Using molecular dynamics simulations, we show that the G3.5 PAMAM dendrimers are able to chelate Ca2+ at similar proportions to EDTA, providing support to the hypothesis that oral formulations of PAMAM dendrimers could use this high chelating efficiency as a potential mechanism for permeating the tight junctions of the intestines if other formulation barriers could be overcome.Poly(amido amine) (PAMAM) dendrimers have been considered as possible delivery systems for anticancer drugs. One potential advantage of these carriers would be their use in oral formulations, which will require absorption in the intestinal lumen. This may require the opening of tight junctions which may be enabled by reducing the Ca A particular mechanism of interest is the pathway by which orally taken PAMAM dendrimers could reach their target location when used as nanocarriers of anticancer drugs. Specifically, there is interest in understanding how these particles would be able to permeate the tight junctions of the intestinal lumen (Poly(amido amine) (PAMAM) dendrimers are complex molecules for which their biochemical activity al lumen .2+) and magnesium for their integrity and function  simulations are routinely used to provide understanding and testing of novel hypotheses at the molecular scale for small molecule drug delivery research . With reEDTA and G3.5 PAMAM dendrimer structures were manually constructed using MarvinSketch by 2+ in water, G3.5 PAMAM dendrimer with Ca2+ in water, EDTA with Ca2+ in a buffer solution, and G3.5 PAMAM with Ca2+ in the same buffer solution. The water model used in all the simulations was the TIP3P  at a concentration of approximately 0.115 M. The concentrations were calculated with respect to the volume of the simulation box and those used in the buffer simulations are representative of the experimental solutions used to examine the ability of EDTA to chelate Ca2+ and increase epithelial absorption by 2+ chelation by EDTA and G3.5 PAMAM dendrimers, when a competing ion (Na+) is present in the solution. For the EDTA simulations the volume of the water box was approximately 75,000 Å3, while for the simulations with G3.5 PAMAM dendrimer the volume of the water box was approximately 460,000 Å3. All the simulations were performed using periodic boundary conditions and a non-bonded cutoff of 9.0 Å in the NPT ensemble. Specifically, the following number of molecules were used in the simulations: the EDTA and Ca2+ in water MD simulation has 1 EDTA molecule, 6 Cl− ions, 5 Ca2+ ions, and 2,141 water molecules; the EDTA and Ca2+ in a buffer MD simulation has 1 EDTA molecule, 6 Na+ ions, 6 Cl− ions, 2 Ca2+ ions, and 2,220 water molecules; the G3.5 PAMAM dendrimer and Ca2+ in water MD simulation has 1 G3.5 PAMAM dendrimer molecule, 32 Ca2+ ions, and 13,297 water molecules; the G3.5 PAMAM dendrimer and Ca2+ in a buffer MD simulation has 1 G3.5 PAMAM dendrimer molecule, 32 Na+ ions, 16 Ca2+ ions, and 13,035 water molecules.For all simulations, the ff12sb force fields were used along with the general AMBER force field (GAFF) to represent the EDTA and G3.5 PAMAM dendrimer . Four dihe TIP3P . For botThree independent MD simulations were performed for each system to obtain ensemble averages of multiple MD runs, which are those presented in the paper. The corresponding results for each of the simulations are provided in the −1. Following the equilibration, we performed a 30-ns MD simulation consisting of 1.5 × 107 steps with a time step of 2 fs. During the MD simulation the temperature and pressure were held constant at 300.0 K and 1.0 bar, respectively, and Langevin dynamics was used with a collision frequency of 1.0 ps−1. The 30-ns MD simulation time was selected according to literature precedence  within each run of the MD simulations. The results reported here correspond to the average radial distributions over the three simulation runs performed for each system considered here .The radial distribution functions were calculated within cpptraj for each set of counter ions (Cahttps://www.flux.utah.edu/project/apt). We used our research project as a demonstration on how to use a computationally intensive application in a cloud computing environment. This study shows that it is possible to perform computationally significant drug delivery research in such an environment, which may provide researchers an appealing and more cost-effective alternative