While primary cilia have been implicated in osteocyte mechanotransduction, the molecular mechanism that mediates this process is not fully understood. We directed a fluorescence resonance energy transfer (FRET)-based Ca2+ biosensor to the cilium by fusing the biosensor sequence to the sequence of the primary cilium-specific protein Arl13b. Using this tool, we investigated the role of several Ca2+-permeable channels that may mediate flow-induced Ca2+ entry: PC2, TRPV4, and PIEZO1.The primary cilium is an antenna-like, nonmotile structure that extends from the surface of most mammalian cell types and is critical for chemosensing and mechanosensing in a variety of tissues including cartilage, bone, and kidney. Flow-induced intracellular calcium ion  contains supplementary material, which is available to authorized users. Despite how universal Ca2+ is, discrete intracellular signaling mechanisms occur because Ca2+ gradients are spatiotemporal and do not comprise one general pool that changes uniformly. For example, there are discrete microdomain Ca2+ signals including “Ca2+ sparks,” “Ca2+ sparklets,” and “scraps” that modulate constriction and relaxation in vascular smooth muscle cells [2+ increases in many cell types including osteocytes, osteoblasts, neurons, and kidney cells. Ca2+ mobilization is required for flow-induced prostaglandin E2 (PGE2) release and flow-induced osteopontin gene regulation in osteocytes, demonstrating that Ca2+ is upstream of mechanotransduction activities and paracrine signaling [Mechanotransduction is a process by which cells sense and convert mechanical signals into biochemical and transcriptional changes. The calcium ion  associates with the mechanosensitive protein polycystin-1 and localizes to the primary cilium. Jin et al. reported that flow-induced Ca2+ elevations occur first in the primary cilium and are followed by cytosolic Ca2+ mobilization. Both ciliary and cytosolic Ca2+ increases were dependent on PC2 [2+ increases without affecting the flow-induced ciliary Ca2+ response [2+ enters and triggers ryanodine receptors in Ca2+-induced Ca2+ release.Recent advances in monitoring ciliary Caal cells ,13,15. Tal cells -18. Su e+ levels ,18. The response . Collect2+ increases in MLO-Y4 osteocyte-like cells are independent of primary cilia and stretch-activated channels, which is different from kidney cells [2+ levels, it is unknown if the local primary cilium Ca2+ environment is distinct from the cytosol. We hypothesized that the osteocyte primary cilium mediates mechanotransduction by forming a distinct Ca2+ microdomain. Therefore, the objective of this study was to monitor flow-induced ciliary Ca2+ levels and elucidate the intricate role of the osteocyte primary cilium as a biochemical and mechanical signaling nexus.Current knowledge of the osteocyte primary cilium-mediated mechanism of mechanotransduction is relatively poor compared with recent progress in kidney epithelia primary cilium mechanotransduction research. Our group previously used the fluorescent dye Fura 2-AM to demonstrate that flow-induced Caey cells . While t2+ biosensor to the primary cilium by fusing a biosensor sequence to the sequence of the primary cilium-specific protein Arl13b. The modified YC3.6 Ca2+-sensitive FRET-based biosensor with the ECFP-YPet donor-acceptor pair contains a calmodulin (CaM) region with four Ca2+-binding domains [2+ results in a conformational change that increases FRET signal, which is characterized by decreased ECFP and increased YPet fluorescence [2+ biosensor containing ECFP and YPet and the diffusive Ca2+ indicator dye Fura Red, we detected ciliary and cytosolic Ca2+ increases within individual MLO-Y4 cells exposed to fluid flow stimulation. Additionally, we examined the role of several Ca2+-permeable channels on the primary cilium: PC2, transient receptor potential vanilloid 4 (TRPV4), and PIEZO1. Coste et al. recently characterized mechanically activated current in neuroblastoma cells and proposed that the multipass transmembrane ion channels PIEZO1 and PIEZO2 mediate mechanically activated cation activity, leading us to explore the role of PIEZO channels in 223osteocyte mechanotransduction [2+ increases and a loading-induced osteogenic response at the transcriptional level. Collectively, our study demonstrates that the osteocyte primary cilium microdomain is distinct from the cytosol and that sources of loading-induced ciliary Ca2+ mobilization are different in kidney epithelia and osteocytes. These are the first measurements of Ca2+ signaling within the osteocyte primary cilium, and we anticipate this work is a starting point for more studies investigating the role of TRPV4 in osteocyte mechanotransduction [In this study, we directed a fluorescence resonance energy transfer (FRET)-based Ca domains . Bindingrescence . YPet asrescence . Using aArl13b gene. We fused Arl13b to the N terminus of CaB using a 15-amino-acid-long flexible linker to form Arl13b-L-CaB (ALC)[3 and Phe17 in the M13 region were performed to block changes in FRET during Ca2+ increases, serving as negative controls of CaB and ALC (mutCaB and mutALC) [Drs. Yingxiao Peter Wang and Mingxing Ouyang previously developed a calcium-sensitive FRET-based biosensor composed of an ECFP donor, calmodulin region, M13 calmodulin-binding region, and YPet acceptor (CaB). Drs. Kenji Kontani and Kristen Verhey generously shared with us the CaB (ALC). Deletio mutALC) .2. Using the BTX 360 with a 300-V, 100-Ω, 1,000-μF pulse, 1.25 million cells were transfected with 10 μg plasmid by electroporation. Cells were co-transfected with the ALC plasmid and 0.5 nmol siRNA. siRNA sequences included Pkd2 (5′-CCUCUUGGCAGUUUCAGCCUGUAAA-3′), Trpv4 (5′-GAUGGACUGCUCUCCUUCUU GUUGA-3′), and Piezo1 (5′-CACCGGCAUCUACGUCAAAUA-3′) [2 and cultured for 3 days in reduced serum containing 2.5% FBS and 2.5% CS. IMCD cells  were cultured in DMEM (Life Technologies) with 10% FBS and 1% PS. Transfected IMCD cells were seeded onto fibronectin-coated glass slides at a density of 8,000 cells/cm2 and cultured for 3 days in 1% FBS. Prior to imaging, cells were incubated in 20 μM Fura Red-AM  with 0.1% Pluronic® F-127 (20% solution in DMSO) (Life Technologies) for 1 h at room temperature to label cytosolic Ca2+.MLO-Y4 osteocyte-like cells  were cultured in MEM alpha (Life Technologies) with 5% FBS, 5% CS, and 1% PS at 37°C in 5% COAAUA-3′) . Transfe2 shear stress, and IMCD cells were stimulated with steady fluid flow resulting in a 1-dyn/cm2 shear stress, both within physiologic range for each cell type. Thapsigargin was added to the imaging media at a final concentration of 10 μM for appropriate samples for a 20-min rest period and maintained at the same concentration during flow. A separate syringe with 10 μM ionomycin was connected to the chamber to verify cell viability after the initial flow stimulus.Slides were placed in the RC-30 Confocal Imaging Chamber (Warner Instruments) and attached to a syringe containing phenol red-free alpha MEM with 1% FBS and 1% CS for MLO-Y4 cells or phenol red-free DMEM with 1% FBS and 1% PS for IMCD cells. MLO-Y4 cells were stimulated with oscillatory fluid flow resulting in a 10-dyn/cm2 at a final concentration of 0.1, 0.25, and 0.5 mM. Baseline signal was recorded for 30 s followed by 5 min of flow. Images were taken at 4 Hz with a 150-ms exposure.One fluorescing cell per slide was selected at random for imaging. The selected cilium was focused on, with the cell body captured within the field of view on an Olympus IX71 inverted epifluorescence microscope with a 1.30 N.A. ×40 oil immersion objective. Donor excitation was achieved on a xenon lamp using a 430/24-nm filter while images were collected simultaneously using a Quad-View system  with emission filters of 470/28, 530/30, and 641/75 nm. Cells were left to rest for 20 min prior to imaging. For calibration studies, we added ionomycin at a final concentration of 5 μM and CaCl2+ peak height was defined as the peak amplitude that occurred during flow exposure. Multiple Ca2+ peaks may occur during the 5-min period of flow. A sample was considered responsive if a Ca2+ peak height was equal to or greater than 1.5 times the maximum baseline oscillation amplitude [Images of each sample were trimmed to regions of interest that tightly enclosed the primary cilium or cell during imaging. Using MATLAB, images were corrected for background and bleedthrough and a pixel-by-pixel basis, resulting in signal only in the cilium and cytosol. Specifically, using MetaMorph, the same background region for ECFP and YPet images was selected from the untrimmed image far from the sample within the field of view, and bleedthrough coefficients were calculated prior to performing experiments . To aid mplitude ,28. Not We used the following primary antibodies: rabbit anti-polycystin-2 , rabbit anti-sera to TRPV4 (generously provided by Heller group), rabbit anti-PIEZO1 (Novus NBP1-78537 for immunostaining and NBP2-10504 for Western blot), mouse anti-acetylated alpha tubulin , and mouse anti-actin . We used the following secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG , Alexa Fluor 568 goat anti-mouse IgG , goat anti-rabbit IgG-HRP , and HRP goat anti-mouse Ig .2, respectively. Upon reaching 80%–90% confluence after 2 days of culture, cells were fixed with 10% formalin, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum and 1% BSA in PBS. Cells were labeled with primary antibodies for PC2, TRPV4, PIEZO1, and acetylated alpha tubulin followed by incubation in appropriate Alexa Fluor-labeled secondary antibodies. Nucleic stain was achieved using DAPI . Confocal z-stack images were obtained on a Leica SP5 using a 1.46 N.A. ×100 oil immersion objective. Maximum X-Z projections were constructed with Leica software.MLO-Y4 and IMCD cells were seeded on 35-mm glass-bottom dishes at approximately 1,000 and 2,000 cells/cmPkd2-, Trpv4-, Piezo1-, and MedGC-scrambled siRNA (Life Technologies) were lysed in RIPA buffer  supplemented with sodium orthovanadate, PMSF, and protease inhibitor cocktail 3 days post electroporation. Five micrograms of each protein sample was run through NuPAGE® Novex® 4%–12% Bis-Tris gels (Life Technologies). After electrophoresis, proteins were transferred to Invitrolon™ PVDF membranes (Life Technologies). The membranes were cut in half to separately label actin bands. Membranes for PC2, PIEZO1, and actin were blocked with 5% BSA (Sigma-Aldrich), and membranes for TRPV4 were blocked with 5% nonfat milk. HRP-conjugated antibodies were detected with chemiluminescence  on a Fujifilm LAS-4000 biomolecular imager.Cells transfected with 2 and cultured for 3 days. Slides were placed in larger, custom-made parallel-plate flow chambers (56 × 24 × 0.28 mm) so that sufficient amounts of RNA are isolated for gene expression analysis. Chambers with slides were incubated for 30 min and then exposed to 5 min of oscillatory fluid flow (OFF) at 1 Hz with a peak shear stress of 10 dyn/cm2. Slides were removed from chambers after 55 min, and RNA was isolated immediately.Transfected MLO-Y4 cells were seeded onto collagen I-coated glass slides at approximately 5,000 cells/cmTrpv4 (Mm00499025_m1), Pkd2 (Mm00435829_m1), Piezo1 (Mm01241570_g1), Piezo2 (Mm01262433_m1), Cox-2 (Mm00478374_m1), and Gapdh (4352339E) primers and probes (Applied Biosystems) by quantitative real-time RT-PCR using the ABI PRISM 7900 detection system (Applied Biosystems). Samples and standards were run in triplicate and were normalized to the endogenous Gapdh expression. Relative gene levels between samples were determined using the relative standard curve method .RNA was extracted from cells using TriReagent (Sigma-Aldrich) and isolated, followed by cDNA synthesis using TaqMan reverse transcriptase (Applied Biosystems). cDNA samples were amplified with t-tests (two-tailed) were used to analyze differences between treated and untreated groups. Comparisons of multiple groups were performed using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test or Bonferroni’s multiple comparison post hoc test for Cox-2 mRNA level comparisons. For all tests, p < 0.05 was considered significant.Results are shown as mean ± SEM. Unpaired 2+-sensitive FRET-based biosensor (CaB) containing ECFP and YPet fused to ARL13B. First, we established that ARL13B localizes to primary cilia in MLO-Y4 osteocyte-like cells and verified ARL13B localization to IMCD primary cilia  enabled us to detect Ca2+ levels within the primary cilium separate from the cytosol. The addition of 5 μM ionomycin, a Ca2+ ionophore, in media containing 1.8 mM Ca2+ led to detectable increases in FRET (represented by the emission ratio of YPet:ECFP fluorescence intensity)  added with ionomycin in Ca2+-free media compared with higher concentrations of CaCl2 . On an individual cell basis, 57% of Ca2+ peaks occurred in the primary cilium prior to the cytosol of the same cell. We also stimulated cells transfected with the inactive biosensor mutALC to verify that the observed peaks during 10 dyn/cm2 OFF were indeed due to increased Ca2+ levels . As expected, we found that Ca2+ release from internal stores is a substantial source of flow-induced Ca2+ mobilization in the cytosol. Under thapsigargin treatment, there was a 45% decrease in responsive samples and a 25% magnitude reduction averaged from those fewer responsive samples in response to flow  vs. control mRNA levels (n = 10), p < 0.005). Upon an initial general analysis of primary cilia from all viable cells (ionomycin-responsive), neither thapsigargin nor any of the siRNA-mediated knockdowns of Pkd2, Trpv4, or Piezo1 exhibited a significant difference in flow-induced Ca2+ peak amplitude relative to untreated samples. Similarly, the only significant difference in peak amplitude relative to untreated samples occurred in the cytosol of cells treated with thapsigargin  for all treatment groups. Thus, a flow-induced peak was considered a response if its amplitude was 1.5× greater than the maximum baseline oscillation amplitude, and we demonstrated that the percentage of cells responding decreased as a peak was considered a larger multiple of maximum baseline oscillation. Out of all the candidate Ca2+-permeable channels, only the loss of Trpv4 clearly impaired the percent of responding cells . We analyzed the response of Trpv4 siRNA-treated cells further and found that 75% of untreated cells had an intense response, a Ca2+ peak height at least five times the maximum baseline oscillation amplitude, compared with only 30% of Trpv4 siRNA-treated cells that had intense ciliary Ca2+ peaks. Pkd2 and Piezo1 deficiencies did not inhibit the percent of ciliary or cytosolic Ca2+ peaks. Interestingly, Piezo1 knockdown sensitized the cytosolic Ca2+ response to flow, perhaps because of a different Ca2+ permeable channel.PC2 mediates mechanically induced ciliary and cytosolic Cas Figure A. Trpv4 s Figure B. Additis Figure B, n = 6.2+ concentrations relative to higher Ca2+ concentrations. Compared with untreated samples, ciliary and cystolic Ca2+ peaks were delayed in MLO-Y4 cells treated with thapsigargin or deficient in Trpv4 and Piezo1 . Scrambled siRNA-transfected cells demonstrated a flow-induced increase in Cox-2 mRNA expression levels, and this flow-induced response was blocked in cells lacking Trpv4 (1.3 ± 0.3-fold (control) vs. 1.0 ± 0.1-fold (Trpv4) increase) ) blocked steady flow-induced ciliary and cytosolic Ca2+ increases in IMCDs normalized to baseline value, and these differences were statistically significant  appears throughout the cell, which is consistent with TRPV4 and PC2 immunostaining in the literature [2+ flux compared with the flux into the primary cilium, it is likely that the machinery and spatiotemporal molecular pathways unique to the primary cilium play a role in downstream mechanotransduction. Our flow experiments revealed that Trpv4 knockdown lowered flow-induced ciliary Ca2+ peaks but did not impair cytosolic Ca2+ peaks. Unlike kidney epithelia, where Ca2+ reportedly enters the primary cilium through PC2 and induces CICR via ryanodine receptor activation [Trpv4 deficiency in osteocytes did not affect cytosolic Ca2+ mobilization. This result suggests that the TRPV4-mediated ciliary Ca2+ microdomain does not regulate CICR in osteocytes. This is different from astrocytes, where TRPV4-mediated CICR regulates neurovascular coupling in an IP3R-regulated system [2+-permeable channels compensate for the loss of TRPV4 function in the cytosol, which is consistent with data from this study showing that the loss of Piezo1 may sensitize cells to flow. Knockdown of PC2, TRPV4, and PIEZO1 channels did not impair the flow-induced cytosolic Ca2+ response, which suggests that a different mechanism is in play that maintains normal cytosolic Ca2+ levels. Taken together, TRPV4’s location in an area of high membrane strain on the primary cilium and dependence of the flow-induced ciliary Ca2+ peak on TRPV4 suggest that the primary cilium acts as a Ca2+ and mechanical signaling nexus dependent on TRPV4.In this study, we present evidence that fluid flow activates TRPV4 on the primary cilium membrane and mediates Caterature -35. Whiltivation , Trpv4 dd system . It is a2+ entry into MC3T3 osteoblasts using gadolinium chloride, which did not eliminate the flow-induced cytosolic Ca2+ flux [2+ flux is not dependent on extracellular Ca2+ entry through stretch-activated channels, Malone et al. also reported that the removal of primary cilia did not affect flow-induced cytosolic Ca2+ flux in osteocytes. The results in this study, here, are consistent with the previous paper and provide additional insight to the understanding of osteocyte mechanotransduction. Using advanced imaging techniques that provide enhanced resolution, our data demonstrate that flow-induced cytosolic Ca2+ flux is independent of extracellular Ca2+ entry through stretch-activated channels; furthermore, flow-induced ciliary Ca2+ flux is dependent on the Ca2+-permeable, stretch-activated TRPV4 channel. Thus, our data suggest that Ca2+ mobilization occurs differently in the cytosol versus the primary cilium during osteocyte mechanotransduction.Consistent with these results, previously, Malone et al. blocked flow-induced extracellular Caa2+ flux . In addiOur understanding of primary cilium bending mechanics and mechanical forces on the plasma membrane covering the primary cilium is essential to elucidating the molecular mechanism of mechanotransduction mediated by the primary cilium. Recently, Young et al. studied the tension force distribution along a primary cilium under flow and suggested that stretch-activated ion channels are likely to be activated and open near the base of the primary cilium where tension force is the highest . While pWe anticipate that TRPV4 will be an attractive pharmacologic target for treating disuse-induced bone loss due to its role mediating osteocyte mechanotransduction and its sensitivity to existing biochemical agents  -43. ThusTrpv4 knockout reduces unloading-induced bone loss due to suppressed osteoclast numbers and impaired bone resorption [Trpv4 is expressed by osteocytes. Osteocytes are mechanosensing cells in bone that regulate osteoclasts’ resorption activities, and it will be important to understand the role of TRPV4 in the population of cells involved in mechanotransduction. The effect of an osteocyte-specific Trpv4 deletion in loading-induced bone formation in vivo would provide evidence indicating whether TRPV4 is involved in the mechanotransduction process versus restricted to mediating osteoclast numbers and bone resorption. Additionally, it is important to recognize that primary cilia play important roles in development; however, in this study, we have not examined how TRPV4 plays a role in developmental ciliary pathways such as Hedgehog and Wnt signaling pathways.Other groups have suggested that TRPV4 is a candidate therapeutic target for bone loss disease. Mizoguchi et al. and Masuyama et al. have examined TRPV4 deficiency in unloading-induced bone formation, and they determined that sorption ,48. Inte2+ mobilization occurs in primary cilia before cytosolic Ca2+ increases in kidney epithelial cells, our flow studies do not demonstrate a clear difference in the timing between ciliary and cytosolic Ca2+ peaks in osteocytes [2+ peaks occurring before cytosolic Ca2+ peaks is numerically higher than the number of ciliary Ca2+ peaks occurring after cytosolic Ca2+ peaks, it is unclear if ciliary Ca2+ triggers cytosolic Ca2+ increases. Unlike the side-dimension imaging method leveraged by Jin et al. to capture calcium signaling along the length of the primary cilium, our imaging method collected signal from only a part of the primary cilium that was in plane. Thus, we are unable to characterize a relationship between ciliary and cytosolic Ca2+ peak timing.While Jin et al. reported that flow-induced Cateocytes . While t2+ mobilization is dependent on PC2 in kidney epithelia [2+ mobilization is dependent on TRPV4, and not PC2, in osteocytes. We conducted similar flow studies with kidney epithelial cells to verify that this difference was due to cell type and not the experimental approach. Our studies with IMCD cells also showed that flow-induced ciliary and cytosolic Ca2+ increases depend on PC2. The consistency in our results suggests that the mechanism of mechanotransduction mediated by primary cilia varies across different tissue contexts. Another difference between the MLO-Y4 and IMCD flow studies was the type of flow regime, consisting of OFF resulting in a 10-dyn/cm2 shear stress for MLO-Y4 cells and steady flow resulting in a 5-dyn/cm2 shear stress for IMCD cells. Previously, Malone et al. determined that flow-induced Ca2+ flux differences in MC3T3-E1 osteoblasts and MDCK kidney cells did not depend on these specific flow