The coordination of growth within a tissue layer is of critical importance for tissue morphogenesis. For example, cells within the epidermis undergo stereotypic cell divisions that are oriented along the plane of the layer (planar growth), thereby propagating the layered epidermal structure. Little is known about the developmental control that regulates such planar growth in plants. Recent evidence suggested that the Arabidopsis AGC VIII protein kinase UNICORN (UCN) maintains planar growth by suppressing the formation of ectopic multicellular protrusions in several floral tissues including integuments. In the current model UCN controls this process during integument development by directly interacting with the ABERRANT TESTA SHAPE (ATS) protein, a member of the KANADI (KAN) family of transcription factors, thereby repressing its activity. Here we report on the further characterization of the UCN mechanism.ucn-1 plants impaired in floral homeotic gene activity revealed that any of the four floral whorls could produce organs carrying ucn-1 protrusions. The ectopic outgrowths of ucn integuments did not accumulate detectable signals of the auxin and cytokinin reporters DR5rev::GFP and ARR5::GUS, respectively. Furthermore, wild-type and ucn-1 seedlings showed similarly strong callus formation upon in vitro culture on callus-inducing medium. We also show that ovules of ucn-1 plants carrying the dominant ats allele sk21-D exhibited more pronounced protrusion formation. Finally ovules of ucn-1 ett-1 double mutants and ucn-1 ett-1 arf4-1 triple mutants displayed an additive phenotype.Phenotypic analysis of flowers of UCN-mediated control of planar growth during integument development. The presented evidence indicates that UCN downstream signaling does not involve the control of auxin or cytokinin homeostasis. The results also reveal that UCN interacts with ATS independently of an ATS/ETT complex required for integument initiation and they further emphasize the necessity to balance UCN and ATS proteins during maintenance of planar growth in integuments.These data deepen the molecular insight into the  In plant tissue morphogenesis the control of cell division patterns is crucial for the establishment and propagation of tissue layers. Spatially restricted asymmetric cell divisions frequently generate new cell layers. Subsequently, symmetric cell divisions maintain a cell layer, often by aligning the division planes along the plane of the layer (planar growth). The regulation of asymmetric cell divisions is under intense scrutiny -5. By coKAN genes, in conjunction with auxin response factor genes ETTIN (ETT) and ARF4, direct abaxial cell fate and lamina outgrowth in leaves [There is evidence for a link between the control of adaxial-abaxial polarity and the laminar growth of the leaf blade. Leaves are lateral determinate organs and are characterized by a distinct adaxial-abaxial or dorsal-ventral polarity across the whole multi-layered organ. Outgrowth of the developing leaf lamina is believed to require stimulation of cells located at the adaxial-abaxial boundary . The conn leaves ,14-16. In leaves ,17. Simin leaves ,19.Evidence for a connection between the regulation of adaxial-abaxial polarity and planar growth also comes from studies using Arabidopsis integuments as model system ,21. InteUNICORN (UCN) [UCN encodes a functional protein kinase that belongs to the AGC2 subclass of the plant-specific AGC VIII family [ucn mutants exhibit local disorganized growth resulting in the formation of one to several multicellular protrusions containing cells with at least partial integument identity. Similar protrusions are also present on stamens and petals. At the cellular level, the earliest detectable defects are local periclinal or oblique cell divisions in individual cell layers. They are clearly distinct from the typical anticlinal cell divisions that maintain planar growth of integuments. In addition, ucn proembryos show altered cell division planes and double mutants carrying null alleles of UCN and its closest homolog UNICORN-LIKE (UCNL) are embryo lethal. These observations indicated that UCN suppresses ectopic growth by influencing division planes in symmetrically dividing cells.Recently it was discovered that maintenance of planar growth of integuments is under control of RN (UCN) ,21. UCN I family -27. InteUCN maintains planar growth during integument outgrowth by interacting with ABERRANT TESTA SHAPE (ATS) [ATS is a KAN gene required for several processes of integument development including, integument boundary formation, inner integument outgrowth and the control of adaxial-abaxial polarity [ucn ats double mutants is strongly diminished indicating that UCN represses ATS[ATS transcript levels are unaltered in ucn mutants and recombinant UCN protein is able to phosphorylate ATS in in vitro kinase assays. Moreover, bimolecular fluorescence complementation (BiFC) analysis further supports direct physical interaction between UCN and ATS [PE (ATS) . ATS is polarity -31. In apolarity  to contrpolarity . Protrusesses ATS. This ne and ATS . Thus, bUCN-mediated maintenance of planar growth during integument development. We provide evidence that UCN functions in an organ-specific manner and that UCN does not influence auxin and cytokinin homeostasis. Our data further suggest that UCN and ATS protein levels must be balanced and that repression of ATS by UCN does not involve either ETT or the ATS/ETT complex.Here we further characterize ucn mutants show protrusions in petals, stamen and ovules  mutants carry sepals in whorl 2 and carpels in whorl 3 [ucn-1 ap3-3 flowers did not show ucn-like protrusions  exhibit petals in the third whorl and an additional flower in the fourth whorl [ucn-1 ag-1 still showed protrusions  mutants unfused carpels with ovules and stamens develop in the first whorl and second whorl, respectively [ucn-1 ap2-8 double mutants were characterized by first-whorl carpels devoid of protrusions but that included ucn-like ovules , encoding a putative component of a chromatin-remodeling complex, result in callus formation upon addition of auxin or cytokinin [AINTEGUMENTA (ANT), encoding an AP2-class transcription factor involved in the control of cell division and organ initiation [ANT acts downstream of auxin and regulates meristematic competence during organogenesis [The maintenance of plant tissue morphogenesis and the prevention of aberrant growth and tumor formation is under hormonal and genetic control -43. For ytokinin . In addiitiation -57, resuitiation . ANT actogenesis ,59.UCN and auxin as well as cytokinin we tested whether the integument protrusions of ucn-1 expressed the well-characterized reporters DR5rev::GFP or ARR5::GUS that act as proxies for the presence of auxin and cytokinin, respectively [DR5rev::GFP signal distribution as reported previously in developing ovules, such as the tip of the ovule primordium or the micropylar end of the young embryo sac [ucn-1 integuments  we detected no difference in size or number of formed calli  failed to form protrusions. These results indicate that auxin contributes to the formation of bel1 outgrowths [ucn-1 integuments one might expect enlarged protrusion formation in ucn bel1 double mutants. However, this is not the case, as ucn bel1 double mutants did not show a noticeable increase in protrusion size [ucn ant double mutants [The reporter-based experiments described above need to be interpreted with caution. Still, the results are also in accordance with previous genetic data. For example, the additive ovule phenotype of  mutants  further elopment ,67-69. Preporter , used toreporter ,71. Furttgrowths . If auxiion size . In addi mutants .CRE1, AHK2, and AHK3[cre1 ahk2 ahk3 triple mutants. These affected ovules may have still suffered from defects occurring during prior primordium outgrowth. The absence of detectable ARR5::GUS expression in ucn-1 protrusions, consisting of at least partially differentiated integument cells, may thus reflect the minor role of cytokinin in integument outgrowth.Available evidence suggests that cytokinin may be of little importance for the control of integument outgrowth. Signaling mediated by the three cytokinin receptor genes  and AHK3 appears  and AHK3 and embr and AHK3,73. Howe and AHK3 were repUCN growth suppression do not involve the regulation of auxin and cytokinin homeostasis.Taken together the available evidence suggests that processes functioning downstream of ATS transcript levels within its normal spatial expression domain in the activation tagging mutant sk21-D[ucn-like protrusion formation in integuments [UCN is a negative regulator of ATS. One interpretation of the sk21-D integument phenotype includes the assumption that elevated ATS transcript levels lead to higher than normal amounts of ATS protein, which may titrate out available UCN. If this notion is correct one might expect that upon reduction of UCN function even more exaggerated protrusion formation should take place in sk21-D plants.The current model states that UCN maintains planar growth in integuments by directly repressing the activity of the ATS protein implying that the balance between UCN and ATS proteins may be crucial in this process. Previously we could show that an about 45-fold increase of nt sk21-D is accomucn-1/+ plants do not show protrusions [sk21-D/+ heterozygous plants revealed less prominent protrusion formation than in ovules of sk21-D homozygous plants  gene ETTIN (ETT) and its closest homolog ARF4 are required for the control of adaxial-abaxial leaf polarity in conjunction with KAN1 and KAN2[ats and ett should show a similar genetic behavior with respect to ucn.Genetic studies indicated that  and KAN2. Further and KAN2. Our pre and KAN2. If inteucn-1 ett-1 double and ucn-1 ett-1 arf4-1 triple mutants and analyzed their ovule phenotypes  Heynh. var. Columbia (Col-0) and var. Landsberg (erecta mutant) (Ler) were used as wild-type strains. Plants were grown essentially as described previously [ag-1[ap2-8[ap3-3[arf4-1[ett-1[sk21-D[ucn-1[ucn-1 sk21-D/+ or ucn-1 sk21-D/sk21-D plants were genotyped using primers sk21-D (GT)_F :GAGAATTAGTACAATGTAATG, sk21-D (GT)_R :GTGATTTAACCCTTCTCAAGTGC and pSKI (GT)_F: CCACCCACGAGGAACATCGTG.eviously . The folsly [ag-1,75; ap2-g-1[ap2-8, ap3-3[72-8[ap3-3, arf4-1[-3[arf4-1,77,78, e4-1[ett-1,79, sk21-1[sk21-D, ucn-1[21-D[ucn-1,21. In dSeedlings were grown in a growth room under constant light conditions at 23°C. For induction of callus freshly germinated seedlings were grown on MS plates for 6 days. Seedlings were then transferred onto MS plates supplemented with 3 μg/ml each of the auxin 1-naphthalene acetic acid (NAA) (Sigma) and the cytokinin kinetin (Sigma) .Preparation and analysis of samples for light microscopy, scanning electron microscopy, and histochemical localization of β-glucuronidase (GUS) activity in whole-mount tissue was done essentially as described ,80. OvulBE conceived of the study, participated in the design, and carried out experiments. KS conceived of the study, participated in the design and coordination and wrote the manuscript. All authors read and approved the final manuscript.