to traditional, dedicated high-performance computing (HPC) environments.All calculations were performed on the Tangent cluster at the Center for High Performance Computing. Tangent is a part of the Adaptable Profile-Driven Testbed (Apt), an initiative by the Flux Research Group to provide a flexible, on-demand cloud computing environment targeted at researchers and scientists  included in the simulations. The average values and their standard deviations are the average over all the ions of the same type over all the trajectories from the three MD simulations performed here for this system.Results from the EDTA and Ca2+ in water. As it can be seen in 2+ ions shows that in all the simulations the Ca2+ ions have migrated, at least temporarily, from a distance of 8.00 Å to a distance within 3.00 Å of the surface of the EDTA molecule. The average distance of the Ca2+ ions to the surface of the EDTA molecule is comparably large, but this is largely due to the fact that EDTA has only four terminal COOH groups. This allows for only two Ca2+ ions to bind to EDTA. To ensure that the same concentration of Ca2+ ions was used for all the MD simulation studies in water, five Ca2+ ions were used in the EDTA and Ca2+ in water MD simulations, therefore not all the Ca2+ ions can be in close proximity to the EDTA molecule, resulting in a larger average distance and standard deviation. This is also apparent from 2+ ions show secondary peaks corresponding to second and third absorption layers. The other two Ca2+ ions are at an average distance of ~2.5 Å, which corresponds to a situation in which the ions are bound to EDTA. This is confirmed by the average percentage dwell time for Ca2+ ions, which shows that for most of the simulation, 80% of the time, at least two of the Ca2+ ions are interacting with the surface of EDTA. These simulations’ results agree with the experimental results showing the large affinity of EDTA to chelate Ca2+  in the buffer solution, as well as their standard deviations of these values when averaged over all the trajectories in the three simulations performed here for this system.Results from the MD simulation study of EDTA and Ca2+ ions to the surface of the EDTA molecule is 2.54 Å, slightly smaller than the one observed in the water simulation and within 3.00 Å of the surface of the EDTA molecule. The average distance of the Ca2+ ions is 9.05 Å, also slightly smaller than in the water simulations. Its relatively large value can be explained by the same argument used for the simulations in water. The average dwell time for the Ca2+ ions also indicates that on average two Ca2+ ions are interacting with the surface of EDTA for a majority of the MD simulations. The results in the table show that the Na+ ions are much less likely to be close to the EDTA surface. It is not surprising that the minimum distance of Na+ (2.02 Å) is closer than that observed in the Ca2+, the Na+ ions are smaller and they only interact with one surface group on the EDTA molecule, whereas the Ca2+ ions commonly interact with two surface groups on the EDTA molecule. The average distance and the average percentage dwell time are considerably larger and smaller, respectively. These results show that while the Na+ is able to get in proximity of the EDTA, its interaction is less favorable than for Ca2+ as demonstrated by the larger average distance and smaller percentage dwell time. The corresponding radial distribution from this simulation (not shown) is similar to the one for the EDTA Ca2+ in water confirming the results reported in The average minimum distance of Ca2+ in water. While it can be observed in the simulations that there is some competition for binding sites on EDTA between the six Na+ ions and two Ca2+ ions in the simulations, when comparing the results in 2+. This indicates that the ability of EDTA to chelate Ca2+ is not affected by the presence of a competing ion (Na+) in the simulation environment. The results of the MD simulations are consistent with experimental results discussed above . Also, the average distance of the Ca2+ ions to the surface of the G3.5 PAMAM dendrimer molecule, 4.13 Å, is much smaller than that observed with EDTA (10.34 Å) in water consistent with the fact that in this system all Ca2+ ions can be bound to the G3.5 PAMAM at the same time. The average percentage dwell time of 0.86 shows that the G3.5 PAMAM dendrimer is nearly