regimes, which suggests that primary cilium-mediated mechanosensation in osteoblasts and kidney cells is indeed different [Several other groups have demonstrated that flow-induced ciliary Capithelia ,18,31. Oifferent . Thus, t2+ flux of seconds and minutes. Here, we studied the early signaling response to flow occurring within seconds after exposure to flow. Thus, our finding that PC2 was independent of mechanically induced ciliary and cytosolic Ca2+ signaling in osteocytes does not contradict the findings by Xu et al. It is possible that PC2 is involved in the later downstream signaling response to flow. While we found that PC2 may not be involved in the early Ca2+ response to flow, it is nonetheless an important channel in bone. Pkd2 mutations have been implicated in skeletal development [Pkd1, encoding the other subunit of the polycystin complex, have impaired both skeletal development and adaptation [Interestingly, in addition to being well-established in kidney epithelial mechanotransduction ,18,31, Pelopment ,51, and aptation .2+ mobilization is different between kidney epithelia and osteocytes. Strikingly, the osteocyte primary cilium forms a distinct microdomain from the cytosol during mechanical loading. The primary cilium microdomain may help maintain spatiotemporal organization within the cell which allows numerous molecular mechanisms to occur with just a limited number of signaling molecules. We expect that the Ca2+-permeable channel TRPV4 will be an attractive therapeutic target for bone loss disease because of its location in an area of high membrane strain on the primary cilium, demonstrated role as a physical transducer in osteocytes in this study and chondrocytes in a recent study by O’Conor et al., and sensitivity to existing biochemical agents [Trpv4 will elucidate how TRPV4-mediated Ca2+ entry in the primary cilium microdomain regulates osteogenic responses to mechanical loads.In conclusion, this study highlights the specialization of primary cilium mechanisms across different tissue contexts. Here, we demonstrate that mechanically stimulated ciliary Cal agents . Further


Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2  to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca Our results support this hypothesis, since a stimulatory glucose concentration generated ROS that increased RyR S-glutathionylation, while RyR inhibition or the antioxidant N-acetyl cysteine (NAC) significantly decreased or abolished GSIS. The main findings of this work were previously presented in abstract form , R-115).In other cell types, ROS stimulate RyR-mediated CICR . Given t2DCFDA), Dispase-EDTA, Dulbecco modified Eagle’s medium, BODIPY-FL-X Ryanodine (BODIPY-Rya) and Calcium Calibration Kit 1 with Magnesium were from Invitrogen . Ryanodine was from Alexis Biochemical , and H2O2 from Merck . The Duolink II red starter kit was from Olink-Bioscience .All reagents used were of analytical grade. Caffeine, NAC, polylysine, RPMI 1640 culture medium and carbamylcholine chloride  were from Sigma-Aldrich Chemical . Fura-2 acetoxymethyl ester (fura-2-AM), Fluo-4 acetoxymethyl ester (Fluo-4-AM), 5-(6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate acetyl ester .Anti-insulin antibodies were from Dako , anti-RyR2 from Affinity BioReagents  or Millipore Corp.  and anti-calnexin from Sigma . The secondary antibodies used were anti-guinea pig FITC from Jackson Immuno Research . Alexa Fluor 635 anti-mouse IgG, and Alexa Fluor 635 anti-rabbit IgG, were both from Invitrogen . Antibodies against Male Sprague-Dawley rats weighing 250–300 g (45–60 days old) were obtained from the Central Animal Facility of the Faculty of Medicine, Universidad de Chile. The animals were kept at 23°C under a 12 h light–dark cycle, with free access to food and water. The Bioethics Committee for Animal Research, Faculty of Medicine, Universidad de Chile, approved all experimental protocols used in this work.2/5% CO2. The RPMI 1640 culture medium was supplemented with 5 mM glucose, 10% fetal bovine serum (FBS), 10 mM HEPES, 100 units/ml penicillin, 100 μg/ml streptomycin. Cell viability was evaluated as described in detail elsewhere i would cause the increase in insulin secretion induced by H2O2. To test this hypothesis, we measured [Ca2+]i with the fluorescent probe fura-2  to a value of 324 ± 5.4 nM . This increase occurred as a consequence of RyR-mediated Ca2+ release since overnight incubation with inhibitory ryanodine prevented the fast [Ca2+]i increase produced by H2O2  that generates a fluorescence signal if the targets lie within an optimal distance of 30–40 nm. In this particular case, the two targets were the RyR2 protein and S-glutathionylated protein adducts. Isolated β-cells stimulated for 1 h with 16.7 mM glucose displayed a significant increase in fluorescent dot density  stimulates insulin secretion , while 1While ROS are damaging to cells when present in excess, controlled ROS generation plays a central role in cell signaling , 51. Pre2+ when key cysteine residues are in the reduced state i to the levels required for insulin secretion. This proposed mechanism is supported by the present results, showing RyR-dependent [Ca2+]i increase after addition of H2O2, as discussed below, and by results obtained in other cell types, where addition of exogenous H2O2 promotes RyR redox modifications and specifically stimulates RyR-mediated Ca2+ release i, which reached values close to 400 nM after H2O2 addition. These levels are within the range of the [Ca2+]i increases elicited by depolarization of human β-cells [2O2 at basal glucose concentration is due to an increase in [Ca2+]i, and extends previous reports showing that H2O2 increases [Ca2+]i to similar levels in islets and β-cell lines through a process that implicates Ca2+ release from the ER i in a dose-dependent manner; this increase is partially sensitive to blockers of L-type channels and is abolished by thapsigargin i to levels that promote exocytosis of insulin-containing granules, albeit the source of Ca2+ remained undefined. Our findings suggest that H2O2-induced RyR-mediated Ca2+ release is a major contributor to the increase in [Ca2+]i, since H2O2 did not increase [Ca2+]i in cells pre-incubated overnight with inhibitory ryanodine. The present results provide the first evidence that RyR channels are involved in the [Ca2+]i increase induced by H2O2 in β-cells.In summary, there is consensus that at basal glucose concentration H2+]i increase produced by Ca2+ entry from the extracellular medium in response to glucose-induced β-cell depolarization. Although not directly tested here, the glucose-induced increase in ATP concentration may also contribute to enhance RyR channel activation by Ca2+, as reported in single RyR channels from neuronal cells [2+]i increase required for insulin secretion. Our hypothesis, presenting GSIS as the combined result of glucose-induced Ca2+ entry and glucose-induced ROS generation leading to enhanced RyR-mediated CICR, adds a new concept to the physiology of the pancreatic β-cell. Our results may also explain why prolonged glucose elevations, which promote oxidative stress [According to the model proposed in this study , the incal cells . The rese stress , adverseS1 Fig(A) MIN6 cells. Immunostaining directed against RyR2 (green) and the ER marker calnexin (red). The right hand panel illustrates the combined red and green fluorescence plus the blue (Hoechst) nuclear staining. (B) Images were collected from a single pancreatic β-cell. Immunostaining directed against RyR2 (green) and the ER marker calnexin (red). The image at right shows the superposition of green and red fluorescence. Bars indicate 20 μm.(TIF)Click here for additional data file.S2 Fig(A) RyR2 mRNA was determined by conventional PCR, using the following primer sequences, which are specific for the RyR2 isoform: RyR2sense: 5'-CTACTCAGGATGAGGTCGGA-3'; RyR2antisense: 5'-CTCTCTTCAGATCCAAGCCA-3'. Lane ST: standard; lanes 1, 2, 5 and 6: RNA extracted from rat primary hippocampal neurons. Lanes 3 and 4: RNA extracted from rat pancreatic islets. Lanes 5 and 9: negative controls. The amplified fragment for RyR2 corresponds to 157 bp. (B) RyR2 protein levels in primary hippocampal neurons and MIN6 cells were assayed by Western blot analysis as described in the text.(TIF)Click here for additional data file.S3 FigImages were acquired after incubation of pancreatic islets with this probe for 1 h (A) or 12 h (B); both images were obtained by confocal microscopy with identical acquisition parameters, allowing qualitative comparisons. The images at left correspond to fluorescence and at right to transmitted light. Calibration bars: 50 μm.(TIF)Click here for additional data file.S4 Fig2+-free solution just before starting the record. Fluorescence values are expressed as (F/F0), where F0 represents the basal fluorescence recorded before addition of thapsigargin. Addition of 5 μM thapsigargin  elicited similar Ca2+ signals in controls (upper panel) as in isolated β-cells pre-incubated with 200 μM ryanodine for 1 h (middle panel) or overnight (bottom panel). (B) Quantification of the areas under the curve. (C) Quantification of maximum fluorescence intensity. In A to C, values represent Mean ± SEM, (N = 3–6 cells from 2 rats). Statistical significance was determined with one-way ANOVA followed by Tukey's multiple comparison test. ns: no significant differences. (D). Representative fluorescence images (upper) of islets loaded with 10 μM CM-H2DCFDA, collected by confocal microscopy; at bottom, light-contrast images. (E) Quantification of H2DCFDA fluorescence intensity determined in control islets, in islets pre-incubated with 200 μM ryanodine for 1 h or overnight, or treated with 0.5 mM H2O2 for 1 h. N = 4–10 islets. ***: p < 0.001, determined by statistical analysis with One-way ANOVA, followed by Tukey’s post-hoc test.(A). Time course of Fluo-4 fluorescence recorded from isolated β-cells before and after addition of thapsigargin to cultures loaded with Fluo-4 AM and transferred to Ca(TIF)Click here for additional data file.S5 FigThe effects of NAC were tested in either basal (2.8 mM) or stimulatory (27.7 mM) glucose (G) concentrations. Values represent Mean ± SEM, N = 3. Statistical significance was determined with one-way ANOVA followed by Tukey's Multiple Comparison Test. *: p <0.05; ***: p <0.001; ns: no significant differences.(TIF)Click here for additional data file.S6 Fig2O2 (100 μM) or stimulatory glucose (16.7 mM) plus NAC (10 mM).The figure displays representative confocal images acquired in disaggregated β-cells from islets, showing PLA labeling (red), insulin immunostaining (green) and the merged images. From left to right, images were taken at different depths, from the bottom to the top of cells incubated in basal glucose (2.8 mM), stimulatory glucose (16.7 mM), basal glucose (2.8 mM) plus H(JPG)Click here for additional data file.


