constitutive NO synthase on Ca2+2+ and NO signalling in interstitial cells Alternative oscillatory mechanism, driving the propagation of NO and Ca2+-oscillatory mechanisms, a very interesting kinetic model linking the NO→cGMP→PKG and cADPR→RyR→Ca2+ signalling pathways was offered some years later 2+ signalling via RyR in non-excitable cells. Taking all this into consideration, the main goal of this work is to show that ACh induces Ca2+ oscillations implicating both: Ca2+→NO→ cGMP→cADPR→Ca2+ long PFL and a short PFL based on CICR via RyR, with the possible involvement of other reinforcing PFLs. We additionally consider the modulator role of NE and of atrial natriuretic peptide (ANP) on this basic mechanism.Irrespective of CaL-glutamine, 4 nM insulin, 0.004% gentamicin and 25 µg/ml sodium ascorbate (Sigma). The obtained suspension contained preadipocytes, since mature adipocytes carry vesicles of fat and do not precipitate under the given conditions.All studies were approved by the Animal Ethic committee of the Institute of theoretical and experimental biophysics. NMRI mice (aged 3–5 weeks) were decapitated after a brief (45–60 sec) anesthesia with carbon dioxide before sacrifice. Mice were subjected to cervical dislocation and disinfected with 70% ethanol prior to dissection. All operations were performed in a sterile environment on ice. White adipose tissue was removed from the epididymal fat depot and placed in a Petri dish with cold DMEM medium . Scissor-minced white adipose tissue was transferred into a tube containing sterile DMEM with 7 mg collagenase II (Sigma) and 4% bovine serum albumin . Then the tissue was incubated for 18 min at 37°C. To stop the enzymatic reaction, the tube was chilled on ice for 20 min with intermittent shaking followed by filtration through 250 µm filter and centrifugation at 1000 g for 10 min. The pellet was then resuspended in cold DMEM medium, filtered through 50 µm filters and centrifuged at 1000 g for 10 min. Finally, the pellet was resuspended in cultural medium containing: DMEM (Sigma), 10% fetal bovine serum , 4 mM 4 preadipocytes were placed on round coverglasses (25 mm in diameter), which were then transferred into 35 mm Petri dishes. 6 hours after adhesion of the cells to the glass, additional culture medium was added to the Petri dishes. On the third day the medium in the dishes was replaced with a fresh portion of medium, which included 10 nM cytosine arabinoside (Sigma) to suppress the proliferation of fibroblasts, and incubation in CO2 atmosphere was continued for 8 hours. After that the medium was replaced with fresh culture medium. On the ninth day of culture, the cells form a monolayer and become differentiated. An important morphological difference of mature white adipocytes from other cells is formation of fat droplets , a ratiometric fluorescent calcium indicator  for 20 min to complete the deesterification of the dyes.NO production in white adipocytes was evaluated using the fluorescent probe DAF-FM diacetate , oxidation of which by NO dramatically increases the quantum yield of the dye 2+-bound and Ca2+-free forms of Fura-2, respectively. The emission of both DAF-FM and Fura-2 was recorded at 530±25 nm. In the case of double staining (Fura-2 and DAF-FM), the total exposure time per three-channel frame was 20 s. Collected 8-bit time-lapse images were analyzed using ImageJ software with Time Series Analyzer and RatioPlus plugins.Dye-loaded cells were visualized using a Cell Observer imaging system . DAF-FM fluorescence was excited using a BP 475/40 filter. BP 340/30 and BP 387/15 filters were used to excite the Ca2+]i are presented as the 340/380 ratio obtained from time-lapse images after background subtraction. DAF-FM fluorescence is presented as ΔF/F0, where ΔF = F – F0 . The experimentally obtained curves were smoothed to decrease the effect of noises. The curve of ΔF/F0 characterizes the fluorescence intensity of benzotriazole, a product of nitrosylation of DAF-FM accumulated in the cells. For a more precise estimation of the changes in NO concentration, the derivative d(ΔF/F0)/dt was used, assuming that the rate of benzotriazole dissociation is very slow in comparison with life time of free NO and therefore NO concentration is proportional to the derivative d(ΔF/F0)/dt. All imaging experiments were performed at room temperature (25–26°C).The changes in i by plasma membrane Ca2+-ATPase (PMCA) is a prerequisite for maintaining the oscillations, but does not have any significant role in the mechanisms underlying generation of the oscillations.The steady modes of Ca (SERCA) . This in3R, Xestospongine C .Furthermore, monitoring those cells with steady modes of Caesponses . The fre2+ oscillations, the