always, almost 90% of the time, binding Ca2+ ions. Note that the decrease in the average distance and dwell time is also associated with the fact that the calcium ions in this simulation are in the correct stoichiometric ratio, such that all the ions can be binding to the G3.5 PAMAM dendrimer at the same time.Both the figure and table present results that clearly show the ability of G3.5 PAMAM dendrimer to chelate Ca2+ in a buffer are presented in + and Ca2+) present in the buffer solution, as well as their standard deviations calculated over all trajectories from the three independent MD simulations performed in this system.Results from the MD simulation study of the G3.5 PAMAM dendrimer and Ca2+ system in water and the G3.5 PAMAM dendrimer in a buffer solution. It is apparent from the figure that (i) all the Ca2+ ions are clearly attached to the G3.5 PAMAM dendrimer and (ii) that the introduction of potentially competitive Na+ ions does not change the chelation ability of Ca2+ ions by the G3.5 PAMAM dendrimers. The ability of Na+ to bind to the G3.5 PAMAM was independently verified by simulations without Ca2+ ions  is shorter than that observed in the Ca2+, in agreement with the results discussed above for the simulation of EDTA in buffer. Results of the average percentage dwell time of the Na+ and Ca2+ ions show a relatively large difference, 0.60 for Na+ and 0.91 for Ca2+ ions, which is similar to that observed in the simulation in water. This indicates that the Ca2+ ions appear to be more frequently bound to the G3.5 PAMAM dendrimer than the Na+ ions. Results show that Ca2+ is an effective chelation agent for G3.5 PAMAM dendrimer, indicating that under the simulation conditions the G3.5 dendrimer exhibits high chelating efficiency of Ca2+ in both water and in a buffer solution.The average minimum distance of NaThe use of AMBER in the Apt HPC environment has been extremely successful. During the course of these studies we were able to secure all the needed resources for the project, and because Apt is able to mirror a standard HPC environment, the migration to this cloud-based computing environment has been straightforward. Moreover, the overhead for using the Apt environment has been minimal. The only overhead incurred when using Apt is the instantiation and de-provisioning of the infrastructure, which is typically less than 15 min, a small fraction of a typical AMBER simulation.2+ comparable to the EDTA efficiency and therefore they may be capable of using this mechanism to open the tight junctions in the intestines. We have demonstrated that computationally intensive applications of interest in drug delivery research can effectively use cloud computing environments like Apt.Using MD simulations, validated by the agreement with existing experimental results in EDTA, we have shown that in MD simulations G3.5 PAMAM dendrimers exhibit a high chelating efficiency of CaSupplemental Materials


FLT3-ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates FLT3-ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show FLT3-ITD occurs within the most primitive Lin-/CD33(+)/CD45dim/CD34+CD38- LIC compartment. Crenolanib alone could not target FLT3-ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC in vitro and severely impaired engraftment in NSG mice. Strikingly, FLT3-mutated samples harboring TET2 mutations were completely resistant to crenolanib whereas neither NPM1 nor DNMT3A mutations influenced response. Conversely, primary AML LIC harboring either TET2, DNMT3A or NPM1 mutations did not show increased sensitivity to AZA.Effectively targeting leukemia-initiating cells (LIC) in FLT3-ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of FLT3-ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.In summary, resistance of  FLT3-ITD) remains a disease with dismal prognosis. Despite intensive chemotherapy and allogeneic stem cell transplantation as post-remission therapy, relapses are frequent. First, second and third generation tyrosine kinase inhibitors (TKI) have been evaluated for FLT3-mutated AML with varying degrees of success and only transient responses as single agents [Acute myeloid leukemia (AML) with internal tandem duplications in the FLT3 tyrosine kinase (e agents –4.FLT3-mutated AML are diverse. Prolonged treatment with TKI leads to emergence of secondary kinase resistance mutations [+CD38- bone marrow (BM) compartment and