Nowadays, monitoring focuses on the primary compounds and does not include degradation products formed during various biological and chemical processes. Transformation products may have the same effects to human health and the environment or sometimes they can be more toxic than the parent compound. Unfortunately, knowledge about the formation of degradation products is still limited, however, can be very important for the environmental risk assessment. Firstly, the photodegradation kinetic of amlodipine was investigated in two experimental conditions: during the exposure to solar radiation and during the exposure to the light emitted by the xenon lamp. In all cases degradation of amlodipine followed a pseudo-first-order kinetics. In the next step, identification of transformation products of amlodipine formed during the exposure to xenon lamp irradiation was performed using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). As a result sixteen photoproducts were identified, their structures were elucidated and ultimately the transformation pathway was proposed. Fifteen compounds (out of 16 photoproducts) were newly identified and reported here for the first time; some of those compounds were formed from the first photoproduct, amlodipine pyridine derivative. Several analytes were formed only in acidic or basic conditions. Furthermore, the occurrence of amlodipine and its identified degradation products was investigated in environmental waters. Only one out of 16 compounds was found in wastewater effluent. The possibility of the sorption of examined analytes to sewage sludge particles was discussed based on QSAR. There are many research which focus on the presence of pharmaceuticals in the aquatic environment and the removal efficiency of these emerging contaminants during wastewater processes in wastewater treatment plants (WWTP) is limited The result of the chronic exposure to low doses of pharmaceuticals and their TPs still remain unknown. Their level in the environment is not regulated, and thus these compounds are considered environmentally hazardous −1Hydra vulgaris at the exposure level of 10 µg L−1Ceriodaphnia dubiaThe compound selected for degradation study was amlodipine - a long-acting calcium channel blocker used to lower blood pressure and to treat anginal chest pain 1O2, ROO•, 3DOM*, e−aq generated by photosensitizers (indirect photolysis) The most important processes which may occur in the aquatic environment include biodegradation, photodegradation and hydrolysis. Biological processes allow only a limited transformation of pharmaceuticals due to their biopersistance Nowadays, investigation of suspected compounds e.g. TPs, their structure elucidation and/or confirmation requires application of highly accurate, selective and reliable techniques. To meet the present expectations, the hyphenated techniques based on liquid chromatography and high resolution mass spectrometry (LC-HRMS) have become a powerful tools In light of these concerns and the lack of scientific literature on phototransformation of a widely prescribed drug, amlodipine, the main aim of this study was to identify transfromation products formed during the continuous exposure of amlodipine water solution to UV/VIS irradiation emitted by xenon lamp. Furthermore, studies on photodegradation kinetics with the simulation of the conditions in natural aqueous waters were undertaken. The final analysis were performed with the application of LC-QTOF-MS technique providing reliable results with mass error <5 ppm. To the best of our knowledge, this is the most extensive study on photodegradation products of amlodipine including kinetics and structure elucidation followed by the proposal of its phototransformation pathway. As a result 16 degradation products were identified. The first product identified was pyridine derivative of amlodipine (known in the literature), and the other fifteen were compounds formed from either the parent molecule or its first degradation product and were reported here for the first time. The proposed structures of newly identified compound were elucidated and verified on the basis of data obtained during LC-QTOF-MS analysis including fragmentation mass spectra of (pseudo-)molecular ions, accurate mass measurements and isotope patterns of each compound. Finally, the presence of amlodipine and its transformation products was investigated in various environmental waters. The application of QSAR allowed the estimation of possible sorption of target compounds on sewage sludge particles.−1 was prepared in methanol and was stored at −80°C.Analytical standards of amlodipine (AML) were purchased from Sigma-Aldrich . LC-MS grade methanol (MeOH) was obtained from Sigma-Aldrich . Formic acid was from Merck . Ultrapure water was obtained from a HLP5 system . MeOH used for extraction were LC grade and were purchased from Merck . Individual stock solution of AML at concentration level of 1 g LDegradation experiments were performed using a small-scale system that consisted of a cylindrical photoreactor (25 mL) equipped with quartz window and cooling system, external light system and optical filters. The radiation was emitted by 1000W xenon lamp  which transmitted light of wavelength ranged from 250 to 1000 nm. Optical filters  were used for cutting off the IR irradiation. This equipment was previously applied for identification of phototransformation products of three other pharmaceuticals  in river water samples −1 since higher initial concentration provides TPs in higher amount (this increases the possibility of proper identification and structure elucidation of TPs). 25 mL of aqueous sample containing AML was stirred magnetically during the irradiation experiment. Aliquots of about 500 µL were collected during the experiment every 15 minutes, filtered through a 0.45 µm nylon syringe filter  and directly subjected to UHPLC-QTOF-MS analysis for further data evaluation.The temperature of the samples during the experiment was maintained at 10°C. The photodegradation experiment was performed in river water (free from the targets) at three different pH . The concentration of AML was 1 mg LPhotodegradation kinetics were designed in two ways: (1) as a result of an exposure to xenon lamp light , (2) as a result of a continuous exposure to solar radiation .−1. The samples were kept in glass vials (25 mL) exposed to natural sunlight; the experiment was performed during the summer 2013 in order to provide sufficient sunlight intensity. The aim was to investigate the influence of the matrix and pH on degradation rate of AML.Degradation under continuous exposure to solar radiation was performed for samples characterized by eight different matrices: wastewater influent and effluent, untreated and treated water, river water, methanol, ultrapure water for pH 3 and pH 10. For this purpose, specific matrix was spiked with AML at the concentration level of 1 mg LDegradation under xenon lamp irradiation was performed only for river water samples (as well as for the identification of TPs). To evaluate the degree of degradation of AML, UPLC-QTOF-MS analysis was performed.−1 in gradient elution . The column was maintained at 22°C and the injection volume was 5 µL.Both chromatographic conditions and mass spectrometer parameters were previously described in other publication −1, capillary voltage 4000 V, nebulizer pressure 20 psig, drying gas 10 L min−1, gas temperature 325°C, skimmer voltage 45 V, octopole RF peak 750 V and fragmentor voltage 100 V. Analysis were performed using MS/MS or TargetMS/MS mode with variable collision energies  and in mass range 50–1000 m/z. The instrument was operated in the 4 GHz high-resolution mode and acquisition rate was 1.5 spectra per second. Acquisition data were processed with Agilent MassHunter Workstation software.The UHPLC system was coupled to a hybrid quadrupole time-of-flight (QTOF) mass spectrometer (Agilent 6540 Series Accurate Mass QTOF-MS) with Dual ESI interface and operated in positive ion mode. Operation conditions were as follow: sheath gas temperature 400°C at flow rate of 12 L minAqueous samples collected from the Gdańsk area were: influent and effluent water samples from a wastewater treatment plant (WWTP), and river water from Kacza River exposed to anthropogenic activity. Samples were collected in pre-rinsed amber glass bottles, filled to the brim in order to reduce analytes transition to the gas phase. They were stored at 4°C and analyzed within 24 h for the presence of AML and identified photoproducts.−1 . Later, the sorbent was dried for 15 min. The analytes retained on sorbent were eluted with 6 mL (3×2 mL) of MeOH and obtained extracts were evaporated to dryness in a stream of N2. Dry residues were redissolved in 1 mL of MeOH/H2O  and 5 µL of the final extract was injected into the UHPLC-QTOF-MS system.Samples were prepared by solid phase extraction (SPE) with the application of Oasis HLB cartridges . SPE cartridges were preconditioned with 5 mL of MeOH and 5 mL of ultrapure water. 1 mL 0.1 M EDTA/100 mL sample was added before loading. A volume of 100 mL, 200 mL and 500 mL of influent, effluent and river water, respectively, were loaded on the sorbent at a flow rate of 5 mL minThe reaction kinetics included the calculation of the degradation rate and the assessment of the influence of external factors on this parameter. This was determined based on the dependence of the product formation/substrate decrease rate on initial concentration of the substrate and the obtained relation allowed determining the reaction kinetic equation. The degradation reaction kinetics, i.e. the reaction order, the rate constant and the half-life were determined for AML for the degradation process induced by xenon lamp irradiation, as well as the degradation process caused by continuous exposure to sunlight, for samples characterized by various matrices. All experiments lasted until the decrease of AML concentration at least below 10% of the initial value. The results are presented in k) was determined according to the Eq. (1):tC is the concentration of the compound at the irradiation time t and 0C is the initial concentration of the analyte.The photodegradation of AML in river water induced by the irradiation with xenon lamp followed a pseudo-first-order kinetic , which w1/2t was calculated using Eq. (2) which was derived by modifying Eq. (1) and replacing tC with 0/2C:Half-life 1/2t = 8.8 min), the experiment was performed for additional 30 min  in order to degrade the pyridine derivative of AML, which was created after 15 min of the exposure, and to identify additional transformation products. The obtained results are consistent with the literature since the photodegradation reactions induced by a constant irradiation are usually followed by the first-order kinetics Even though the signal from AML was not observed after 60 min of the exposure to xenon lamp light  which may adversely affect the identification procedure. Not every wavelength reaches the Earth at the same intensity and the contribution of the low wavelengths (below ∼250 nm) is limited and dependent inter alia on the latitude. In the aquatic environment direct and indirect photolysis occurs simultaneously. The occurrence of direct photolysis depends on the overlap of the absorption spectrum of the compound with the wavelength range emitted by the radiation source, and therefore, compounds with the absorption maxima in the range of low wavelengths will more probably undergo to indirect photolysis. However, in the case of a xenon lamp irradiation the occurrence of direct photolysis is equally possible because it emits radiation of constant intensity. Amlodipine has two absorption maxima: 240 and 360 nm  and evenThe identification of photoproducts was performed only in river water samples exposed to xenon lamp radiation. This matrix was chosen for several reasons: river water is representative for the aquatic environment, it is a source of drinking water, a place of wastewater discharges and popular recreational place for people. Identification of photoproducts was performed by UHPLC-QTOF-MS analysis of river water sample spiked with AML exposed to a xenon lamp radiation and collected at specified intervals. Firstly, (pseudo-)molecular ions in the full scan acquisition mode which occurred during the experiment and could respond to probable photodegradation products were selected. Later, the analysis in TargetMS/MS mode was performed in order to obtain the fragmentation spectra of selected compounds. As a result, it was possible to verify the pre-selected compounds and elucidate the structures of photodegradation products. In order to provide reliable results acceptable accuracy mass measurement error was below 5 ppm for (pseudo-)molecular ions and 10 ppm for fragment ions. This analytical approach, including specific identification evidence  as indicated by other authors + transformation compounds ranged from −4.5 to 5.3 ppm. The accurate mass MS/MS spectra for identified photoproducts were obtained by fragmenting the selected protonated molecule [M+H]+ at specific m/z value. Based on the fragmentation patterns observed in the MS/MS spectra the molecular formula and structure of the products were then tentatively assigned. These accurate mass measurements of both precursor and product ions together with the fragmentation pattern of each photoproducts obtained by UHPLC-QTOF-MS analysis were of high importance while verifying the structures and elucidating the transformation pathway of amlodipine transformation. In the main article only two exemplary MS/MS spectra (for AML 1 and 2) are presented; the other spectra (for AML 3 – AML 16) are presented in supplementary materials (Xenon lamp irradiation of the studied AML resulted in the formation of new chromatographic peaks corresponding to transformation products generated during the photolysis process. Xenon lamp irradiation of AML produced a further sixteen new products (AML 1–16), and fifteen of them are reported here for the first time. All photoproducts had lower molecular weights