Psoroptes cuniculi can parasitise the ear canal of the rabbit, and cause the afflicted animals to cease feeding and become severely debilitated, sometimes resulting in death. In this study, we examined the oxidative status and inflammatory level of the peripheral blood of rabbits infested with Psoroptes cuniculi and investigated the pathogenesis of this disease.A total of 24 rabbits were divided into a healthy rabbit group and two infested rabbit groups. After weighing the rabbits, approximately 5 ml of blood was obtained from each animal. Then, the blood serum was extracted and used to assess the levels of antioxidant enzymes and inflammatory factors.2, interleukin-6, interleukin-8 and transforming growth factor-β1 were increased in peripheral blood.Compared to the healthy rabbits, the activities of catalase and glutathione-S-transferase and the level of malonyldialdehyde were increased, but the activity of superoxide dismutase was reduced in the infested rabbits. At the same time, a variety of inflammatory cells were activated, and the levels of inflammatory factors such as prostaglandin EPsoroptes cuniculi infestation in rabbits and can help guide the effectual treatment of this disease in clinics.Animal acariasis was associated with immunosuppressive disorders and inflammatory reaction. These results advance our understanding of the pathogenesis of  Psoroptes parasitizes the body surface or the epidermis of sheep, horse, rabbit, goat, cattle and buffalo, etc., causing the afflicted animals to cease feeding and become severely debilitated. In rabbits, Psoroptes cuniculi can damage the pineal layer of the rabbit ear. Infestation can occur by direct contact with infested rabbits or by contact with infected bedding, especially for young rabbits and peaked rabbits  [As we know, once animals are infested with es (ROS) ,9. When es (ROS) ,11. At tes (ROS) . HoweverIn this paper, we studied the oxidative status and inflammation levels of rabbits with psoroptic mange by determining the activities of the antioxidant enzymes and the levels of inflammatory factors in the peripheral blood. Our goal was to develop a better understanding of the pathogenesis of this disease.Rabbits (80–100 days of age) were obtained from the Experimental Animal Center, Lanzhou Institute of Biologicals . The experiments complied with the rulings of the Gansu Experimental Animal Center  and were officially approved by the Ministry of Health, P.R. China in accordance with NIH guidelines.In this study, all of the diseased rabbits were divided into two groups (scores of 3 and 6). Each group consisted of 8 rabbits, and 8 healthy rabbits were used as the healthy control group. They were kept in cages where they had free access to food and water, and they were maintained on a 12 h light/dark cycle.et al. and Shang et al. [Psoroptes cuniculi and suffered from the clinical disease for at least 10 days before presentation. None of the rabbits had been treated with ectoparasiticides or steroidal anti-inflammatory drugs in the 30 days before the blood samples were drawn. The degree of infestation was evaluated according to a previously described scoring system: 0 indicated an absence of scabs and or mites; 0.5 was irritation in the ear canal but no mites observed; 1 was a small number of scabs in the ear canal, mites present; 2 was external ear canal filled with scabs, mites present; 3 was scabs in ear canal and proximal 1/4 of the pinna, mites present; 4 was pinna half-filled with scabs, mites present; 5 was 3/4 of the pinna filled with scabs, mites present; 6 was the entire internal surface of the pinna covered with scabs, mites present [The inclusion criteria for the animals selected for this study have been previously described by Fichi g et al. ,14. Brie present . In thisAfter careful laboratory and clinical examination, all of the rabbits were weighed. Then, 3% pentobarbital sodium (30 mg/kg) was used for the sedation of rabbits and approximately 5 ml of blood was obtained from the auricular vein of each animal using a sterile needle into tubes. After standing for 30 min, the serum was extracted from the blood samples and stored at −20°C for up to 5 days. It was used to assay the oxidative stress and inflammatory factors promptly.2O2 as a substrate with CAT assay kit (Lot. 20130506), and the absorbance was scanned at 405 nm by ultraviolet spectrophotometer. GST activity was determined by assaying the concentration of GSH with GST assay kit (Lot. 20130528), and the absorbance was scanned at 412 nm by ultraviolet spectrophotometer. The concentration of MDA, a reliable marker of lipid peroxidation, was estimated in blood serum following the manual of Nanjing Jiancheng MDA assay kit (Lot. 20130407). Optical density was measured using an ultraviolet spectrophotometer at 532 nm against blanks prepared by using distilled water. The activities of SOD, CAT and GST and the level of MDA were calculated from the resulting absorbance values.After collecting the blood serum, 100 μl aliquots were used to determine the activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and malonyldialdehyde (MDA) with a Nanjing Jiancheng assay kit . Detailed, SOD activity in blood serum was measured by using nitro blue tetrazolium as a substrate after suitable dilution with SOD assay kit (Lot. 20130424), the increase in absorbance was scanned with an ultraviolet spectrophotometer  at 550 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited autooxidation by 50% under the given experimental condition and the values were expressed as U/ml. CAT activity in blood serum was estimated by using H2 (PGE2), interleukin-6 (IL-6), interleukin-8 (IL-8) and transforming growth factor-β1(TGF-β1) with rabbit ELISA PGE2 assay kit, rabbit ELISA IL-6 assay kits, rabbit ELISA IL-8 assay kits and rabbit ELISA TGF-β1 assay kits according to the manufacturer’s instructions , respectively. Briefly: firstly 10 μl sample serum with 40 μl sample diluents were added to 96-well plates (50 μl standard solutions). Following incubation for 30 min at 37°C, the solutions were removed and the plate was washed 5 times with wash solution. 50 μl HRP-conjugate reagents were then added to each well, and after incubating for 30 min for 37°C and washing 5 times again 50 μl chromogen solutions A and B were added and incubated for 10 min at 37°C in the dark. Finally, 50 μl of stop solution was added to each well, and the optical density was measured at 450 nm within 15 min using a microplate reader Multiskan MK3 , and the levels of PGE2, IL-6, IL-8 and TGF-β1 were calculated from the resulting absorbance values.After collecting the blood serum, 10 μl aliquots were used to determine the levels of prostaglandin Et-test for the comparison between test and control, and Dunnett’s test was used when the data involved three or more groups. P-values of less than 0.05 (P < 0.05) were considered significant.The data obtained were analyzed using SPSS software version 13.0 and expressed as the mean ± SD. The data were analyzed by a one-way ANOVA followed by Student’s two-tailed P. cuniculi with clinical scores of 6 and 3 were significantly decreased to 1.35 ± 0.404 kg (P < 0.01) and 1.64 ± 0.302 kg (P < 0.05), respectively. This reduction in weight may be related to the oxidative stress and inflammation induced by the mites and if the degree of disease was more serious, the weight of rabbits was lighter.We weighed all of the rabbits included in this study, which were selected for 80–100 days of age. The results showed that the healthy rabbits had an average weight of 2.15 ± 0.410 kg, and the weights of the rabbits infested with P < 0.01). Meanwhile, the CAT activity in the infested rabbits with scores of 6 (26.258 ± 1.175 U/ml) was increased compared to the healthy controls (21.335 ± 1.135 U/ml) (P < 0.01). This was most likely due to the increased biosynthesis of the enzyme in response to H2O2 and other free radicals associated with P. cuniculi infestations. The MDA levels were assessed to evaluate the degree of lipid peroxidation in the body and to determine the potential role of lipid peroxidation in the pathogenesis of the infested rabbits. The result showed that the level of MDA in the infested rabbit with scores of 3 (4.675 ± 0.244 nm/ml) and 6 (5.195 ± 0.396 nm/ml) was markedly increased compared to the healthy rabbits (2.835 ± 0.180 nm/ml) (P < 0.01)  and 6 (1.831 ± 0.087 U/ml) was weakly inhibited but not significantly different from that observed in the healthy rabbits (1.918 ± 0.061 U/ml). However, the GST activity was significantly elevated in the infested rabbits with scores of 3 (79.938 ± 3.256 U/ml) and 6 (125.138 ± 4.094 U/ml) compared to the healthy controls (67.783 ± 3.694 U/ml) (P < 0.01). The levels of PGE2 and IL-8 were only significantly elevated in the infested rabbits with a score of 6  compared to the healthy controls (0.2670 ± 0.0131 mg/L and 0.0330 ± 0.0083 mg/L) (P < 0.01) , the levels of the inflammatory factors IL-6 and TGF-β1 were markedly increased in the peripheral blood of the infested rabbits with scores of 3  and 6  , hypochlorite, and oxygen radicals. These reactive oxygen substances produced by cells of the immune system show potent cytotoxic effects on parasites and other pathogenic organisms [It is known that inflammatory cells are activated as a result of inflammation in animals with mange, and recruit neutrophils and macrophages with reactive oxidants, such as hydrogen peroxide  systemic inflammatory response. In our study, the results indicated that psoroptic mange was associated with immunosuppressive disorders and inflammatory reaction, and the oxidant/antioxidant balance and the inflammatory factors of the rabbits infested with P. cuniculi were disrupted and released in the development of this disease, respectively. Based on the results, the administration of antioxidants, anti-inflammatory drugs or vitamins in conjunction with acaricides may improve the outcome of the disease. Similarly, treatment that is initiated at the early stage of the disease results in a better outcome.Now, more and more people are aware that animal acariasis induced by mites is not just the topical disease; but can also lead to some systemic disease, especially the oxidative response. In 2008 and 2012, Singh enhanced . Then, Bd [et al. thought P. cuniculi lost a dramatic amount of weight, demonstrated oxidative stress and released inflammatory factors into the peripheral blood. This caused extensive cellular damage throughout the body and resulted in compromised immune and inflammatory reactions. These processes may contribute to the pathogenesis of psoroptic mange.In summary, the rabbits infested with All authors declare that they have no competing interests.XS and DW conceived the study, XW and XM determined the index, XS, JL and ZY wrote the manuscript, HP performed statistical analyses. All the authors read and approved the final version of the manuscript.