responsible for disease propagation [+ LIC in FLT3-ITD AML persist and expand during treatment with first- and second generation TKI owing to their selective protection by stromal cells [Reasons for the limited success of TKI in utations , 6. Autoutations . Importapagation , 8. LIC al cells .FLT3-ITD AML [Developing more effective TKI and combining TKI with other novel agents such as epigenetic drugs are strategies currently being pursued in -ITD AML –12. SeveIn vitro studies have found creno to have superior cytotoxic effects compared to sorafenib (sora) or quizartinib and the compound is currently being evaluated in phase II trials in FLT3-ITD AML [FLT3-mutated LIC to TKI may be mediated by co-mutations, the most frequent being either NPM1 or epigenetic mutations  [As a more potent FLT3 inhibitor, we chose crenolanib (creno), a promising new third-generation type I FLT3 inhibitor with high selectivity towards FLT3 , 14. In -ITD AML . Azaciti-ITD AML , 17. Pre-ITD AML . In cont-ITD AML , 19. Fur IDH1/2) , 21, whi IDH1/2) –25. Last IDH1/2) –29.in vitro as well as in an in vivo xenograft model, we investigated whether creno in combination with AZA can eliminate LIC protected by stromal contact in FLT3-mutated AML and determined whether treatment response is dependent on co-existing genetic mutations in DNMT3A, NPM1 and TET2.Here, using primary AML samples DNMT3A, TET2 and IDH1/2 occur at a very early pre-leukemic stage within the primitive hematopoietic stem/progenitor cell compartment. In contrast, FLT3-ITD is considered a late event in leukemogenesis, but may often be the crucial initiator for leukemic expansion [FLT3-ITD in LIC and sophisticated sorting was not performed in these studies, nor were results correlated to epigenetic mutations (unknown at the time) [FLT3-ITD in distinct phenotypically defined leukemic stem/progenitor subpopulations in relation to pre-leukemic and early leukemic hits, we analyzed ten FLT3-ITD AML samples  data that mutations in epigenetic regulators such as xpansion . Only lihe time) , 32. ThuNPM1 mutations were found in 7/10 patients. In 7/10 patients, epigenetic lesions in either TET2, IDH1, IDH2 or DNMT3A were found. IDH1/2 mutations appeared exclusive, whereas two patients (#4 and #9) with DNMT3A mutations also carried a TET2 mutation. Only one patient (#5) had FLT3-ITD as the sole mutation, but showed an aberrant t karyotype /CD45dim/CD34+CD38-) in all CD34+ AML samples  was dominated by multilymphoid progenitors , whereas in CD34- AML, we also found multipotent progenitors  and hematopoietic stem cells , consistent with published reports  hierarchically increased from there /CD45dim/CD34+CD38- LIC compartments. We can therefore ascertain that FLT3-ITD is indeed an appropriate TKI target for LIC elimination in AML. However, co-occurrence of NPM1, TET2 and DNMT3A mutations in this same compartment may influence response of FLT3-mutated LIC to TKI.In summary, we demonstrate that FLT3-ITD LIC, we confirmed the FLT3-ITD-selective effects of creno in leukemia cell lines. Creno was cytotoxic to murine Ba/F3 cells transduced with human FLT3-ITD (Ba/F3_FLT3-ITD) as well as FLT3-ITD human leukemia cell lines MV4-11 and MOLM-13 in a dose-dependent manner whereas no effect was observed on Ba/F3 cells transduced with FLT3 wild-type receptor (Ba/F3_FLT3-WT) or the leukemia cell line RS4;11  to determine short- as well as long-term proliferative potential characteristic of LIC  while levels of LIC-derived FL were unchanged  AML mouse model. PDX models faithfully mimic patient characteristics and thus are a powerful tool to define drug efficacy and resistance in leukemia [FLT3-ITD AML samples were serially transplanted into NSG mice with reliable and reproducible engraftment for several passages, proving presence of LIC. Targeted resequencing of PDX cells demonstrated that driver mutations present in the founding clone of the original AML were preserved during passaging [in vivo engraftment capacity of two separate FLT3-ITD-mutated PDX samples .To recapitulate our leukemia , 47. Priassaging . We testes Table  after inh Figure . Of noteFLT3-ITD/NPM1mut/DNMT3Amut), engraftment was monitored by serial PB sampling. At 7 weeks post-transplant, PDX cells (mCD45-/hCD45+/hCD33+) were detected in PB of all mice in the DMSO cohort , at which point all mice were sacrificed. PDX cells in PB were also detected in the AZA , creno  and AZA + creno  groups. Strikingly, PDX infiltration of BM as assessed by FACS and IHC was significantly reduced in mice transplanted with AZA + creno-treated FLT3-ITD cells compared to DMSO control or AZA alone  stable expression of luciferase enabled monitoring of engraftment by luciferin-induced bioluminescence. BLI signals were first detected in the BM of extremities and sternum 4 weeks post-transplantation. Notably, during the first 35 days, leukemic expansion of FLT3-ITD/NPM1mut/TET2mut cells was significantly accelerated in the creno treatment group compared to DMSO controls /CD45dim/CD34+CD38- LIC compartment beginning at the level of HSC or MPP, i.e. in the same early compartment as mutations in NPM1, DNMT3A or TET2 inferred by NGS data. This is in line with recent data demonstrating that either DNMT3A or TET2 mutations together with FLT3-ITD alone are sufficient to transform murine HSC and induce AML in mouse models [FLT3-ITD may be subclonal in some leukemias and is sometimes lost at relapse, our data ascertain the validity of FLT3-ITD as a TKI target to eliminate LIC.In order to cure AML, any effective therapy must eliminate the chemo-resistant, quiescent and self-renewing LIC enriched within the CD34e models , 50. ThuFLT3-ITD LIC in our in vitro BM model. Although LIC with higher FLT3-ITD/WT ratios were more sensitive to creno, this only held true for samples without TET2 mutations. FLT3-ITD/TET2mut LIC were completely resistant to creno as a single agent in the presence of stroma and expanded upon TKI treatment with CFC results corroborated by our PDX transplant data. In support of our primary human data, TKI insensitivity has been observed in FLT3-ITD/TET2-/- murine progenitors as well [FLT3-ITD AML.Nevertheless, the highly FLT3-selective TKI crenolanib alone could not effectively target stroma-protected  as well . This obIDH mutations inhibit TET2 function and induce a similar leukemic signature as TET2 mutations [IDH mutations may also confer resistance to TKI in FLT3-ITD AML. Unfortunately, we were unable to investigate this due to the limited number of IDH-mutated AML samples in this study.Since utations , it is tFLT3-ITD LIC in vitro, resulting in significantly reduced expansion in CFC assays despite concurrent NPM1, DNMT3A or TET2 mutations. In addition, engraftment ability of in vitro-treated FLT3-ITD PDX cells was severely impaired, providing further evidence we are targeting FLT3-ITD LIC with long-term proliferative potential. Translated to the clinical setting, this would suggest that the combination of AZA + creno could be employed as maintenance therapy to suppress residual LIC.Strikingly, addition of AZA to creno effectively overcame niche protection of DNMT3A or TET2 mutations in retrospective analyses, but data from prospective trials have not been reported thus far [FLT3-ITD LIC, the presence of NPM1, DNMT3A or TET2 mutations did not confer a better response to AZA alone, arguing against a direct link between AZA efficacy and epigenetic mutations. Furthermore, despite many efforts, a clear correlation between DNA demethylation and response to AZA has not been able to be demonstrated. The fact that AZA is incorporated to a large part into RNA (up to 90%) and only to a smaller percentage into DNA [FLT3-ITD LIC may also be mediated via the RNA level and not only by DNA demethylation.Conflicting data has been reported on the correlation between clinical response to AZA and the presence of thus far , 52, 53.into DNA , 54, sugin vitro co-culture assays. However, at least in our experimental setup, TKI resistance was not mediated by reactive FL secretion as has been described previously [FLT3-ITD LIC in their niche. It therefore seems unlikely that targeting a singular pathway involved in stromal protection may recapitulate this effect.Indeed, our RNA-seq data suggest that several pathways involved in the complex interaction between LIC and their niche were actively disrupted by AZA that warrant further investigation. Crosstalk between LIC and the niche is not dependent on one single mechanism but is a dynamic process mediated by diverse mechanisms including cellular and soluble mediators. Surely, we were not able to depict the entire complexity of stromal TKI resistance of LIC in our eviously  or nicheFLT3-ITD AML. We demonstrate that efficacy of TKI therapy in FLT3-mutated AML is highly dependent on the mutational landscape