than the parent compound and photoinduced dimerization was not observed. Detailed results presenting the identified compounds are listed in aterials .The first photoproduct found was amlodipine pyridine derivative . Formation of this product was explained on the basis of a radical cation intermediate according to Fasani's investigation Xenon lamp irradiation of AML solution caused after 15 minutes formation of further seven new products. Formation of AML 2 (m/z 389.1722) is postulated to occur from AML 1 by loss of chlorine atom and hydroxylation of benzene ring by hydroxyl radicals present in the aqueous environment under photolysis Another photoproduct detected after 15 minutes of the exposure of water solution to xenon lamp is AML 5. This product with the m/z value 348.1008 may correspond to the derivative after loss of 2-aminoethenol fragment from AML 1 compound. The most important fragment in the mass spectrum of product AML 6 is m/z 330.1344 and may correspond to the disconnection of chlorine atom and etheneamine from the AML 1 product. Compound AML 7 with the m/z value 339.1351 was probably formed by dehalogenation, oxidation and elimination of formaldehyde molecule from AML 1 product, whereas AML 8 (m/z 343.1298) results by loss of methyl substituent and transformation of the methyl ester to carboxylic acid derivative, followed by dehydrogenation.Compound AML 9, which structure corresponds to the AML 3 byproduct after elimination of the furanylmethyl group (m/z 298.1074), was identified in the sample taken after 30 minutes of continuous exposure of the amlodipine solution. Further irradiation of amlodipine (45 min) revealed formation of another degradation product . This compound was assumed to be the result of elimination of methyl substituent from AML 9 photoproduct.In the mass spectrum of product AML 11, the most important fragment is the m/z 329.1142, which may corresponds to the hydrolysis of ethyl ester of AML 4 photoproduct.The irradiation of a sample containing amlodipine in a water solution acidified to the pH 3 led to the formation of another compound AML 12. This product with the m/z value 381.1212 may correspond to amlodipine derivative after hydrolysis of ethyl ester.Exposure of this drug solution to xenon lamp light in the water at pH 10 caused formation of four photoproducts. Compound AML 13 and AML 14 originated from amlodipine after aromatization (AML 1); however, AML 15 and AML 16 were formed directly from amlodipine. The most important fragment in the mass spectrum of product AML 13 is the ion of m/z 379.1035, which corresponds to hydrolysis of ethyl ester from the parent molecule. The structure of m/z value 37.1261 (AML 14) may correspond to AML 1 product after transformation of the methyl ester group to aldehyde moiety. In the mass spectrum of product AML 15, the most important fragment is the m/z 366.1107, which may correspond to the elimination of etheneamine fragment from the AML. The final compound detected in this solution  was probably formed by hydrolysis of methyl ester from the AML 15 molecule.Irradiation of amlodipine solution by the xenon lamp has been terminating after 90 minutes. No signals originate from AML was recorded, whereas the presence of several degradation products were still detected in the examined solution.On the basis of the degradation products identified in this experiment the hypothetical amlodipine degradation pathway was suggested, and schematically shown in The presence of amlodipine and identified compounds of its phototransformation was investigated in wastewater (influent and effluent) and river water in order to estimate the actual pollution of the environment. The analysis was performed using UHPLC-QTOF-MS system in TargetMS/MS mode. Confirmation of the presence of degradation products was done by using information obtained during the analysis and MS/MS fragmentation of (pseudo-)molecular ion (compliance of retention time (deviation <2%), mass accuracy of (pseudo-)molecular ion (<5 ppm) and minimum two fragment ions (<10 ppm), and relative intensity of fragment ions (deviation <5%)).−1Photodegradation products of amlodipine were not detected in the tested samples, except for AML 2 which was present in the wastewater effluent (average peak area ± SD (n = 4)  = 1,1E+05±2,0E+04). These results can be interpreted in various ways. Amlodipine as a parent compound is rarely detected in surface water and wastewater samples ow, are lower than 4, which indicate high solubility in water of most of the examined compounds (except for AML 5 which log Kow was 4.47). The calculations of soil adsorption coefficient, log Koc, gave more specific results suggesting low or negligible sorption to sewage sludge and moderate or rapid migration to ground water for most of the compounds. The exception was observed for AML 5 (log Koc = 3.44) indicating its moderate sorption do sludge particles, and it was also observed in STP model, which estimated its removal by sewage sludge on a level of 54%. An increased sorption was noticed for AML 15 and AML 16 as well, however, it was considered as insufficient to conclude that the sorption process was the most dominant one in their removal.However, there is a possibility that both parent compound and TPs are partially adsorbed on the sewage sludge particles, which are known to sorb certain organic compounds on their surface Although, the predicted removal of AML and its photoproducts through the sorption to sewage sludge particles was estimated on the level below 6% for most of the analytes, the presence of AML in the sludge was reported previously The occurrence of only the photoproduct AML 2 may suggest that the amlodipine was present in the wastewater; however, it degraded or migrated to other parts of the environment e.g. ground water. Ongoing research confirms that tracking the fate of not only amlodipine but also other pharmaceuticals in the aquatic environment wider the knowledge of the environmental pollution.k) and half-lives (1/2t) were determined for both experiments. The identification of degradation products was the main aim of this research, and it was performed with the application of xenon lamp irradiation in order to independent the experiment from the external conditions, which may influence the experiment, and to provide a constant light source with a wavelength range overlapping the natural sunlight wavelength. As a result sixteen transformation products were identified, and 15 TPs are new compounds which structure was elucidated and reported here for the first time. It was discovered that the degradation process do not stop after the formation of the first photoproduct (amlodipine pyridine derivative), but it is progressing and reveal the existence of various compounds, some of them are created from the first photoproduct. It was also discovered that some TPs are formed in acidic (pH 3) or basic (pH 10) conditions. As a result, transformation pathway of amlodipine was proposed.The photodegradation of amlodipine was investigated using UHPLC-QTOF-MS. The degradation kinetics of AML was determined for two types of experiments: with the exposure of the samples to solar radiation, and with the application of xenon lamp radiation. It was observed that all the reactions follow a pseudo-first-order kinetics, and the time-based pseudo-first-order rate constants . Only AML 2 was detected in four wastewater effluent samples. Performing the QSAR proved that sewage sludge adsorption is not a dominant process in AML removal . In general, the values of log KUHPLC-QTOF-MS is a very powerful tool for the identification of TPs in the aqueous environment providing accurate mass measurements, MS/MS fragmentation or isotope profiling which are of a high significance while identifying degradation products and elucidating/verifying their structure. To the best of our knowledge this is the most extensive study on amlodipine photodegradation published so far.Figure S1Absorbance spectrum of amlodipine.(TIF)Click here for additional data file.Figure S2MS/MS fragmentation spectra of amlodipine and its 16 photoproducts identified by UHPLC-QTOF-MS.(TIF)Click here for additional data file.Table S1Detailed data used for photodegradation kinetics calculations .(DOCX)Click here for additional data file.


Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. Genome integrity is constantly threatened by DNA lesions resulting from both endogenous stress and exogenous insult. Of the many types of genomic damage, double strand breaks (DSBs) present the greatest threat to genomic health. Left unrepaired, a DSB can result in the loss of substantial genetic material. Alternatively, if DSBs are repaired incorrectly this may result in chromosomal structural abnormalities; including dicentric chromosomes that drive further genome instability through a breakage-fusion-bridge cycle. Eukaryotic cells have thus evolved a sophisticated DNA damage response (DDR) that controls DNA repair and cell cycle arrest to cope with the dangers presented by genotoxic stress While it is critical that cells engage in efficient DSB repair, it is just as important that DSB repair is silenced under certain conditions. One such condition is at the naturally occurring chromosome ends where DSB repair is prevented by specialized nucleoprotein structures called telomeres Spatiotemporal localization of DDR factors during DSB repair is routinely analyzed through observation of cytological ionizing radiation induced foci IRIF, . In brieIn addition to regulating DSB repair, the DDR also controls interphase cell cycle arrest in response to genotoxic stress While there were indications that the DDR differed in mitotic and interphase cells Orthwein et al. Like RNF8 pT198, 53BP1 pT1609 and pS1618 are residues phosphorylated specifically during mitosis Both studies found that generating a double phosphomimetic allele (53BP1 T1609E S1618E or “53BP1 TESE”) resulted in the exclusion of 53BP1 TESE from mitotic and interphase IRIF Mitotic cells are sensitive to DSBs, presumably because DSB repair is silenced Discussed below is the current understanding of how the telomeres cooperate normally with the DDR to mediate cell cycle arrest and the implications of the findings by Orthwein et al. on telomere-dependent tumor suppression.DDR activity is modulated at chromosome ends by the telomere-specific “shelterin” protein complex However, an emerging theme in telomere biology is that telomere deprotection under physiological or pathological conditions is different from telomere dysfunction induced by genetically deleting shelterin subunits We now understand that human telomeres adopt three distinct protective states in relation to DDR activation and DSB repair Observation of spontaneous intermediate-state telomeres in immortalized or cancer cells indicated that TRF2 possessed independent protective functions which (1) inhibited DDR activation and (2) suppressed NHEJ Also reminiscent of the mitotic DDR, intermediate-state telomeres in asynchronous cultures induce muted ATM checkpoint signaling where ATM is activated but CHK2 and NBS1 are not phosphorylated In the recent study using TRF2 knockdown to induce intermediate- and uncapped-state telomeres it was observed that the telomere DDR is refractory to G2/M checkpoint activation The initial static observations of spontaneous telomere deprotection revealed that γ-H2AX, MRE11 and MDC1, but not 53BP1, localized to metaphase-TIF  . For DSBWhy intermediate-state telomeres fuse when mitotic DSB repair is activated is not immediately clear, as some TRF2 remains bound at metaphase-TIF in aged cells and during prolonged mitotic arrest The passage of intermediate-state telomeres through mitosis is likely a critical feature of telomere-dependent tumor suppression Intermediate-state telomeres routinely pass through mitosis in increasing abundance during cellular aging, until sufficient numbers accumulate to induce senescence When DSB repair competent mitotic cells were irradiated to induce non-specific genome wide damage, this resulted in telomere-fusion-dependent genomic instability A potential answer is that Aurora B activation of the telomere DDR during prolonged mitosis is a general response to accumulating stress in mitotic cells. This is supported by the observation that increasing duration of mitotic arrest corresponds with increasing numbers of metaphase-TIF The recent discoveries described here highlight our emerging understanding of the complex relationship between telomeres, mitosis, and the DDR. Suppressing DSB repair during mitosis appears to ensure that intermediate-state telomeres pass through cell division to facilitate telomere-dependent growth arrest in stable diploid G1 phase cells without inducing genomic instability. However, much about the connectivity between mitosis, DSB repair and telomeres remains to be understood. For example, what is the molecular mechanism that facilitates telomere fusion in DSB repair competent mitotic cells? Also, what is the mechanism linking genomic breaks induced during mitosis to the Aurora B dependent mitotic telomere DDR, and how does this impact cells with silenced mitotic DSB repair? It is also unclear if 53BP1 dissociation from TIF at the G2/M boundary is controlled by the same mechanism that prevents 53BP1 from associating with mitotic IRIF or if there is another independent level of control regulating mitotic DSB repair that remains to be discovered.An appealing prospect raised by the discovery of the mechanism inhibiting mitotic DSB repair is that the spontaneous occurrence of abundant intermediate-state telomeres in many cancer cells may present a vulnerability to be exploited for therapeutic means. A combinatorial approach that arrests cancer cells in mitosis while simultaneously targeting mitotic kinases and/or the DDR may hold promise to induce telomere fusions that kill tumor cells. It will be exciting to unravel this surprising and complex relationship between telomeres, mitosis, and DSB repair and to identify the implications of this relationship on human aging and oncogenesis.