Evidence accumulated over the past decade indicates a pivotal role of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release in the regulation of cytosolic and nuclear Ca2+ signals. IP3 is generated by stimulation of plasma membrane receptors that couple to phospholipase C (PLC), liberating IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2). An intriguing aspect of IP3 signaling is the presence of the entire PIP2-PLC-IP3 signaling cascade as well as the presence of IP3Rs at the inner and outer membranes of the nuclear envelope (NE) which functions as a Ca2+ store. The observation that the nucleus is surrounded by its own putative Ca2+ store raises the possibility that nuclear IP3-dependent Ca2+ release plays a critical role in ETC. This provides a potential mechanism of regulation that acts locally and autonomously from the global cytosolic Ca2+ signal underlying ECC. Moreover, there is evidence that: (i) the sarcoplasmic reticulum (SR) and NE are a single contiguous Ca2+ store; (ii) the nuclear pore complex is the major gateway for Ca2+ and macromolecules to pass between the cytosol and the nucleoplasm; (iii) the inner membrane of the NE hosts key Ca2+ handling proteins including the Na+/Ca2+ exchanger (NCX)/GM1 complex, ryanodine receptors (RyRs), nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs), Na+/K+ ATPase, and Na+/H+ exchanger. Thus, it appears that the nucleus represents a Ca2+ signaling domain equipped with its own ion channels and transporters that allow for complex local Ca2+ signals. Many experimental and modeling approaches have been used for the study of intracellular Ca2+ signaling but the key to the understanding of the dual role of Ca2+ mediating ECC and ECT lays in quantitative differences of local [Ca2+] in the nuclear and cytosolic compartment. In this review, we discuss the state of knowledge regarding the origin and the physiological implications of nuclear Ca2+ transients in different cardiac cell types  as well as experimental and mathematical approaches to study Ca2+ and IP3 signaling in the cytosol and nucleus. In particular, we focus on the concept that highly localized Ca2+ signals are required to translocate and activate Ca2+-dependent transcription factors  through phosphorylation/dephosphorylation processes.Calcium plays a crucial role in excitation-contraction coupling (ECC), but it is also a pivotal second messenger activating Ca The modulation of [Ca2+]is a crucial factor for a variety of physiological functions of living cells. In cardiac myocytes, including ventricular and atrial cells, Ca2+ release through channels located in the sarcoplasmic reticulum (SR) membrane and termed ryanodine receptors (RyRs), is a key event linking membrane depolarization and mechanical activity during excitation-contraction coupling (ECC) . IP3 freely diffuses within the cytoplasm to bind to a second type of SR Ca2+ release channels, the inositol 1,4,5-trisphosphate receptor (IP3R) , are expressed in the SR membrane and nuclear envelope (NE)  and pro-hypertrophic signaling , whereas the cytosolic Ca2+ is regulated separately via sarcolemmal GPCR signaling and IP3R-mediated SR Ca2+ release in conjunction with Ca2+ release and removal by the set of proteins involved in ECC . Sarcolemmal GPCRs allow for paracrine signaling and positive inotropic effects mediated by hormonal stimulation ,  Bers, . The amo2+-induced Ca2+ release (CICR) providing sufficient Ca2+ for the activation of the proteins regulating muscle contraction and to induce active muscle force  which triggers CICR and thereby amplifies the cytosolic Ca2+ signal to levels required for the activation of the contractile proteins. An important feature of all ventricular myocytes, setting them apart from most atrial cells, is the presence of plasma membrane invaginations throughout the cytosol (transverse or t-tubules), putting LTCC in close vicinity to RyRs  during a ventricular cell twitch   has been reported for the modulation of cytosolic Ca2+ transients in a variety of animal models   triggered Cace Bers, . Membrans Figure . The SR h Figure . Unlike t Figure   to diffuse along the gradient between the cytosol and nucleoplasm. It has been proposed that NPCs can act as diffusion filter and introduce a kinetic delay in the equilibration of nucleoplasmic Ca2+ concentration ([Ca2+]nuc) and [Ca2+]i , RyRs, NAADPR (nicotinic acid adenine dinucleotide phosphate receptor), Na+/K+ ATPase and Na+/H+ exchanger  and degraded over time  are important to generate highly localized Ca2+ signals to control Ca2+-dependent transcription  and promote histone deacetylases (HDAC) phosphorylation  dephosphorylation via the Ca2+ sensitive phosphatase calcineurin (CaN). This ultimately leads to the activation of different sets of transcription factors, e.g., myocyte enhancer factor 2 (MEF2) for HDAC and GATA for NFAT  as a measure of Ca2+ binding affinity is crucial for the selection of the appropriate Ca2+ dye for a particular cellular compartment of interest. Low affinity, high Kd dyes (like Fluo-5N) are used for the visualization of changes in SR [Ca2+] or nuclear envelope [Ca2+], whereas, e.g., Fluo-4 (Kd of 345 nM) is one of the preferred dyes for imaging of changes in cytosolic free [Ca2+], which varies roughly between 100 nM and values at times exceeding 1 μM during ECC. Since the nucleoplasm and the cytoplasm are interconnected compartments with similar global [Ca2+] characteristics, dyes suitable to show changes in [Ca2+]i can be used for the detection of changes in [Ca2+]nuc as well. Using Ca2+ sensitive dyes, Zima and Blatter (3R-mediated Ca2+ release events (Ca2+ puffs) and show a positive inotropic effect of neurohumoral stimulation with Endothelin-1 in cardiac myocytes. As mentioned above, the same group was also able to show changes of local nuclear envelope [Ca2+] in isolated nuclei upon stimulation with IP3, using Fluo-5N  or heparin are widely used IP3R blockers to study the GPCR/PLC/IP3 pathway. More recent molecular techniques and the generation of transgenic animals complement these tools. Noteworthy are the generation of IP3R knock-out and IP3R overexpressing mice as well as the development of IP3-sponges that allows the cellular overexpression of IP3 buffering proteins. The generation of IP3R overexpressing mice combined with the adenoviral expression of an IP3 sponge provided novel insights into the importance of this pathway in cardiac physiology and pathophysiology. Ca2+ transients in IP3R overexpressing mice were increased and showed a higher potential for arrhythmias after Endothelin-1 treatment. These effects were abrogated after expression of the IP3 sponge  between cyan and yellow fluorescent protein (CFP and YFP) upon binding of IP3. For that purpose FIRE contains a fusion protein of CFP, YFP, and the IP3 binding domain of the IP3 receptor type 1, 2, or 3 and can be targeted to the cytosolic or nuclear compartment. An increase in [IP3] is detected by an increase in FRET signals and a change in the YFP/CFP fluorescence ratio.An approach to directly visualize cellular . Moreover Figure 3 uncaging on different cellular compartments in atrial and ventricular cells. Figure 3 uncaging on local cytosolic and nuclear Ca2+ transients in field stimulated (0.5 Hz) ventricular cells (Fluo-4). Only prolonged exposure to the IP3 uncaging signal (100 ms laser illumination) has immediate visible effects on local Ca2+ release  and the overall effect on cytosolic, nuclear and subsarcolemmal Ca2+ transient amplitudes is higher upon IP3 uncaging. Note also the altered Ca2+ transient kinetics with a prolongation of the Ca2+ transient's amplitude following IP3 uncaging . Figure 2Cb shows the effect of the IP3R blocker 2-APB (10 μM). The effect of IP3 uncaging on Ca2+ transients in an atrial cell, pre-incubated with 2-APB, was abolished.Further progress in the study of IPe Figure . The IP3g Figure a. Figure2+/IP3 signaling regulate cardiac ECC. A number of deterministic models of ventricular and atrial myocyte electrophysiology, intracellular Ca2+ handling and bioenergetics have been published. For a more complete review on successes and failures in these modeling pursuits we refer the reader to some excellent recently published articles  receptors and kinetics associated with pre-coupling the receptors. In 1992, De Young and Keizer  geometry from electron-tomography series. This model simultaneously tracks the changes in cytoplasmic and ER [Ca2+], includes luminal and cytoplasmic Ca2+ buffers, plasma membrane Ca2+ fluxes, SERCA, ER leakage, and type-2 IP3R. A unique feature of the model is the inclusion of the stochastic behavior of type-2 IP3R. The results showed that IP3Rs in close proximity modulate the activity of their neighbors through local Ca2+ feedback effects. Finally, in 1999 an analysis performed by fluorescence measurements of [Ca2+]i and [Ca2+]nuc in ventricular myocytes revealed that [Ca2+]nuc increases concomitantly with [Ca2+]i upon electrical stimulation, but the pattern of [Ca2+]nuc increase was biphasic (rapid and slow)  this tool would provide fundamental new information on the role of IP3R-mediated Ca2+ signaling during ECC for arrhythmogenesis, for electrophysiological changes and for nuclear Ca2+ signaling in normal and failing cardiac cells; (b) as more experimental details on the complexity of IP3 regulation in myocytes accumulates, the intuitive interpretation of new findings becomes increasingly impractical and sometimes controversial. In pursuing this goal we extended the Shannon-Bers model in rabbit ventricular myocytes  were incorporated  are stable during 10 min stimulation at 0.5, 1, or 2 Hz. The model mimics also the frequency-dependent increases in the diastolic [Ca2+]i  were slower in the nucleus as compared to the cytosol. The physiological utility of the model was tested further by applying different frequencies to simulate the positive force-frequency relationship . Moreover we compared experimentally the influence of IP3 uncaging on different compartments  and were able to show that ventricular cells need a stronger IP3 stimulus to elicit a nuclear response, whereas atrial cells display substantial increases in nuclear and cytosolic Ca2+ transient amplitude upon a weaker IP3 uncaging stimulus, consistent with their higher total expression of IP3Rs as compared to ventricle. The recent development of FRET-based probes