within each leukemia and provide a valuable framework for designing rational combination therapy in FLT3-ITD AML. The recently reported encouraging results of the RATIFY trial show that combining the unselective TKI midostaurin with intensive chemotherapy results in a survival benefit for FLT3-ITD AML patients, but it is uncertain how much of this effect is due to inhibition of FLT3 as opposed to off-target effects of midostaurin [FLT3-ITD AML for TET2 mutations should be considered before initiation of more selective FLT3-TKI therapy and that the combination of AZA + TKI could be employed as a means to suppress residual LIC.To our knowledge, this is the first work studying the effect of TKI and AZA on different mutational genotypes in primary human ostaurin . Our datFLT3-WT and Ba/F3_FLT3-ITD cells were generated as described previously [MV4-11, MOLM-13 and RS4;11 cell lines were obtained from and propagated as suggested by the German Collection of Microorganisms and Cell Cultures (DMSZ), were authenticated by DMSZ by DNA typing and PCR analysis as well as cytogenetic testing, and passaged for <6 months after receipt. EL08-1D2 stromal cells were used up to passage 12 and cultured as described . Ba/F3_Feviously . All cytFLT3-ITD, DNMT3A, NPM1, TET2 and IDH1/2 mutations as described [BM samples were obtained from newly diagnosed AML patients recruited to the German AMLSG trials or from the Munich Leukemia Laboratory (MLL). Written informed consent in accordance with the Declaration of Helsinki was obtained from all patients according to protocols approved by the Ethics Committee of the TUM. Samples were screened for presence of escribed . MononucFLT3-ITD AML BM samples were stained for lineage markers using biotinylated antibodies: CD4 , CD8a , CD19 , CD41 , CD235alpha , CD56 . Cells were then stained with the following fluorochrome-conjugated antibodies: CD34-FITC , CD90-PE , CD33-PC5.5 , CD45RA-APC Cy7 , Streptavidin-eFluor 450 (eBioscience), CD38-APC , CD45-APC-Cy7 . PI was added as live/dead marker. Cell sorting was performed on a FACS Aria II . Sorting purity of >98% was routinely obtained.MNC from 5 cells and the QIAamp DNA Micro Kit  for >2 x 105 cells according to the manufacturers’ instructions.gDNA was isolated from sorted AML subpopulations using the ZR Viral DNA Kit™  for <2 x 10BCOR, BCORL1, CDKN2A, CEBPA, CUX1, DNMT3A, ETV6, EZH2, IKZF1, KDM6A, PHF6, RAD21, RUNX1, STAG2 and ZRSR2 and oncogenic hotspots of ABL1, ASXL1, ATRX, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CSF3R, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK2, JAK3, KIT, KRAS, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PTEN, PTPN11, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, TET2, TP53, U2AF1 and WT1. For preparation of target enrichment libraries, 50 ng of genomic DNA were used and prepared as recommended in the manufacturers’ protocol . Samples were paired-end sequenced (2x225 bp) on a MiSeq sequencer using MiSeq Reagent Kits V3 (Illumina). Sequence data alignment of demultiplexed FastQ files, variant calling and filtering was done using the Sequence Pilot software package  with default settings and a 5% variant allele frequency (VAF) mutation calling cut-off. Detection of large insertions and deletions was performed using PINDEL algorithm following BWA-MEM mapping with default settings [Molecular alterations in the gDNA from AML bulk samples (AML samples #1-10) were evaluated by targeted sequencing using the TruSight Myeloid assay  which covers the following 54 genes or gene hotspots related to myeloid neoplasms: settings , 58. HumAnalysis in gDNA extracted from sorted AML subpopulations  was done using targeted resequencing based on individual amplicons generated specifically for the affected regions. Deep sequencing was performed on MiSeq (Illumina) or IonTorrent PGM  platforms with a minimum coverage of 10,000 reads.2, and APC-Annexin V (BD Biosciences) and PI was added. Flow cytometric analyses were performed on a CyAn ADP Lx P8 (Beckman Coulter). Data were processed with FlowJo software .Dose-dependent cytotoxicity was determined by colorimetric quantification of dimethyl-thiazole diphenyl tetrazolium bromide  according to the manufacturer’s instructions. To determine cell death, cells were washed and stained in Annexin/PI buffer containing 1 M Hepes, 2.5 M NaCl, 1.62 mM CaClSDS-PAGE and immunoblotting were performed as