The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.2+ handling in heart failure remains unclear.The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca2+ transient (CaT), cell shortening (CS), the frequency of diastolic Ca2+ sparks (CaSF), and sarcoplasmic reticulum Ca2+ concentration ({Ca2+}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).We investigated the effect of milrinone plus landiolol on intracellular Ca2+}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca2+}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca2+ leak.A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca Morerhythmia , 2. Therrhythmia , 2, 3–6.2+ overload [Short-term inotropic therapy may benefit patients with acute decompensated heart failure (ADHF) corresponding to Forrester subset IV by reducing symptoms and improving endo-organ perfusion , 8. Howeoverload , 11.1/2: 4 min) and β1-selective adrenergic receptor blocker (β1/β2 = 255), similar to esmolol, with a significant chronotropic effect and little or no negative inotropic effect at low doses [The use of a β-blocker in combination with inotropic agents to treat ADHF has been contraindicated. In cases where acute heart failure with tachycardia is refractory to standard treatments such as diuretics, vasodilators, and milrinone , a low-dose β-blocker might be effective for treating ADHF, if it has modest negative chronotropic but few cardiosuppressive effects. Landiolol  is the most ultrashort-acting intravenous  used in the present study, were all female beagle dogs . Housing and husbandry conditions at Science Research Center at Yamaguchi University are as follows see .Housing: A large separate gage (D90cm x W85cm x H80cm) were given to each dog (number of gage was 12)Husbandry condition: light /dark cycle (12hrs/12hrs) 7am–7pm; temperature 70°F ± 2°F; Food; food for experimental animals  was given every day. Water; drinking water.Health check was performed by stuffs every day before and after pacemaker implantation in both operated group and non-sham operated group. If necessary, animal doctors saw dogs and treated them.In 6 adult beagle dogs (10–13 kg), heart failure was induced by continuous application of rapid right ventricular pacing at 250 bpm using an externally programmable miniature pacemaker  for 28 days, as described previously , 25. Dog2/5%CO2 -bubbled Minimal Essential Medium (Sigma) supplemented with 50 μM Ca2+, 0.5 mg/mL and 0.02 mg/mL protease type XIV). Then, antegrade perfusion from the coronary artery branch was performed for 1 hour with perfusion buffer with collagenase  supplemented with 50 μM Ca2+, 0.5 mg/mL collagenase B, 0.5 mg/mL, collagenase D and 0.02 mg/mL protease type XIV). The temperature of the perfusion buffer kept 37°C. Finally, the perfused LV was minced with scissors and rod-shaped adult canine cardiomyocytes were prepared. The Ca2+ concentration in the incubation medium was gradually increased to a final concentration of 1 mM . The isolated cardiomyocytes were transferred to laminin-coated glass culture dishes and incubated for 12 h at 37°C in a 95% O2/5% CO2 atmosphere. It took 6 hours to finish the isolation of cardiomyocytes since the measurement of cardiac function and LV geometry by echocardiography. Measurement of cell shortening and Ca2+ transient, Ca2+ spark assay were started after 12 hour-incubation (overnight), and all measurements were finished within 8 hours.Cardiomyocytes were isolated from the LV free wall of the beagles with a little modification as described previously , 24, 25.2+ transients (CaT) were measured using Fura-2 AM as described previously [2+-induced fluorescence at 505 nm was measured by excitation at 340 and 380 nm using a dual-excitation spectrofluorometer. The intracellular calcium concentration was calculated as the ratio of the fluorescence emission intensities at these 2 excitation wavelengths [Cardiomyocyte cell shortening (CS) and intracellular Caeviously , 24, 25.elengths , 24, 25.To determine the dose-dependent effect of landiolol on CS in isolated normal and failing cardiomyocytes, we measured CS with various doses of landiolol (from 0 nM to 1000 nM).2+ sparks were measured as previously described [2+ sparks were recorded from 30 sec to 40 sec after start of pacing at the rate of 0.5 Hz. Thus, Ca2+ spark frequency for each image  was measured in the same scanning window to exclude the possibility that different Ca2+ spark frequency caused by different laser scanning time. Each cardiomyocyte was scanned repeatedly at 325.7 Hz along a line parallel to the longitudinal axis of the cell to avoid nuclei. The data were analyzed with SparkMaster, an automated analysis program that allows rapid and reliable Ca2+ spark analysis in confocal line-scan images, as described previously [Caescribed , 25, 26,eviously , 25, 26.2+ transient was measured by first applying a stimulation train at 0.5 Hz for 60 sec and then rapidly switching the superfusion solution to a solution containing 20 mM caffeine for 5–6 s, as previously described [A caffeine-induced Caescribed , 25, 26.In canine cardiomyocytes, a fluorescent probe, 2,7-dichlorofluorescin diacetate , was used to assess intracellular reactive oxygen species (ROS) formation, as described previously , 28. FluWe performed immunoblot analyses using specific antibodies against ryanodine receptor 2 , Ser2808-phosphorylated RyR2 , phospholamban , Ser16-phosphorylated PLB , and Thr17-phosphorylated PLB  as previously described , 29.P values less than 0.05 were considered statistically significant.The chi-squared test was used to compare prevalence or frequencies. The significance of differences between 2 groups was determined by post-hoc tests with Least Significant Difference algorithms following repeated-measures analysis of variance to evaluate the dose-dependence of landiolol on cell shortening in isolated cardiomyocytes. Comparisons across milrinone(+/-), landiolol(+/-), and heart failure(+/-) were independently verified with multivariate analysis of variance in experimental studies. Kruskal Wallis ANOVA was used to evaluate the antioxidative effect of landiolol on intact cardiomyocytes. All analyses were performed with SPSS 18.0 software . After 4 weeks-rapid pacing, decreased left ventricular ejection fraction (LVEF), dilated left ventricular end-diastolic dimension (LVDD) and dilated left ventricular end-systolic dimension (LVDS) were confirmed in HF group as compared with non-sham operated controls . There w2+ handling experiments, as described previously [2+ sparks (CaSF) increased significantly, and both peak CaT and CS decreased markedly compared with normal cardiomyocytes  in failing cardiomyocytes  slightly increased the phosphorylation levels of RyR2, Ser2808, and PLB Thr17 and markedly increased that of PLB Ser16 . The addIn failing cardiomyocytes, the baseline RyR2 phosphorylation level was abnormally elevated, as described previously , 33, 34.2O2, whereas it was restored to normal levels in the presence of 100 μM edaravone, which is a radical scavenger. By contrast, fluorescence intensity was not altered in the presence of 10 nmol/L landiolol.  milrinone monotherapy enhanced Ca2+ leakage from failing RyR2, while adding low-dose β1-blocker to milrinone suppressed this milrinone-induced Ca2+ leakage, leading to greater improvement in cardiomyocyte function; and 3) low-dose landiolol prevented mechanical alternans in failing myocardiocytes. This report is the first to demonstrate that a low-dose pure β1-blocker in combination with milrinone can acutely benefit abnormal intracellular Ca2+ handling. Our results  landiolol, a pure β1-blocker, inhibited Ca results  suggest function .2+ handling during heart failure. To test this hypothesis, we examined the effect of low-dose landiolol on Ca2+ release through RyR2 and CS by electrically pacing isolated cardiomyocytes. Alternans of Ca2+ transient and cell shortening appeared in 30% of intact failing cardiomyocytes, and not at all in intact normal cardiomyocytes. Addition of low-dose landiolol significantly diminished the alternans of Ca2+ transient and CS -dependent protein kinase A (PKA) phosphorylation via β-adrenergic stimulation , 34. Howrylation , 34. A l failure . Therefo2+leakage through failing RyR2, which was enhanced by milrinone, with a low-dose β1-blocker might improve cardiac function and suppress arrhythmogenesis [In heart failure, the difference in phosphorylation level between RyR2 and PLB might arise from the compartmentation of the PKA signaling cascade –40. Indeogenesis 2+ overload and enhanced myocardial oxidative stress [2+ leakage from SR. Therefore, low-dose landiolol in combination with milrinone may be a novel strategy to prevent lethal arrhythmia in patients with acute heart failure.Tachycardia itself complicated acute heart failure-induced intracellular Cae stress . Therefo2+ release through RyR2 is the oxidation of RyR2 due to ROS [2O2  blocker plays a role through its blocking β1AR, the model used in the present study is the cultured cells where there is no any catecholamine in the medium. How does the β1AR play the role in regulation of intracellular Caocardium .2+ leak from SR in failing cardiomyocytes [Are the phenomena which landiolol induced, landiolol-specific? Other β blockers might have similar effects to greater or lesser degree. The reasons are as follows; 1) β blockers such as nebivolol, bisoprolol, metoprorol, carvediolol, and bucindolol have inverse agonism effect , 2) β blmyocytes , 33.2+ leakage from SR, which causes intracellular Ca2+ overload and decreases {Ca2+}SR. Second, a low-dose β1-blocker selectively suppresses RyR2 Ser2808 hyperphosphorylation to inhibit Ca2+ leakage from SR but leave Ca2+ uptake through the sarco/endoplasmic reticulum Ca2+-ATPase unchanged. Third, monotherapy with milrinone selectively increases phosphorylation of PLB Ser16 and Thr17, but not to the extent of RyR2 Ser2808. Additionally, Ca2+ leakage from SR increases proportionally to increasing Ca2+ uptake. Eventually, the peak Ca2+ transient is slightly elevated. Fourth, combination therapy with milrinone and a low-dose β-blocker increases phosphorylation of PLB Ser16 and Thr17 and suppresses that of RyR2 Ser2808. These drugs also increase Ca2+ uptake and decrease Ca2+ leakage, which increases {Ca2+}SR and the peak Ca2+ transient.On the basis of our results, we propose the following model for the molecular basis of low-dose β-blocker treatment of ADHF . First, 2+ leakage from the failing SR has been suggested to arise in part from selective inhibition of phosphorylated RyR2 (Ser 2808), the target amino acid of cAMP-dependent PKA. In the present study, however, we did not directly examine the effect of low-dose landiolol on phosphorylation of RyR2 (Thr 2814), the target amino acid of Ca2+/calmodulin-dependent protein kinase II (CaMK II). Recently, several reports indicated that CaMK II, rather than PKA, plays a critical role in diastolic Ca2+ leak through RyR2 [2+ leak may also involve inhibition of RyR2 (Thr 2814) phosphorylation.Inhibition of milrinone-induced diastolic Caugh RyR2 , 44. The2+ handling through PKA phosphorylated site. Xiao B et al. reported that RyR2-Ser2030 site was the major phosphorylation site in RyR2 responding to PKA activation upon β adrenergic stimulation in normal and failing rat hearts [2+ leakage through RyR2 may be due to the inhibition of phosphorylation of RyR2-Ser2030 as well as the inhibition of phosphorylation of RyR2-Ser2808. Further research is needed to clarify these possibilities.The phosphorylation level for PLB-Ser16 (PKA phosphorylated site) is much larger than PLB-Thr17 (CaMKII phosphorylated site) after addition of milrinone, which may suggest that milrinone affects Cat hearts . In the 2+ handling and significantly restored mechanical alternation by inhibiting diastolic Ca2+ leakage from SR. Thus, the molecular mechanism by which a low-dose β1-blocker can suppress milrinone-induced Ca2+ leakage from SR is very important for the treatment of ADHF.In failing cardiomyocytes, the addition of a low-dose β1-blocker to milrinone improved intracellular CaS1 ARRIVE Checklist(DOC)Click here for additional data file.