used for the detection of [IP3] as well as approaches to alter nuclear and/or cytosolic [IP3] provide experimental tools for the study of IP3-dependent Ca2+ release and its importance in ECC and ETC.In this review we discussed the current state of experimental and modeling approaches to investigate nuclear and cytosolic Ca2+ signaling model that integrates three different cellular subsystems  and couples Ca2+ dynamics in the cytosol and nucleus. This new tool is under development and will undergo further testing in its prediction of experimental [Ca2+]nuc and [Ca2+]i data in rabbit ventricular cells. The proposed model will also be extended to investigate how the complex dynamics of type-2 IP3 receptors (Sneyd and Dufour, 3R channel (Fraiman and Dawson, 3 signal transduction pathway with neurohumoral agonists (Cooling et al., 3-induced positive inotropy in cardiomyocytes continue to be controversial with numerous cellular targets being implicated in the response, including L-type Ca2+ channels, K+ channels, and Na+/Ca2+ exchange (Lauer et al., 2+, IP3, and CaMKII signaling pathways contribute to heart failure phenotypes. Finally, the tools and insights our group develops will be useful to investigate how perturbations in cytosolic and nuclear Ca2+ and IP3 signaling affect ECC and ETC in atrial myocytes (Grandi et al., We also presented our recent efforts of a first attempt to develop an electrophysiological and CaDesigned the work: Anushka P. Michailova and Felix Hohendanner. Performed the experiments: Felix Hohendanner and Lothar A. Blatter. Performed the simulations: Anushka P. Michailova. Analyzed the data: Anushka P. Michailova, Felix Hohendanner, Lothar A. Blatter, and Andrew D. McCulloch. Contributed reagents/materials/analysis tools: Anushka P. Michailova, Felix Hohendanner, and Lothar A. Blatter. Wrote the paper: Anushka P. Michailova, Felix Hohendanner, Lothar A. Blatter, and Andrew D. McCulloch.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NMDA receptor-dependent intrinsic oscillations require Ca2+-dependent K+ (KCa2) channels for burst termination. However, the location of Ca2+ entry mediating KCa2 channel activation, and type of Ca2+ channel – which includes NMDA receptors and voltage-gated Ca2+ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca2+ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca2+ entry is not similarly constrained. Where Ca2+ enters relative to KCa2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca2+ permeating NMDA receptors is the dominant source of Ca2+ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca2+ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate KCa2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca2+ entry but leaves membrane potential and Ca2+ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca2+ chelation. Taken together, these results demonstrate that KCa2 channels are closely located to NMDA receptor-dependent Ca2+ entry. The close spatial relationship between NMDA receptors and KCa2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca Throughout the central nervous system oscillatory activity requires the precise temporal activation and inactivation of neurons. This may be achieved through intercellular inhibition, but also through mechanisms intrinsic to individual neurons. The intrinsic neuronal and synaptic properties that are utilized during oscillatory activity shape the behaviors neurons subserve at the network level. Rhythmic behaviors, such as locomotion, require the transformation of an excitatory command from brainstem neurons into an oscillatory output patterns of motoneuron bursting 2+ entry to activation of an outward Ca2+-activated K+ channel . The animals were anesthetized with tricaine methanesulfonate , decapitated, and dissected in cold saline solution of the following composition (in mM): 130 NaCl, 2.1 KCl, 2.6 CaCl2, 1.8 MgCl2 or 1.8 MgSO4, 4 glucose, 5 HEPES, adjusted to a pH of 7.60 with NaOH and to a final osmolarity of 270±5 mOsm. The spinal cord was isolated, removed from the protective meninx primitiva and placed in a cooled, small-volume chamber with a sylgard  floor that is inserted onto the stage of an upright microscope. At minimum, a 15-segment section of spinal cord was pinned to the sylgard. In experiments involving whole-cell patch recording, a 10–20 µm slice of tissue was removed from the surface of the spinal cord over the ventral horn using a vibratome tissue slicer. Patch pipettes were then readily introduced to the cut ventral surface Experiments were performed on isolated spinal cords of late stage larval (voltage clamp step experiments) and recently transformed lampreys  or KOH (for oscillation and evoked EPSC experiments) to a final osmolarity of 250±2 mOsm. In voltage clamp experiments, 1 mM phosphocreatine  was added to the patch solution to prevent rundown of VGCCs. Cell types were identified by their location in the tissue and the electrode positioned on individual neurons under visual guidance. Pipettes had open-tip resistances of 5–10 MΩ. Series resistance compensation was performed for voltage step experiments and was monitored continuously by giving a 10 mV voltage step before each episode. Cells were discarded if the series resistance changed by more than 10%. Voltage steps were given at 5 s intervals to minimize current rundown. After whole-cell access was achieved, 10 min was allowed for diffusion of Ca2+ buffers from the pipette (unless stated otherwise) or replacement of Ca2+ with Ba2+ in the extracellular solution to allow currents to reach maximum amplitude before drug application. Due to the large size of ventral horn neurons in recently transformed lampreys, neurons from larval lampreys were used to minimize space clamp error generated from voltage steps. As an exception, transformer neurons were used for all experiments where membrane NMDA receptor-dependent membrane potential oscillations were induced in current clamp prior to switching to voltage clamp to perform step protocols. In adult transformer lampreys, paired recordings were made between presynaptic reticulospinal (RS) axons recorded with sharp microelectrodes and postsynaptic spinal neurons recorded under whole-cell voltage clamp in Mg2+-free ringer (to circumvent NMDA receptor block) while clamping between −70 mV and −60 mV. Sharp microelectrode were made conventionally with thin-walled glass with tip resistances of 20–50 MΩ. The pipette solution for recording and stimulating RS axons was 3 M KCl unless otherwise stated. Action potentials were evoked in the presynaptic axons at 15 s intervals to prevent postsynaptic current rundown. Drugs were only added after a stable baseline was achieved (10–15 min).Ventral horn neurons (motoneurons or interneurons) were recorded in whole-cell voltage clamp configuration, blind to cell identity. The patch pipette solution contained (in mM): 102.5 cesium or potassium methane sulfonate, 1 NaCl, 1 MgClSelective, extracellular excitatory interneuron (EIN) stimulation was achieved using custom made tungsten stimulating electrodes. Tungsten wire  was electrochemically etched to make fine tips, inserted into a glass capillary tube and then secured in place by glue. The glass was then pulled using an electrode puller to expose the tips and sylgard was used for added insulation, only exposing the very tip of the wire. EPSCs were evoked using an isolated stimulator  to deliver low intensity (5–10 µA), highly localized stimulation, 1–2 segments rostral and ipsilateral to the whole-cell voltage clamped cell. Strychnine was simultaneously applied to prevent glycinergic inhibition from coincident stimulation of inhibitory interneurons. Selective EIN excitation and reticulospinal axon avoidance was achieved through several factors: (1) reticulospinal axons have a very high membrane capacitance and require substantial focal current injection to induce an action potential. This means that the axons require more charge than the tungsten electrode delivers at low stimulation intensity, (2) extracellular stimulation is insufficient to cause spikes in reticulospinal axons recorded with a microelectrode, and (3) the majority of reticulospinal axons passing over cell somata are removed from the slice preparation, while remaining medial or lateral axons can be visually avoided. Stimuli were given at 15 s intervals to prevent postsynaptic current rundown and drugs were only added after a stable baseline was achieved (10–15 min).2+-sensitive dye, 10,000 MW Oregon Green 488 BAPTA-1 dextran (Life Technologies (Invitrogen), Grand Island, NY), was injected into the body musculature  to allow uptake into motoneuron axon terminals. The animal was then allowed to recover in a cooled aquarium, allowing the dye time to be retrogradely transported into somata and dendrites. After 48 h, the animal was decapitated under anesthesia, and the spinal cord adjacent to the injection site was removed. Imaging revealed Ca2+-sensitive dye labeling selectively in motoneurons of corresponding hemisegments.Recently transformed lampreys were anesthetized. The dextran-conjugated Cahttp://alford.bios.uic.edu/Research/software.html).Recently transformed lampreys were used for all imaging experiments. Confocal imaging was performed using a modified Bio-Rad  MRC 600 confocal microscope. Two laser excitation wavelengths were used (488 nm argon ion and 568 nm krypton–argon) through an acousto-optic tunable filter-coupled fiber optic launch . Excitation was applied through a custom dichroic mirror with sharp excitation bands matching the two laser wavelengths . Two detectors were placed after a second dichroic, with a transmission band from 500–560 nm and long-pass reflection from 580 nm. Emission filters were bandpass (500–560 nm) and long pass (above 580 nm). The photomultiplier outputs were amplified with low-noise current amplifiers  and digitized to 12 bits with a National Instruments  board and custom software written under Matlab . The scan-head mirrors were driven though the MRC 600 scan-head amplifiers with the same custom software, available on our website . Acquisition was performed using µManager software Simultaneous fluorescence imaging combined with electrophysiological recordings were performed with a CCD system  mounted onto a compound microscope  equipped with a rapidly switchable Xenon source  and emission filter wheel . A 490 