previously described . Cells wFLT3-ITD BM samples were enriched for CD34+ cells by magnetic bead separation . CD34+ cells were cultured on EL08-1D2 in serum-free medium (SFM) with 5 growth factors (5 GF): kit ligand (KL), Flt3-ligand (FL), thrombopoietin (TPO), interleukin-6 (IL-6) and interleukin-3 (IL-3) as described [+ cells were harvested and assayed for short-term colony-forming units (CFU) and long-term culture-derived colony-forming cells (LTC) as described [MNC from escribed . Cells wescribed .g-Prkdcscid IL2rgtm1Wjl/Sz mice  were maintained at the animal facility of the TUM.Animal studies were performed in agreement with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health , in compliance with German law on the protection of animals, and with approval of the responsible regional authorities. NOD.CFLT3-ITD AML patient derived xenograft (PDX) cells and transgenic PDX cells which express firefly luciferase were generated by serial passaging in NSG mice as described [in vitro. Cells were harvested and viable cells were enumerated by trypan blue exclusion. A total of 2 x 105 viable cells (per mouse) in 200 μl PBS were injected into the tail vein of non-irradiated 6 to 16-week-old NSG mice. To monitor engraftment, peripheral blood (PB) samples were collected every other week beginning week 6 after PDX injection from the facial vein of NSG mice and analyzed by flow cytometry.escribed . PDX celin vivo imaging (BLI). Mice were narcotized and injected with D-luciferin  intraperitoneally to activate recombinant luciferase. Images were taken using a charge-coupled device camera, equipped with an image intensifier for 10-120 seconds, bin size 2, gain 900  as previously described [Transgenic PDX engraftment in NSG mice was repetitively monitored by bioluminescence escribed . The SimMice were sacrificed when PB sampling or BLI indicated systemic AML in the DMSO-treated mice. PDX cells were re-isolated from PB, femurs, tibiae and spines. After erythrocyte lysis, cells were stained with the following antibodies for flow cytometry: hCD45 APC-Cy7 , hCD34-FITC , hCD33-PC5.5 , mCD45-APC , Cell Trace Violet (Life Technologies). Flow cytometric analyses were performed on a CyAn ADP Lx P8 . Data were processed with FlowJo software .Tissue sections were deparaffinized. Antigen retrieval was carried out by pressure cooking in citrate buffer (pH 6) for 7 minutes. Anti-human CD45 was detected after 1-hour incubation at room temperature by the Mouse on Mouse kit (Abcam) according to the manufacturer's protocol. Slides were evaluated using an Olympus BX53 microscope  and images taken with an Olympus DP26 camera (Olympus).Total RNA was isolated from murine EL08-1D2 and human PDX cells using RNeasy Mini Kit (Qiagen). RNA sequencing (RNA-seq) library preparation and data processing was performed according to the Unique Molecular Identifiers (UMI) sequencing protocol, a modified bulk single-cell RNA barcoding and sequencing (SCRB)-seq protocol .Briefly, cDNA libraries were prepared from 5 ng RNA per sample. Unique molecular identifiers (UMIs) and sample barcodes were introduced during reverse transcription. Next, cDNA of all samples were pooled, unincorporated primers were digested with Exonuclease I and pre-amplified by single-primer PCR (12 cycles). After quality control of amplified cDNA, the sequencing library was constructed from 1 ng full-length cDNA using the Nextera XT Kit (Illumina). To enrich for barcoded 3’ ends in the Nextera XT library, a custom i5 primer was used. Library pools were sequenced on an Illumina HiSeq1500 on a rapid flow-cell with paired-end layout. The first read contained the sample barcode and the UMI sequences (16 cycles), while the second read contained the cDNA fragment (50 cycles). Note that UMI sequencing is a 3' method to count the number of unique transcripts. This procedure has the advantage that not the whole gene length has to be covered and a smaller number of reads compared to full-length RNA sequencing methods is sufficient , 61.All samples were mapped against concatenated human (hg19) and mouse (mm10) genomes and gene features were quantified using Ensembl gene models (v75). Gene expression of PDX samples (reads mapped to human) and EL08 samples (reads mapped to mouse) was analyzed separately.The total number of mapped reads and the number of reads per species (human or mouse) were calculated for each sample. In