Here, we report the development of mouse models of t-AML/MDS. First, we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU), resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia, myelodysplasia, and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second, we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall, we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics.Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their  AML is a heterogeneous disease and is classified by the World Health Organization into several subtypes on the basis of cytogenetic, molecular, and phenotypic characteristics . Therapyde novo AML is composed of a cellular hierarchy initiated and maintained by self-renewing leukemia stem cells (LSC) that are functionally defined by their ability to reconstitute AML in xenograft models [A growing body of evidence indicates that t models . The celt models . Recent t models –17. Sevet models –20. One t models . The clit models , 23.Numerous mouse models of AML and MDS have been described in order to improve understanding of disease pathogenesis and test novel therapeutic approaches –28. XenoHere, we report the development of new mouse models of t-AML/MDS using both wild type mice and human patient samples. We treated wild type adult mice with ENU and monitored for development of t-AML/MDS. Moribund mice were diagnosed with AML, MDS, or other diseases through extensive pathologic characterization and in accordance with guidelines from the Mouse Models of Human Cancers Consortium (MMHCC) , 47. In Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and followed protocols approved by the Stanford University Institutional Animal Care and Use Committee (Protocol # 22264). Mice were housed in sterile cages with sterile bedding and subjected to daily light/dark cycles. There were no more than five mice per cage. Mice were maintained on sterile water and chow containing trimethoprim-sulfamethoxazole. Mice were inspected daily by the veterinary staff. Fifty wild type 9–12 week-old DBA/2J (n = 40) or SWR/J mice (n = 10)  were treated with two weekly intraperitoneal doses of N-ethyl-N-nitrosurea  totaling 100–300 mg/kg per animal. For all human and mouse transplantation experiments, recipient 8–12 week old NOD/SCID/IL2R-gamma-null  mice were conditioned with sublethal X-irradiation (200 rads) followed by tail vein injections containing donor cells. All mice were observed with at least weekly physical exams for signs and symptoms of disease and with monthly weight checks, complete blood count (CBC) using a HemaTrue blood analyzer , peripheral blood (PB) smear, and flow cytometric analysis of PB cell composition. Mice that exhibited ruffled fur, hunched posture, weight loss or severe CBC abnormalities were considered moribund and were euthanized using carbon dioxide asphyxiation. Anesthetization with isofluorane inhalation, in addition to carprofen and normal saline injections, were used to minimize pain associated with experimental procedures.All mouse experiments were carried out in strict accordance with the recommendations in the All human AML samples were obtained from patients at the Stanford Medical Center with written informed consent in accordance with the Stanford University Institutional Review Board (IRB) approved protocols (Stanford IRB numbers 18329 and 6453). Mononuclear cells from each sample were isolated by Ficoll separation and cryopreserved in liquid nitrogen. All experiments used freshly thawed cells.Mice were sacrificed when moribund and necropsy performed to identify any gross pathologic abnormalities and to measure the spleen size. Cell suspensions were then made from the PB, bone marrow (BM), spleen, and any tumors. PB mononuclear cells were obtained from mouse whole blood through a two-step purification process using 2% Dextran followed by incubation with Ammonium-Chloride-Potassium (ACK) Lysing Buffer . BM cells were isolated from the skeleton using a mortar and pestle. BM, spleen, and any tumors were passed through a 100 micron nylon cell strainer  to create single cell suspensions which were then incubated with ACK Lysing Buffer to deplete red blood cells. Mononuclear cells from each sample were isolated by Histopaque (Sigma-Aldrich) separation, analyzed, and cryopreserved in liquid nitrogen. PB smears were also prepared from each mouse at the time of sacrifice. In addition, smears of BM, spleen, liver, and any tumors were prepared by using a 5/0 paintbrush dipped in hypotonic fetal bovine serum to brush cells onto microscope slides. PB and paintbrush smears were fixed and stained using the Harleco Hemacolor staining system . In addition, tissue samples from BM, spleen, liver, kidney, lungs, and brain were fixed in 10% formalin. These samples were sent to HISTO-TEC Laboratory  where they were decalcified, paraffin-embedded, sectioned, and either stained with hematoxylin and eosin or immunostained with MPO or CD3. All slides were evaluated and images obtained using a Leica DM5500B upright microscope and camera system . All diagnoses were made according to guidelines from the MMHCC , 47 and The following anti-mouse monoclonal antibodies were used: Ter119 PE-Cy5 , CD45.1 PE-Cy7 , c-Kit APC-Cy7 and PE-Cy7 , CD34 FITC , CD16/32 A700 , Gr-1 PE-Cy5 , Mac-1 PE-Cy5 , CD42d PE , CD71 FITC , IL-7Ra A700 and PE-Cy5 , B220 APC and PE-Cy5 , CD19 PE and PE-Cy5 , CD43 FITC , TCRb APC , CD3e PE, PE-Cy5 and eFluor450 , CD4 PE-Cy5 and PE-Cy7 , CD8a FITC and PE-Cy5 , and CD5 PE  from eBioscience , and CD45.2 AF488 and BV510 , Sca-1 PB , Flk-2 APC , Gr-1 APC , Mac-1 PB , F4/80 A700 , and IgM PE-Cy7  from Biolegend . The following mouse stains were used: hematopoietic stem and progenitor (HSPC)–Lineage Cocktail , c-Kit (1:50), Sca-1 (1:50), CD34 (1:25), CD16/32 , and Flk-2 (1:50); Myeloid–Ter119, CD45.2, c-Kit, Gr-1, Mac-1, F4/80, CD42d, and CD71 ; B-lymphoid–Ter119, CD45.2, IL-7Ra, IgM, B220, CD19, and CD43 ; T-lymphoid–Ter119, CD45.2, TCRb, CD3e, CD4, CD8a, and CD5 ; and Engraftment lineage–Ter119, CD45.1, CD45.2, c-Kit, Gr-1, Mac-1, B220, and CD3e . The following anti-human monoclonal antibodies were used: CD45 V450 , CD34 APC , CD38 PE-Cy7 , CD19 PE-Cy5 and APC , CD20 PE-Cy5 , CD99 FITC , CD3 APC-Cy7 , and CD33 PE  from BD Biosciences , and Tim3 PE  from eBioscience. The following human stains were used: HSPC–CD34 (1:50), CD38 (1:100), Tim3 (1:5), CD99 (1:5), CD3 (1:25), CD19 (1:50), and CD20 (1:50); and Engraftment–mTer119 (1:200), mCD45.1 (1:200), CD45 (1:100), CD3 (1:50), CD19 (1:100), and CD33 (1:100). Propidium iodide (1:1000) was used to exclude dead cells. Cells were suspended in FACS buffer  and incubated (30-90mins at 4°C) with the appropriate antibody stains. Fluorescence activated cell sorter (FACS) analysis and sorting was performed using a BD FACS Aria II flow cytometer (BD Biosciences).Bulk BM cells, FACS CD3-depleted BM cells, or FACS purified BM subpopulations from ENU-induced mouse diseases were transplanted into NSG recipients. Human therapy-related AML samples were CD3-depleted using Robosep  before undergoing FACS purification of leukemia cell subpopulations and transplantation into NSG recipients. NSG mice were conditioned, injected, and monitored as previously described.Flow cytometry data were analyzed using FlowJo software . Figures and statistical analysis were performed using Prism .A total of fifty 9–12 week old adult mice were administered two weekly intraperitoneal doses of the alkylating agent, ENU. Ten SWR/J and 20 DBA/2J mice received 300 mg/kg, 10 DBA/2J received 200 mg/kg, and 10 DBA/2J received 100 mg/kg ENU. Mice were monitored with weekly physical examinations and monthly evaluations including weight check, CBC, peripheral blood (PB) smears, and PB flow cytometric analysis. Mice were sacrificed when moribund and underwent necropsy, spleen size assessment, and flow cytometric analysis of PB and bone marrow (BM) cell compositions. Smears were prepared from the PB, BM, spleen, liver, and any tumors or enlarged lymph nodes for morphologic analysis. In addition, formalin-fixed paraffin-embedded (FFPE) sections of BM, spleen, liver, kidney, lungs, and brain were stained with H&E. Diagnoses were determined by evaluation of these clinical and pathologic assessments in accordance with guidelines from the MMHCC. Key defining criteria for a diagnosis of AML included: (1) Diffuse involvement of hematopoietic tissues; (2) Cytopenia(s); (3) Increased nonlymphoid hematopoietic cells in the spleen; (4) Neoplastic cells in non-hematopoietic tissues or leukocytosis with at least 20% immature forms/blasts; and (5) Malignant behavior including at least 20% immature forms/blasts in the blood, spleen, or bone marrow, rapidly fatal to the primary animal, or transplantable and rapidly fatal (within eight weeks) to secondary recipients . Key diaOut of a total of 50 ENU-treated mice, 8 mice were diagnosed with AML, 11 mice with MDS, and 16 mice with non-AML/MDS diseases . Non-AMLSurvival from the day of the first ENU injection was determined for each mouse diagnosed with a myeloid or non-AML/MDS disease. Kaplan-Meier survival analysis was then applied to determine median overall survival (OS) of the entire cohort of animals, excluding mice that were not evaluable due to direct ENU toxicity or unanticipated early death. Median OS for the 35 mice diagnosed with AML, MDS, or non-AML/MDS diseases was 162 days from the day of first ENU treatment  or ENU dClinical features, including weight, spleen length, and CBC were collected for each diseased mouse  at the time of sacrifice and are summarized in Furthermore, the ENU-induced mouse AML and MDS cases had morphologic and histologic features of human AML and MDS and fulfilled the diagnostic criteria established by the MMHCC. Wright-Giemsa staining of PB, BM, spleen, and liver smears revealed circulating blasts and infiltrating blast populations in AML cases. Representative images are shown in To complete the clinical and pathologic characterization of ENU-induced AML and MDS cases, the relative compositions of BM lineage positive and HSPC subpopulations were determined by flow