nm or 560 nm bandpass excitation filter and 510 nm long pass or 645 nm bandpass emission filter was used for 488 nm CaΔF/F  = 0. Baseline was defined as the fluorescence at the location analyzed prior to electrical stimulation. For oscillation imaging experiments, this was defined as the fluorescence prior to NMDA application. For figures displaying the location of changes in fluorescence, the entire image sequence was divided by the average of the prestimulus images. This generated a relative fluorescence sequence, the peak of which was averaged and then contrast- and brightness-adjusted to display a black background. For all experiments, cells were discarded if bleaching or response amplitude rundown occurred for the entirety of the control condition. Data are given as means ± SEM and n = number of cells. Student’s paired, one-tailed t test was used to calculate the significance between control and drug conditions.Electrophysiological analysis was performed offline using Axograph X software . Imaging data analysis was performed using ImageJ . For analysis of Oregon Green 488 BAPTA-1 and Fluo-5F, fluorescence intensities after background subtraction were normalized to the baseline (prestimulus), giving a baseline value of All experiments on animals were performed in accordance with Institutional and national guidelines as laid down by AALAC and under an approved University of Illinois at Chicago Animal Care Committee protocol issued to SA (Permit #: 12–023). All surgery was performed under MS-222 anesthesia, and all efforts were made to minimize suffering.2+-dependent 2+ is necessary for the repolarization of the cell via KCa2 channel activation 2+ entry is unclear. We hypothesize that oscillations are driven through local, NMDA receptor-dependent synaptic activity where Ca2+ entry is coupled to KCa2 channels. To determine if the site of Ca2+ entry responsible for repolarization is closely apposed to KCa2 channels, we used whole-cell recordings to dialyze neurons with either BAPTA or EGTA, fast and slow Ca2+ buffers, respectively 2+ before activating KCa2 channels to repolarize the membrane. In contrast, NMDA (50 µM) invariably induced repetitive oscillations with inclusion of EGTA in the pipette, indicating that EGTA does not bind Ca2+ before it can mediate KCa2 channel-dependent repolarization . KCa2 channel blockade significantly and reversibly prolonged the oscillation duration  and in an additional 2 cells reversibly prevented repolarization  and TTX (1 µM) were bath-applied to induce oscillations in whole-cell current clamp  and TEA (5 mM) were applied to block voltage-gated K+ currents. Depolarizing steps were used to activate voltage-dependent currents in 10 mV increments. Despite blocking K+ channels, the true threshold for activation and magnitude of step-evoked inward currents was not measurable with Ca2+ in the Ringer’s solution and EGTA as a Ca2+ buffer in the recording pipette. A late, secondarily activated, outward current was recorded overlaying the inward current. We hypothesized that this is due to KCa2 channel activation 2+ (2.6 mM) with Ba2+ (1 mM) in the superfusate .Addition of NMDA (50–100 µM) to the superfusate hyperpolarized the threshold for current activation to membrane potentials as negative as −80 mV in cells perfused in Baeptor 2+  comparedr 2+  at −90 mV and depolarizing steps applied in TTX, 4AP and TEA as above. Again, addition of NMDA leads to a biphasic current in which the initial inward current is followed by activation of an outward current . Addition of NMDA caused an outward current with steps to −20 mV . In contnt clamp ii and bynt clamp . This de2+ imaging to determine sites and magnitude of Ca2+ entry during voltage steps. Ventral horn neurons were whole-cell clamped with Ca2+-sensitive dye (either Oregon Green 488 BAPTA-1 (OGB1) 50 µM; or Fluo-5F, 100–200 µM combined with 50 µM of the inert dye, Alexa Fluor 568 hydrazide) included in the patch solution. Dye was allowed to diffuse into the cell for 30 minutes following whole-cell access and then imaged using a confocal microscope. From a holding potential of −90 mV in TTX (1 µM), 4-AP (1 mM) and TEA (5 mM), cells were stepped to membrane potentials between −70 mV and 0 mV to define the relationship between voltage-dependent currents and Ca2+ dye fluorescence  and presumably intracellular Ca2+ (Ca2+i) signals that are too small to be detected by voltage clamp current recording alone in these spatially complex neurons.Whole-cell voltage clamp was combined with Carescence –5. In alcurrents . The abicurrents  reflects2+ fluorescence and current. Fluorescent Ca2+ signals  lowered the threshold for step-evoked Ca signals  were evo signals  in all naneously . Again, aneously i and curaneously iii. Addianeously . Thus, C2+ entry is necessary for KCa2-dependent repolarization during membrane potential oscillations recorded in NMDA and TTX, we pharmacologically isolated NMDA receptor-mediated currents. Cd2+ was applied as a non-specific high voltage-activated VGCC antagonist during the step protocol. Cd2+ completely blocks VGCCs at concentrations of 50–200 µM 2+ (>50 µM) reduces NMDA receptor currents in cultured rat hippocampal neurons 2+ blocks VGCCs at 25 µM in situ, neurons were whole-cell voltage clamped  with BAPTA in the patch pipette (9.5 mM) to isolate VGCC currents evoked by stepping from a holding potential of −90 mV as before. Cd2+ (25 µM) significantly inhibited VGCC current  had no effect on either the peak current or area under the curve . At this reduced concentration, Cd2+ is thus selective for VGCCs, having little effect on NMDA receptors.To confirm that this concentration of Cd2+ was then applied to neurons in which TTX-resistant NMDA receptor-activated membrane potential oscillations were recorded 2+ (25 µM) was applied to the superfusate. Cd2+ caused a significant and reversible reduction in the duration of the depolarized phase , on NMDA-dependent TTX-resistant membrane potential and Ca2+ oscillations recorded with fluorescent dyes. ω-CgTxMVIIC (5 and 2 µM) inhibits both VGCCs in lamprey cultured ventral horn neurons in vitro spinal cord preparation, we performed membrane potential step protocols as before . VGCC currents were substantially inhibited to 28±13% of control  in the patch solution. After dye diffusion (∼30 min after whole-cell access), oscillations were induced by bath application of NMDA (50–100 µM) and TTX (1 µM). Image sequences of Ca2+ fluorescence in dendrites were acquired while making simultaneous electrophysiological recordings before and during application of ω-CgTxMVIIC . Thus, despite a profound block of VGCC current, ω-CgTxMVIIC does not impair membrane potential or Ca2+ oscillations.To determine whether ω-CgTxMVIIC affects membrane potential and CaIC 5 µM, i. IndiviCa2 channel activation. Ca2+ entry through either NMDA receptors or VGCCs Ca2 channels. During locomotion, NMDA receptors may be physiologically activated by glutamate release from either descending reticulospinal (RS) neurons Intrinsic rhythmicity of individual cells requires K2+ signals was found as the holding potential was increased from −80 mV to −30 mV  included in the patch solution. After dye diffusion, EIN-mediated synaptic responses were selectively recorded by placing a 2–5 µm stimulating electrode, over the ventro-lateral spinal cord  and using very low intensity stimulation (5–10 µA), which is insufficient to evoke RS axon stimulation , reversibly abolished these evoked increases in Ca2+ fluorescence in all cells imaged , and strychnine (5 µM) added to isolate NMDA receptor EPSCs and glycine (1 µM) to ensure NMDA receptor activation. Superfusion of apamin (5 µM) over the preparation had no significant effect on the amplitude of single, monosynaptic RS-evoked NMDA receptor EPSCs (Both EIN and RS axons evoke CaCa2 channels. Evoked NMDA receptor EPSCs were achieved as before with an extracellular stimulating electrode in the presence of NBQX (5 µM), glycine (1 µM) and strychnine (5 µM) at a holding potential between −60 and −50 mV. Both apamin and the potent KCa2 channel inhibitor, UCL 1684, led to a significant and reversible increase of EPSC amplitude , tempering excitability and synaptic plasticity Nonlinear dynamics, such as oscillations, are found in all regions of the nervous system and underlie activities from encephalographic recordings of coordinated brainwave behavior 2+ entry are central to the generation of membrane potential oscillations in lamprey ventral horn neurons which contribute to the precise rhythmic output of the spinal CPG 2+ block 2+ entry as may VGCCs. However, depending on the location of KCa2 channels with respect to the NMDA receptor 2+ entry to the cytosol may subsequently activate an outward current. The relationship between this Ca2+ entry and secondary effectors will influence oscillatory properties extending from the cell to network level. Thus, it is important to determine the route of Ca2+ entry that drives K+ current needed to terminate bursting activity 2+ release from internal stores has little or no effect on NMDA-induced fictive locomotion 2+ entry to determine their contribution to nonlinear properties of neurons during oscillatory activity.Nonlinear membrane properties in response to NMDA receptor activation and Ca2+ buffering occurs with BAPTA dialysis  and the peak plateau membrane potential reached within the same cell during NMDA-evoked TTX-resistant oscillations revealed a sharp contrast (2+ currents were often more depolarized than the plateau peak (Ca2 channel activation due to either close coupling or the low threshold (EC50 ∼0.3 µM) for Ca2+-dependent KCa2 activation 2+ whereas VGCCs contribute very little Ca2+ within the oscillation range. Ventral horn neurons undergo phase-locked oscillations in Ca2+i with the largest amplitudes in distal dendrites 2+ signals parallel NMDA-induced changes in membrane potential 2+, leaving dendritic Ca2+ oscillation amplitudes unaffected. For these reasons, we hypothesized that NMDA receptors provide the main source of Ca2+ underlying the repolarization phase of the oscillation via KCa2 channel activation, localized to dendritic synapses.In non-spiking conditions (i.e. in TTX or subthreshold oscillations during fictive swimming), NMDA receptors and VGCCs may be activated during the depolarization phase of ventral horn neuron oscillations, yet the