PLC dependent Ca2+-signalling pathway (A), the long PFL based on activation of PLC by Ca2+, and CICR via IP3R do not operate under the conditions of the aforementioned experiments. However, these mechanisms may still be realized in adipocytes, where they are responsible for the generation of a so called “baseline” Ca2+ oscillation observed in the presence of fetal bovine serum 3R and PLC can be controlled by phosphorylation of the adaptor protein IRAG 3R and PLC may correspond to the formation of negative feedback loops (NFLs) under the control of PKG, which could be responsible for switching off Ca2+-signalling pathway (A) under the conditions used in our experiments.These results indicate that the classical mechanisms of CaPreincubation of adipocytes with the selective phosphoinositide 3-kinase gamma (PI3Kγ) inhibitor AS-605290  or with 2+ response, which may be the result of activation of the Gαq→PLC→IP3→ IP3R→Ca2+ signalling pathway (A) under these conditions. It is known that class 1A PI3Kγ is activated by the G protein beta gamma subunits of G proteins (Gβγ-subunits) of Gq and Gi proteins Only a small proportion of the cells retained an impulse-shaped Caβγ→PI3Kγ→PKB→eNOS.GFunctioning of this signalling axis has previously been demonstrated in endothelial cells under the action of endothelin-1 2+ responses in adipocytes, application of 1–10 µM norepinephrine (NE) lead to the generation of steady periodic modes in 55±5,8% of the cells (2+ oscillations (3-cholinergic receptors (In the presence of low concentrations of ACh (less than 50 nM), which cannot induce Cahe cells  whereas llations . The obseceptors .2+-oscillations are also observed upon the addition of an α1-adrenergic receptor selective agonist, phenylephrine  with higher values of [Ca2+]i  and was detected in adipocytes for the first time. No doubt the cells in culture have different sets of parameters (activities of enzymes and channels), being in different stages of their development. This parametric diversity may underlie the versatility of dynamic behaviour observed, although we cannot exclude the possibility that different cells with similar sets of parameters have different concentration ranges for key metabolites . In both cases application of a second agonist promotes various periodic modes or switching phenomena.Such diversity of dynamic behavior in the Ca2+ oscillations in adipocytes promoted by ACh and the Ca2+ impulses arising from the effect of NE or α2-adrenergic receptor agonists - UK-14,304, guanabenz and L-arginine, can be realized with the implication of the following signalling pathway:Previously, using inhibitory analysis 2+ (B)PI3K→PKB→eNOS→sGC→PKG→CD38→RyR→ Ca2+ oscillations and switching phenomena promoted by NE or by α-adrenoreceptor agonists in the presence of low concentrations of ACh , may thus be responsible for the synergic activation of signalling pathway (B) and observed dynamic versatility.The induction of Cas of ACh  provide βγ-proteins may additionally activate PLCβ and PLCδ3-AChRs and of α1- α2-adrenoreceptors switches on both Ca2+ signalling pathways implicating IP3R and RyR:Taking into consideration those Gαq, Gβγ→PLCβ→ IP3→IP3R→Ca2+ (A)Gβγ→PI3Kγ→ PKB→ eNOS→ NO→ sGC→ cGMP→ PKG→ cADPR→RyR→Ca2+ (B)G2+ oscillations induced by ACh may depend on the extent of IP3R inhibition due to phosphorylation of IRAG protein by PKG The impact of the PLC signalling pathway on CaIn turn, PKG activity may strongly depend on the presence of L-arginine in incubation medium 2+-signalling pathway (B) through activation of M3-mAChR (Gq) and α1 (Gq) and α2 (Gi) adrenoreceptors respectively. Active functioning of this pathway may be responsible for the complex nonlinear dynamic behaviour observed  , ACh and NE potentiate Caobserved  and for hway (A) .2+-induced Ca2+-release via RyR The mechanism of Ca2+→ RyR→ Ca2+ (1)Ca2+ dependent calmodulin kinase (CaMKII) The known activation of RyR by Ca2+→CaMKII→RyR→Ca2+ (2)CaThis short PFL(2) may reinforce the functioning of short PFL(1).2+ dependent 2+ release via RyR 2+→NO→Ca2+ positive feedback loop, as was predicted 20 years ago It is well known also that NO synthesis by constitutive NO synthases is strongly Ca2+ signalling pathway (B) represent long PFL(3):This long feedback loop arising in Ca2+→eNOS→NO→sGC→cGMP→PKG→CD38→cADPR→RyR→ Ca2+ (3)Ca2+ which potentiates activation of RyR. Active interplay of these loops may produce various types of Ca2+ oscillations and other non-linear dynamic effects observed  may be prevented by inhibitors of any enzyme in this loop. Preincubation of cells with the PKG inhibitors KT5823 and Rp-8-Br-cGMPS not only leads to the suppression of the observed oscillatory modes in 92±5,4% (±SD) of the