cytometry and compared to age-matched controls. Among lineage positive cell subpopulations, myeloid cells (Gr-1+ and Mac-1+) were increased and B-lymphoid cells (CD19+) decreased in AML and MDS mice compared to control mice , p<0.05.6 bulk BM cells from each primary mouse into 3–5 sublethally irradiated adult NSG mice. In some cases, we first FACS purified CD3-depleted BM from primary mice prior to transplantation. Mice were monitored for signs of disease, and PB engraftment was assayed every four weeks by flow cytometry using congenic CD45.1 (NSG) and CD45.2 (DBA/SWR) markers. By day 60 after transplant, peripheral blood engraftment was detected in at least two mice from each primary AML  cell subpopulations  and hCD45 markers. Composition of engrafted hCD45-positive cells was determined by staining with CD33 , CD19 (B cell), and CD3 (T cell). PB engraftment at the time of NSG mouse death from t-AML or day 120 from transplant is shown in Since there are few previously described human t-AML models, we next attempted to model human t-AML in patient-derived xenograft NSG mouse models to complement our ENU-induced mouse AML/MDS models. We identified seven cases of WHO-defined t-AML from our tissue bank. Clinical information is summarized in Since the immunophenotype of LSC in t-AML is not well-described, we next FACS purified CD34-, CD34+/CD38+, and CD34+/CD38- subpopulations from SU108, SU158, and SU190 and transplanted equal numbers of these cell subpopulations into sublethally irradiated adult NSG mice (3–7 mice per subpopulation per primary AML sample) to identify subpopulations capable of initiating AML xenografts. Mice were monitored as above until the time of death or 180 days from transplant. CD34+CD38+ and CD34+CD38- cells, but not CD34- cells, from SU108 were able to establish xenografts . One moude novo disease, and relatively few t-AML/MDS mouse models have been described. Here, we describe multiple mouse models of t-AML/MDS with features consistent with clinical disease. We first developed models of alkylator-induced t-AML/MDS by treating wild type adult DBA/2J and SWR/J mice with ENU and established a diagnosis of AML or MDS in nineteen mice using the rigorous diagnostic criteria proposed by the MMHCC. We demonstrated that these models have clinical and pathologic characteristics consistent with human t-AML/MDS and are therefore relevant models to study human disease. We also established xenograft models derived from three primary human t-AML samples. These models were characterized by lethality to recipient mice and LSC immunophenotype that varied among the primary t-AML cases.t-AML/MDS is a distinct subgroup of AML that develops years after exposure to chemotherapy and ionizing radiation. These diseases are characterized clinically by recurrent chromosomal alterations and gene mutations, and carry a dismal prognosis with current treatments. Overall, less is known about t-AML/MDS pathogenesis compared to Our mouse alkylator-induced AML and MDS models closely modeled many clinical features of human t-AML/MDS. Mice developing AML or MDS became rapidly moribund with leukocytosis, anemia, and thrombocytopenia in AML cases and macrocytic anemia and thrombocytopenia in MDS cases. The mouse diseases characterized here also had morphologic features consistent with human disease, and the bone marrow was hypercellular with expanded myeloid cell populations as in human AML and MDS. The finding of decreased immunophenotypic HSC in MDS mice without changes in other HSPC compartments differs from prior reports showing expansion of HSC and CMP  or GMP  in highede novo AML, we attempted to establish xenografts with a panel of seven primary human t-AML samples. Three primary samples led to engraftment of aggressive and rapidly lethal disease in NSG mice. Interestingly, these three patients had adverse-risk complex or monosomal karyotypes and refractory disease consistent with prior studies showing that the likelihood of establishing AML xenografts correlates with karyotype and treatment response [Since the AML LSC model has been most well-described in response . When weresponse , and it In summary, we report several new mouse models of t-AML/MDS that are consistent with clinical disease and that could potentially be used to further define disease susceptibility and pathogenesis and test novel therapeutics. For example, future studies could take advantage of the predictable timing of MDS development to evaluate therapeutic and transplant approaches or tailor ENU dosage to study specific hematopoietic diseases. Further analysis of human t-AML LSC should also confirm the heterogeneous immunophenotypic and mouse phenotypic characteristics of human t-AML LSC seen in our models.S1 FileFigure A. KLS positive cells from AML and MDS cases stably engraft NSG mice with lymphoid-biased hematopoiesis over time. Figure B. Human t-AML samples form PDX models in NSG mice. Table A. Strain, Gender, ENU Dose, Diagnosis, Survival, CBC, Necropsy and Immunophenotype for all primary mice. Table B. Characteristics of secondary transplant mice described in (PDF)Click here for additional data file.


Polymalic acid (PMA) has many applications in food and medical industries. However, so far it has not been commercially produced by fermentation. Therefore, it is veryimportant how to develop an economical process for a large scale production of PMA by one step fermentation.Aureobasidium spp. isolated from the mangrove systems in the South of China were screened for their ability to produce Ca2+-polymalate (PMA), it was found that Aureobasidium pullulans var. pullulans MCW strain among them could produce high level of Ca2+-PMA. The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca2+-PMA production. Then, 121.3 g/L of Ca2+-PMA was produced by A. pullulans var. pullulans MCW strain within 120 h at flask level. During 10-L batch fermentation, 152.52 g/L of Ca2+-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium. After purification of the Ca2+-PMA from the culture and acid hydrolysis of the purified Ca2+-PMA, HPLC analysis showed that A.pullulans var. pullulans MCW strain produced only one main component of Ca2+-PMA and the hydrolysate of the purified Ca2+-PMA was mainly composed of l-malic acid. Mw (the apparent molecular weight) of the purified PMA was 2.054 × 105 (g/moL) and the purified PMA was estimated to be composed of 1784 l-malic acids.After over 200 strains of A. pullulans var. pullulans MCW strain obtained in this study could yield 152.52 g/L of Ca2+-PMA within the short time, the produced PMA had the highest molecular weight and the medium for production of Ca2+- PMA by this yeast was very simple.It was found that  Three hundred milliliters of the seed culture were transferred into 6 L of the Ca2+-PMA production medium containing only 140.0 g/L of glucose, 7.5 g/L of CSL and 65.0 g/L of CaCO3. The fermentation was performed under the conditions of the agitation speed of 300 rpm, the aeration rate of 8 L/min, the temperature of 28°C, and the fermentation period of 120 h. During the fermentation, 10.0 mL of the culture was collected in the interval of 12 h and was centrifuged at 6,000×g and 2°C for 10 min, and Ca2+-PMA and reducing sugar (glucose) in the supernatant obtained were determined as described below. The cell dry weight in 10.0 mL of the culture during the 10-L fermentation was also measured as described below.Ca2+-PMA in 10 mL of the supernatant was obtained with the repeated methanol precipitation. Briefly, the first addition of 5.0 mL of methanol was to selectively remove exopolysaccharide (EPS) as a precipitate. After the EPS was removed by centrifugation, 30.0 mL of methanol was added into the supernatant, and the mixture was incubated at 4°C for 12 h. The resulting Ca2+-PMA precipitates were collected by centrifugation at 6,000×g and 2°C for 10 min and dried by evaporating at 50°C. After the dried materials were re-dissolved in 5.0 mL of distilled water, 15.0 mL of methanol was added into the solution, and the mixture was incubated at 4°C for 12 h; the above procedures were repeated several times until the pure Ca2+-PMA was obtained. Finally, the amount of the purified Ca2+-PMA from 10 mL of the supernatant was weighed and the amount of Ca2+-PMA in 1.0 L of the culture was calculated.The culture obtained during the 10-L fermentation was centrifuged at 6,000×g and 2°C for 10 min. CaThe amount of the reducing sugar in the culture was determined according to the Nelson–Somogyi method .The dried biomass in the culture was measured according to the methods described by Chi et al. .2+-PMA was carried out with 0.5 M sulfuric acid in sealed glass tubes at 90°C. Calcium malate and l-malic acid formed during the hydrolysis of the purified Ca2+-PMA were assayed using HPLC as described below.The chemical degradation of the purified Ca2+-PMA and hydrolysate of the purified Ca2+-PMA obtained above were dissolved in distilled water, respectively. The solutions were analyzed using HPLC  for determination of the purity of the Ca2+-PMA. First, the Ca2+-PMA sample was separated on ZORBAXSB-C18 column . The HPLC conditions were that mobile phase was 0.01 M (NH4)2HPO4 in 10.0% methanol solution which pH was adjusted to 2.7 using 1.0 M phosphoric acid and degassed using a microwave; flow rate was 1.0 mL/min; column temperature was 30°C; the sample volume was 20.0 μL; detector was waters 996 Diode-Array Detector; detection wavelength was 210 nm; sensitivity was 0.02 AUFS. The pure calcium malate and l-malic acid purchased from Sigma  were used as the standards.The purified CaThe apparent molecular weight of the purified PMA produced by the yeast strain MCW was evaluated by the Gel Permeation Chromatography (GPC) utilizing a Waters™ 1515 instrument combined with Multi-angle laser light scattering detector (MALLS) and concentration detector (RI) under the following conditions: Solvent: water; Flow rate: 0.500 mL/min; Laser wavelength: 658.0 nm; Calibration constant: 3.7138e-5 1/(V cm); Cell type: K5. The pullulan standards of varying known sizes  were used as the molecular mass references.