relative thresholds for VGCC and NMDA receptor activation have not been examined in neurons within the spinal cord. Direct comparisons between the VGCC activation threshold  around points of entry. However, such synaptic Ca2+ entry is capable of activating a substantial apamin- and UCL 1684-sensitive outward current following a single stimulus of presumed EINs , very little VGCC-dependent current is recorded and, correspondingly, very little Ca2+ dye fluorescence is detected at any somatic or dendritic location (2+ entry over the same voltage range (2+ entry within the oscillation range. Inevitable voltage breakthrough resulting from uncorrectable space clamp errors may contribute to this enhancement in NMDA-evoked currents. However, block of VGCCs with ω-CgTxMVIIC, which selectively inhibits N- and P/Q-subtype VGCCs 2+ oscillations (2+ abolishes VGCC current but did not prolong membrane potential oscillations. Thus, because oscillations persist and do not increase in duration, and Ca2+-sensitive dye fluorescence does not reduce after pharmacological block of VGCCs, it is unlikely that VGCCs contribute substantially to the repolarization. This suggests that the NMDA receptor, which is maximally activated by the peak of the oscillation, is the primary route of Ca2+ entry that subsequently activates KCa2 channels to drive the repolarizing phase of the oscillation.Our imaging data also demonstrates that the majority of Calocation . Howeverge range . This dellations . SimilarCa2 in lamprey spinal neurons Ca2 channels Ca2 channels located in dendrites, closely apposed to NMDA receptors activated by glutamate release from EINs. These KCa2 channels participate in the repolarization of membrane potential oscillations. The location and route of Ca2+ entry is central to the corresponding intrinsic membrane property and subsequent output.Previously, VGCCs have been shown to activate K2+ ionophores whose activation is voltage-dependent in excitatory, synaptically driven neural behaviors. In the case of spinal motor oscillators, their function requires the activation of NMDA receptors, but Ca2+-activated outward currents might arise from a number of distinct systems. We demonstrate that the dominant Ca2+ signal for KCa2 channel activation responsible for nonlinear oscillations underlying rhythmogenesis in lamprey ventral horn neurons is entry through the NMDA receptor itself, particularly those receptors synaptically activated by local spinal EINs.It has proven difficult to sort the physiological roles of various Ca


DMPK. In DM1 expanded CUG repeat RNAs form hairpin secondary structures that bind and aberrantly sequester the RNA splice regulator, MBNL1. RNA splice defects resulting as a consequence of MBNL1 depletion have been shown to play a key role in the development of DM1 pathology. In patient populations, both the number and severity of DM1 symptoms increase broadly as a function of CTG tract length. However significant variability in the DM1 phenotype is observed in patients encoding similar CTG repeat numbers. Here we demonstrate that a gradual decrease in MBNL1 levels results both in the expansion of the repertoire of splice defects and an increase in the severity of the splice alterations. Thus, MBNL1 loss does not have an all or none outcome but rather shows a graded effect on the number and severity of the ensuing splice defects. Our results suggest that once a critical threshold is reached, relatively small dose variations of free MBNL1 levels, which may reflect modest changes in the size of the CUG tract or the extent of hairpin secondary structure formation, can significantly alter the number and severity of splice abnormalities and thus contribute to the phenotype variability observed in DM1 patients.Myotonic dystrophy (DM1) is a highly variable, multi-system disorder resulting from the expansion of an untranslated CTG tract in  DMPKMyotonic dystrophy (DM1) is a multi-system disorder, demonstrating myotonia, skeletal muscle weakness and wasting, cardiac arrhythmias, CNS dysfunction, cataracts, insulin resistance and endocrine disorders in vitro and the stem of such hairpins binds the splice regulator, Muscleblind-like 1 (MBNL1) DMPK 3′UTR with expanded CUG repeats show either the formation of a single large hairpin or two or more shorter hairpins with similar calculated free energy in vivo is unknown, hairpin formation is strongly supported by the fact that expanded CUG repeat encoding RNAs bind and aberrantly sequester MBNL1 to form CUG-protein aggregates or foci within the nucleus Expanded CUG repeat RNA form metastable, slippery hairpin secondary structures DM1 pathology is widely regarded to result from the aberrant splicing of a set of physiologically important RNAs Based on these data we hypothesized that dose variations in free MBNL1 levels could play an important role in disease variability in the DM1 patient population. Using human myoblasts as a model system we show that MBNL1 depletion does not show an all or none effect but rather demonstrates that incremental depletion of MBNL1 results in an increase both in the number and severity of splice defects. Our results therefore support the hypothesis that variations in free MBNL1 levels, which may occur as a function of the size of the CUG tract length expansions or the extent of hairpin secondary structure formation, play an important role in modulating the number and severity of DM1 symptoms.IR), cardiac troponin T (cTNT), Z-band alternatively spliced PDZ-motif protein (ZASP), fibronectin 1 (FN1) and muscleblind like 2 (MBNL2) that are known to be abnormally spliced in DM1, in normal human myoblasts (SkMC) Clcn1) RNA in addition to the 5 sample RNAs detailed above in wild-type, +/Δ3Mbnl1 and  Δ3/Δ3Mbnl1 mouse skeletal muscle tissue CLCN1 RNAs were not examined in human myoblasts as this RNA is not expressed at significant levels in this cell type (data not shown).To examine the hypothesis that incremental loss of MBNL1 is a significant contributing factor to the number and severity of DM1 splice defects, we studied the splicing of 5 sample RNAs: insulin receptor  as the silencing of MBNL1 increases from ∼67% to ∼98%. In the case of FN1, inclusion of the EDB exon increases from ∼34% to ∼60% (Scrambled Control: ∼17–18%) as MBNL1 silencing increases from ∼67% to ∼98%. Similarly, inclusion of IR exon 11 decreases from ∼22% to ∼8% (Scrambled Control: ∼48%) when MBNL1 silencing increases from ∼87% to ∼98%. Lastly, cTNT exon 5 inclusion increases from ∼82% to ∼88% (Scrambled Control: ∼49%) and ZASP exon 11 inclusion increases from ∼70 to ∼81% (Scrambled Control: ∼23%) when MBNL1 decreases from ∼97% to ∼98% , inclusion of exon 9 of Zasp increases from ∼12% to ∼43% (wild-type: ∼4%) and inclusion of the EDB exon in Fn1 increases from ∼24% to ∼59% (wild-type: ∼7%). No significant splice defects in Ir, cTnt and Mbnl2 were observed in +/Δ3Mbnl1 mouse muscle .siRNA oligonucleotides were synthesized by Dharmacon (USA), deprotected and the complementary strands were annealed. The sequences of the siRNAs used in this study were:Scrambled: 5′-GCGCGCUUUGUAGGAUUCGdTdT-3′;MBNL1∶5′-CACUGGAAGUAUGUAGAGAdTdT-3′.MYC and the long-lived 18S RNA were used as controls. In all cases, the relative band intensities were measured by densitometry analyses and 18S RNA levels were used for normalization. RNA half-lives were calculated using previously described methods Myoblasts were cultured and maintained in SKGM media  containing 10% fetal bovine serum and 1% penicillin-streptomycin. siRNA-mediated depletion of MBNL1 was carried out using the methods described in Dansithong et al MBNL2, FN1, IR, ZASP and cTNT splicing using primers and PCR conditions described in To examine splice defects as a function of MBNL1 levels, SkMC samples post-siRNA transfection, were divided into 4 parts where one aliquot was used to measure MBNL1 levels and total RNA was extracted from each of the three other aliquots. Total RNA was isolated using the RNAeasy mini kit (Qiagen), according to the manufacturer’s instructions. RT-PCR analyses of splice isoforms was performed as described in Paul et al ΔE3/ΔE3Mbnl1 mice were a gift from Dr. Swanson (University of Florida College of Medicine). Skeletal muscle tissue from the hind limbs was collected from adult (2–3 month) wild-type (n = 2), +/ΔE3Mbnl1 (n = 3) or ΔE3/ΔE3Mbnl1 (n = 2) mice. The samples were subsequently divided into two aliquots. One aliquot was used to measure Mbnl1 levels by western blot analyses and total RNA was prepared from the second aliquot and RNA splice isoforms were examined. From each set of RNAs, three independent sets of cDNAs (150 ng) were prepared and splicing of Mbnl2, Fn1, Ir, Zasp and cTnt was studied using PCR conditions as described in Clcn1 splicing was examined as described by Kanadia et al Whole-cell and tissue extracts were prepared and equal amounts of protein (5–10 µg) were separated by SDS–PAGE and transferred onto Hybond P membranes . After blocking with 5% skim milk in 0.1% Tween-20 in PBS, the membranes were incubated with the primary antibodies for 2 h at room temp or overnight at 4°C. After incubation, the membranes were washed with 0.1% Tween-20 in PBS and incubated with the corresponding secondary antibodies conjugated with HRP. Signals were detected using the ECL plus detection reagents  according to the manufacturer’s instructions. MB1a monoclonal antibodies at a dilution of 1∶3000 were used to detect MBNL1 Figure S1Alternative RNA splicing in normal myoblasts with a graded decrease in MBNL1 levels from ∼9-67%. MBNL1 depletion in SkMC cells is shown in IR, cTNT and ZASP with GAPDH RNA as an internal control. No significant splice errors were observed in IR, cTNT and ZASP when MBNL1 was depleted from ∼9–67%.(TIF)Click here for additional data file.Figure S2Graphical representation of RNA half-life measurements in SkMC. The average decrease in RNA levels in two independent experiments at the time points shown were used to calculate half lives using semi-log plots as previously described (TIF)Click here for additional data file.Table S1Primers for half-life measurements and PCR parameters.(PDF)Click here for additional data file.Table S2Primers used for human RNA splicing and PCR parameters.(PDF)Click here for additional data file.Table S3Primers used for mouse RNA splicing and PCR parameters.(PDF)Click here for additional data file.