cells, but also results, after a short lag phase, in a slow increase of i as a fast variable of the autocatalytic mechanism, we should expect that a slow variable or variables must also be present within this long PFL. For example, this may be NO or processes linked to NO production (i.e. eNOS or PKG activities).From a mathematical point of view, considering [Ca2+ and NO oscillatory mode with a period of several minutes (2+]i (0)/dt, green curve), we may consider [Ca2+]i as a fast variable and NO (or a process related to NO production) as a slow variable in the oscillatory mechanism. The rise in [Ca2+]i level is followed by an increase in NO production which may indicate the direct activation of the eNOS by Ca2+. The maximal values of NO correspond to the minimal values of [Ca2+]i. This may reflect the acceleration of [Ca2+]i extrusion by Ca2+ ATPases SERCA and PMCA, owing to their possible activation due to phosphorylation by PKG.The slow Ca minutes  confirms2+]i oscillations and the sawtooth shape of NO oscillations also indicate that Ca2+ and NO can be considered as the fast and slow variables of this oscillatory mechanism.The impulse shape of i level.it may participate in the control of the activities of Caβγ→PI3Kγ→PKB by ACh, leading to activation of eNOS upon its phosphorylation by PKB. The intermediates of this long PFL(3) , upon corresponding addition of an NO donor (SNAP), a permeable analog of cGMP (8-Br-cGMP) or a substrate of ADP-ribosyl cyclase (ADPRC) or CD38 (βNAD) may modulate the Ca2+ oscillations generated by short PFL(1) or may produce the oscillations on their own. In this way we can determine the role of PFL(3) in the generation of fast Ca2+ oscillations.The parametric modulation of activity of the long PFL(3) occurs naturally with the implication of the signalling axis G3R, or some other mechanism.If the signalling loop intermediate added in the incubation medium quickly enters the cells and equilibrium is quickly established, this means that clamping (fixation) of the variable of PFL can occur. In this case the long PFL(3) would break and the possible sources of oscillations could only be related to CICR via RyR, or CICR via IPIf, on the other hand, an intermediate of the signalling loop slowly penetrates into the cell, and the influx has nearly constant speed, this would not break the long PFL, but would parametrically modulate its activity. Slow release of NO from SNAP may model influx of NO into the cell (input). Activation of PKG by 8-Br-cGMP or substrate control of ADPRC (or CD38) by βNAD, may represent modulation of the corresponding enzyme activities.In the absence of ACh, addition into incubation medium of SNAP (2+ oscillation) with periods ranging from several seconds to several dozens of seconds  with a higher level of i required for activation of RyR or eNOS, until the moment of complete activation of the long PFL(3). Tandem mechanisms implying a potentiating action of IP3R on RyR have been proposed by several authors 2+-oscillations by ACh in the presence of PLC, IP3R and PKC inhibitors allows us to consider such a possible mechanism of action for the signalling pathway (A) as secondary or unlikely under our conditions.It may be assumed that activation of IP2+ and NO oscillations, observed at slow periodic regimes (2+ (by CaCaM) some slower Ca2+-dependent processes may also participate in the reinforcement of long PFL(3).A phase shift between the maxima of Ca regimes , indicat2+]i level and on the phosphorylation of eNOS by kinases of different types. The enzyme is activated by the CaCaM complex 2+-dependent CaMKII takes part, along with PKB, PKA, AMPK and PKG, in the covalent modification and activation of this enzyme It is well known that the activity of eNOS is strongly dependent on the i level;it may control the activity of PKG and thereby control the activities of Caαq→PLC→IP3→Ca2+ by inhibiting IP3R upon phosphorylation of IRAG by PKG and inhibiting PLC upon phosphorylation of RGS proteins with the participation of PKG.it may act to switch off the signalling chain Gβγ proteins may predominate over selective Gαq signalling by suppressing the IP3R functioning (via PKG) and dissociating Ca2+ and DAG/IP3  signals in the cells.Point (5) assumes that integrated, non-selective (promiscuous) signalling via G2+-signalling system  with enlarged Ca2+-level (stable or average). Considering the parasympathetic control of adipose tissue and a possible role for Ca2+ in the regulation of lipid metabolism, the very complex organization of Ca2+ signalling system may have important implications for the function of this endocrine tissue.In conclusion, the model of Cag system  presenteFigure S1(DOCX)Click here for additional data file.