Acute myeloid leukemia (AML) arises from neoplastic transformation of hematopoietic stem and progenitor cells, and relapsed disease remains one of the greater challenges in treating this hematologic malignancy. This paper focuses on angiogenic aspects of AML including the significance and prognostic value of bone marrow microvessel density and circulating cytokine levels. We show three general mechanisms whereby AML exploits angiogenic pathways, including direct induction of angiogenesis, paracrine regulation, and autocrine stimulation. We also present early evidence that leukemia cells contribute directly to vascular endothelia. Novel treatment strategies are proposed, and a review of relevant antiangiogenic clinical trials is presented. By understanding how blood vessels can serve as a reservoir for refractory and relapsed AML, new diagnostics and promising treatment strategies can be developed. Acute myeloid leukemia (AML) is a cancer of the bone marrow characterized by a mutation in a hematopoietic stem or progenitor cell (HSPC), which develops into a highly proliferative accumulation of dysfunctional and immature myeloid cells. These abnormal cells eventually dominate hematopoietic niches like the bone marrow and result in abnormal peripheral blood counts  . InitialUntil recently, leukemia studies have focused primarily on the leukemia cell. However, with mounting evidence showing the importance of the bone marrow microenvironment in regulating hematopoiesis, it is necessary to broaden the scope of investigation beyond the leukemia cell. A better understanding of the pathobiology surrounding and supporting leukemia cell survival has great potential to lead to promising new therapies.The importance of the cancer microenvironment is widely recognized in solid tumors. Cancer cells interact with the stromal microenvironment in complex ways to promote their own survival and proliferation. However, in the case of hematologic malignancies like AML, the leukemia microenvironment is highly dynamic. The typical leukemia niche is within the bone marrow microenvironment. But AML cells can also migrate systemically to other organs that support hematopoiesis, such as the liver and spleen . MonocytAlthough the complete model of the bone marrow microenvironment is not yet fully understood, it has been simplistically divided into three compartments: an endosteal niche that maintains quiescent hematopoietic stem cells, a vascular niche which regulates entry and exit from the bone marrow, and the central marrow space filled with various hematopoietic progenitors in the process of differentiation  Figure . One of the earlier investigations in the relationship between AML cells and the vascular niche was performed by Fiedler et al. . These iP < 0.001), suggesting a role of angiogenesis in AML. This is particularly significant when considering the strong positive correlation between increased bone marrow vasculature and overall survival of leukemia [Recent evidence indicates that leukemia cells, like tumor cells, depend on angiogenesis in the bone marrow. Clinically, increased angiogenesis has been reported in the bone marrow of patients with AML. Hussong et al. stained bone marrow biopsies for blood vessels in 20 patients with untreated AML, compared with 20 control patients and quantified the number of vessels/mm in each case . They foleukemia , 9. A hileukemia . In vitro studies have shown that upon transwell or direct coculture with HUVECs, AML blasts proliferate to a higher degree and are less susceptible to traditional chemotherapeutic agents such as cytarabine [Endothelial cells support adhesion and transmigration of subsets of normal CD34+ HSPCs. tarabine . We have3H-Thymidine incorporation assays were used to quantify proliferation of endothelial cells. Their results showed enhanced endothelial cell proliferation in transwell coculture with AML blasts [While endothelial cells enhance leukemia proliferation, emerging evidence indicates that leukemia cells may have a reciprocal effect of enhancing endothelial cell proliferation. Hatfield et al. investigated the hypothesis of interdependence using transwell and direct contact experiments between primary AML cells and dermal microvascular endothelial cells (DMVECs). L blasts . At the L blasts , evaluatWith ECs promoting AML cells, and AML cells promoting ECs, a cyclical positive feedback loop is established and strongly favors the potential for refractory and relapsed disease . Clinically, increased levels of circulating angiogenic factors correlate with increased angiogenesis in the bone marrow  and are The ability of leukemia cells to respond to angiogenic signals from endothelial cells suggests a close relationship between the two cell types. In fact, AML cells from patients have been reported to express VEGF and VEGFRs , 13, 18.Given that AML cells can secrete VEGF and express VEGFRs, it stands to reason that AML cells may benefit from an autocrine loop. Indeed, investigators have reported a possible autocrine loop including VEGF produced by leukemia cells and their own VEGF receptors. In particular, VEGFR2+ AML cells were treated with VEGFR2 neutralizing antibodies in serum-free growth conditions. Blocking VEGFR2 resulted in decreased leukemic cell growth, supporting the notion of a VEGF/VEGFR2 autocrine loop .in vitro study by Reikvam et al. found that blocking angiopoietin interactions with the Tie2 receptor using antibodies led to marked decreases in AML cell proliferation [The angiopoietin/Tie2 axis represents another potential autocrine loop in AML. We and others have found that certain myeloid leukemia cell lines, namely, K-562, express angiopoietin-1 (Ang-1), Ang-2, and their receptor, Tie2. Primary human AML specimens have also been reported to express these endothelial cell associated factors and receptor . An in vferation . Interesferation .in vivo model of primary human AML cells into immunocompromised mice. Hepatic sinusoids were lined by hybrid human AML-murine ECs (AML-EC). Moreover, these fused AML-ECs contained separated human DNA and murine DNA typically found in syncytia. Yet to be determined is whether the expression of EC proteins endows AML myeloblasts with resistance to chemotherapy. If AML-EC hybrids are more resistant to chemotherapy and can regenerate and proliferate AML cell population, then these cells may represent sources of refractory and relapsed disease. Another explanation for AML myeloblasts that coexpress EC phenotype is the possibility of fusion. Studies by Skinner et al.  further Blood and blood vessels are closely linked in developmental biology. In the embryo, the hematopoietic and endothelial lineages are generated from a common mesodermal progenitor, the hemangioblast , 26. Our− CD34−) harboring the BCR/ABL gene fusion was identified in the BM of patients with CML [in vitro at the single-cell level. Moreover, when transplanted into NOD/scid mice, these VEGFR2+ CD31− CD34− cells were capable of reproducibly transferring CML to transplanted mice and generating ECs within blood vessels that expressed BCR/ABL. In other studies, it was shown that transformed genotypes including BCR/ABL and the Janus kinase 2 (JAK2) V617F mutation are not readily found in colonies generated in endothelial colony forming cell (ECFC) culture conditions, whereas angiogenic monocytes that form CFU-Hill colonies can harbor such mutations [In one report, a subpopulation of vascular progenitor cells  brings about modest clinical efficacy in the treatment of colon cancer when combined with cytotoxic chemotherapy . In the in vitro and mouse xenograft models. The preliminary in vivo studies of Lal et al. showed that aflibercept slowed disease progression in two systemic human AML mouse xenograft models. Combining aflibercept with doxorubicin enhanced antileukemia effects, decreased microvessels, and induced perivascular apoptosis [Another agent that binds and targets the VEGF ligand, and with higher affinity than bevacizumab, is aflibercept VEGF trap. This fusion protein binds to VEGF-A, VEGF-B, and placental growth factor (PlGF). Aflibercept has only been evaluated in solid tumors for tolerability and shows modest antitumor activity . In AML,poptosis . Another strategy in targeting VEGF activity is to target VEGFRs. In specific, small molecule tyrosine kinase inhibitors (TKIs) have been designed to impair VEGFR phosphorylation activity. Vatalanib, active against VEGFRs and PDGFRs, is well tolerated and has shown clinical activity in multiple solid tumors. Vatalanib has been tested in AML and MDS. A two-armed phase I clinical trial of dose-escalated vatalanib showed that the agent is well tolerated in AML and MDS patients with minimal side effects . Hyperteβ, and VEGFR-3. In AML, a phase I clinical study of cediranib in 35 leukemia patients showed a correlation between cediranib exposure and plasma VEGF levels and dose- and time-dependent reductions of soluble VEGFR-2 [Cediranib, which was designed with even greater affinity for VEGFR-1 and VEGFR-2, has also shown clinical activity in certain solid tumors such as glioblastoma multiforme and nonsmall cell lung cancer , 48. Thi VEGFR-2 . Also, aIn AML, activating mutations in FLT3, especially internal tandem duplications, predict for a higher chance for refractory and relapsed disease. Thus, the current standard of care is to refer patients with FLT3 mutant AML for allogeneic hematopoietic cell transplant , as this is the only potential for cure. However, few older AML patients are candidates for allo-HCT because of comorbidities, difficulties in finding a donor and financial/insurance reasons. Therefore, attempts have been made to target FLT3 activity with inhibitors. n = 5) receiving sorafenib. Prior to the consolidation AraC cycles, there was a trend in slower regeneration of leukocytes and thrombocytes in the sorafenib arm. However, in terms of clinical response, there were no improvements in event-free survival and overall survival compared to the placebo group. This trial suggests that while targeting FLT3 with sorafenib is tolerable in AML, it shows little clinical activity.Sorafenib, a TKI designed to target Ras-Raf/MEK/ERK signaling, but also targets FLT3, has shown clinical activity in renal cell carcinoma and hepatocellular carcinoma , 50. GivSunitinib (SU11248) is another angio-inhibitory TKI that has been tested as an antineoplastic agent . In addi2 twice a week, the drug was well tolerated, with mostly mild to moderately severe adverse events. The most striking adverse event, not seen in solid tumor studies of semaxanib, was severe bone pain, which may be attributed to the activity of the drug in bone marrow. Of 25 patients who were evaluable for clinical response, 1 patient achieved a morphological response followed by a relapse after 8 weeks, and 7 of the 25 patients achieved partial response with decreases in bone marrow and peripheral blood leukemic blasts of at least 50%. In terms of biological response, VEGF levels and bone marrow MVD correlated with each other and decreased after semaxanib treatment. Again, the modest clinical activity of this small-molecule TKI dampens enthusiasm for monotherapy in AML. However, the agent did show biological activity and this merits further study, including combination therapy.The small-molecule TKI semaxanib (SU5416) targets the common VEGF receptors 1 and 2, cKit, and FLT3. In a multicenter phase 2 trial of semaxanib, 42 patients with advanced, c-kit positive AML, either refractory or elderly patients not fit for intensive induction chemotherapy, received at least one dose of treatment . At a doDue to their multitargeted potential, TKIs could open the door to more personalized cancer treatments. However, many are metabolized through hepatic cytochrome P450 enzymes and have the potential to interact with many medications commonly prescribed to patients with AML . It is conceivable that in the future, AML cell surface expression or genetic mutation may dictate what type of TKI to prescribe. This is particularly attractive in older patients with AML who may be ineligible for high intensity chemotherapy. These older patients may fare better with an oral TKI and low intensity chemotherapy. β-tubulin, lead to microtubule depolymerization and selectively target rapidly proliferating ECs. Unlike colchicine, combretastatins exhibit vascular disruption below maximum tolerated dose (MTD). Therefore, these agents are highly attractive for clinical translation. We and others have shown that naturally occurring combretastatins, CA4 and CA1, potently regress AML in xenograft models. We also showed that the combretastatin CA1 results in a VEGF-driven reactive angiogenesis which supports disease relapse. Therefore we devised a strategy to combine combretastatin (CA1) and anti-VEGF antibodies (bevacizumab). This novel strategy resulted in potent regression of AML. We therefore translated this work into a phase I clinical study of CA1 (OXi4503) in patients with relapsed and refractory AML and MDS . After establishing MTD, we plan to combine combretastatins with other angio-inhibitor agents like bevacizumab in future clinical trials.Our own studies have led further efforts to develop multitargeted therapies. Our lab has identified a promising multi-target antivascular treatment strategy, using a novel endothelial cell targeting agent, combretastatin A-1 (OXi4503) , alone aWhereas cancer angiogenesis is classically thought of in context to solid tumors, there is mounting evidence that angiogenesis is also significant in leukemia. With specific regard to leukemia and endothelial cells, there are several aspects to consider. First, endothelial cells can support leukemia cells via secreted factors. Whereas a few key axes have been identified , the full panoply of secreted factors has yet to be defined. Second, leukemia cells can promote endothelial cells. The observation of a codependent relationship creating a vicious cycle of support substantiates the strategy of using endothelial cell targeting agents such as combretastatins. Third, studies identifying critical adhesion molecules between leukemia and endothelial cells are lacking, and this represents an open area of research. These studies will enlighten us of how leukemia cells enter and exit the bone marrow. Understanding how sinusoidal endothelial cells—gatekeeper cells of the marrow—regulate emigration and immigration will lead to novel strategies for mobilizing leukemia out of protective niches and towards heightened sensitivity to treatment. Fourth, leukemia cells can coexpress endothelial cell features. How these features impact sensitivity and protection from conventional treatment need to be better elucidated. Part of these studies will involve inhibiting endothelial cell-associated expression, and it will be important to determine whether this inhibition results in increased sensitivity to treatment. If so, then this would represent a novel strategy for sensitizing leukemia-EC hybrids to treatment. Fifth, the vascular niche in the bone marrow contains many other cell types. Two in particular, perivascular pericytes and intercalated megakaryocytes, participate in the way that the sinusoidal vessels regulate hematopoietic stem and progenitor cell function. Therefore, it is reasonable to consider these other cells types as important players in the governance of leukemia behavior. Finally, beyond cell types, there are many noncellular elements of the bone marrow microenvironment that need to be assessed. These bone marrow conditions include chronic physiological hypoxia, low pH, low glucose levels, and extracellular matrices. All of these factors affect angiogenesis and vice versa; therefore, these factors need to be defined in the context of leukemia pathology to have a more complete picture of how the disease develops, responds to treatment, and relapses.One important clinical translation of a better understanding of the relationship between angiogenesis and leukemia is in the realm of improved diagnostics. Currently bone marrow biopsies and repeated blood draws are the mainstay for diagnosis, prognosis, and response assessment. With advent of new biologics that have antiangiogenic and antivascular activity, there is a calling for novel biomarkers and methods to measure response and predict for responders. Through the development of a pre-treatment biomarker screening method, we may be able to utilize existing and future antiangiogenic agents to their full potential on a more personalized basis. Another clinical translational consideration is how to assess treatment response after administering vascular targeting agents in AML. Currently, there are no established methods. Consider that the primary target is bone marrow blood vessels, it would reason to follow that changes in bone marrow vascularity indicate treatment response. However, there are several methods to quantify bone marrow vascular activity. Serial bone marrow biopsies for measurement of microvessel density, EC function, and angiogenic cytokinesare involve procedures that are painful and possibly affected by sampling area. Furthermore, histologic and biochemical testing takes time and do not provide results in real time. Another method to monitor bone marrow blood vessel activity is via serial computed tomography (CT) scans. However, this method exposes the patient to harmful ionizing irradiation and intravenous contrast carries the risk for nephrotoxic reactions. Magnetic resonance imaging (MRI) is another method to measure changes in bone marrow blood vessels. This method is rapid and does not deliver harmful ionizing irradiation. Moreover, the risks of a contrast (gadolinium) toxicity is lower than with CT scans. Recently, two groups have shown early data that dynamic contrast enhanced- (DCE-) MRI can be used to assess bone marrow vascular perfusion and predict for response to chemotherapy , 52.The last clinical translational consideration is that many of the vascular targeting clinical studies in AML have lacked investigation of mechanisms of action. It is assumed that the administered antivascular agents operated through blood vessel targeting mechanisms, but without an accurate measurement of response this theory has yet to be proven. Thus, it is imperative that rationally designed biomarkers be used to evaluate response to vascular targeting agents. In addition, AML cells themselves can aberrantly express an endothelial-like phenotype, and therefore may be direct targets of vascular targeting agents.Finally, defining how leukemia exploits the bone marrow vascular niche may lead to promising new therapeutics. We have already translated the vascular disrupting agent, CA1P (OXi4503), into the clinic, and there are many more on the way. As with many antiangiogenic and antivascular agents, cytopenias have been observed in early clinical studies. This is to be expected considering the crosstalk between angiogenesis and hematopoiesis. Therefore, the application of these novel agents into the leukemia unit and clinic will require careful administration and an “induction” mindset. Blood product transfusions and prophylactic antibiotics will likely be required in these AML patients. However, with adequate support, these agents may show promising results over time.