addition, the percentage of reads per species (human or mouse) was calculated. To calculate the number of expressed genes per sample, UMI counts were normalized such that each sample had a total number of 1 million counts. The comparability of UMIs across experiments was checked with a box plot of log-transformed UMI values. The minimum UMI greater than 0 from each experiment was added to each value to avoid log(0). A gene was defined as expressed if the UMI count/million was greater or equal than 1. For PDX samples, only reads mapped to human, for EL08 samples, only reads mapped to mouse were considered.Gene expression analysis and visualization was done with R version 3.2.0 on a x86_64-pc-linux-gnu (64-bit) platform under Ubuntu 14.04.2 LTS. Differential gene expression analysis of PDX and EL08-1D2 in co-culture (DMSO) compared to PDX and EL08-1D2 alone (monoculture controls) was performed using DESeq2 . DESeq2 P values are presented in the figures where a statistically significant difference was found: *, P <.05; **, P <.01; ***, P <.001.Statistical analyses were performed by Student’s t-test or repeated measures ANOVA using GraphPad Prism software . Linear regression analyses using Greenhouse-Geisser correction was performed in SPSS 24 software (IBM).


The human islet amyloid polypeptide (hIAPP) is an intrinsically disordered protein that can self-assemble into fibrillar aggregates that play a key role in the pathogenesis of the type II diabetes mellitus. hIAPP can transiently adopt  Cosecreted with the insulin by the islet l muscle ,2. Like l muscle ,4, hIAPPl muscle ,6,7. Alt[β-cells ,9,10. Ho[β-cells . In part[β-cells , includi[β-cells ,14,15 anciations ,17,18.The 37 amino acid sequence of hIAPP is KCNTATCATQactivity , but itsactivity ,22,23,24In the presence of sodium dodecyl sulfate (SDS) micelles, the three-dimensional structure of the non-amidated and amidated hIAPP monomers are quite comparable. Indeed, the NMR structure of the micelle-bound non-amidated hIAPP, solved at pH 4.6 by Patil et al. (PDB ID: 2KB8), exhibits a very stable es 22–28 . Similarnd 21–28 . It coulnd 21–28 , whereasnd 21–28 .In contrast, the monomeric hIAPP three-dimensional structure is less defined in the absence of lipid-like molecules. It is widely considered as a natively unstructured peptide which can transiently adopt ion 5–20 ,28,29,30eriments . HoweverAlternatively, the structural dynamics of hIAPP monomer can be studied using computational approaches. Among them, molecular dynamics (MD) and replica exchange molecular dynamics (REMD) simulations are often used for their ability to explore the biomolecule conformational ensembles. We recently reported a short review of computational studies on full-length hIAPP to reveal the most converging conformational states of its monomeric and oligomeric forms in solution and in interaction with lipid membrane . It was ablished . In ordeThe amidated and non-amidated hIAPP monomer structures were resolved by NMR experiments in the presence of SDS micelles, and, overall, both peptides have helical conformations ,26. In tgyration  indicateFrom the time evolution of the hIAPP secondary structures , it can ted form . Using a peptide , pointinIn order to explain the difference between the conformational ensembles of the amidated and non-amidated hIAPP, the average distances between the C-terminal Tyr37 and all other residues were calculated over the two MD trajectories . In the Despite an aggregation kinetics different from that of the amidated hIAPP, the non-amidated peptide can still form fibrils. Thus, it may have a significant propensity to undergo conformational transitions towards To gain insight into these metastable states, the REMD sampled conformations were clustered based on their structural similarity with an RMSD threshold of 0.2 nm. Over the 560 generated clusters, the 10 most populated ones were characterized by their RMSD with respect to the initial random coil, radius of gyration, contents . A reprecontents . We obsecontents , most ofThe propensity of the non-amidated hIAPP residues to form helical, extended and random coil conformations is detailed in rat IAPP . In addiTo better understand the relationship between the conformations of hIAPP monomer and its aggregation into fibrils, several theoretical studies examined the influence of mutations on the peptide structures and their fibrillization propensity. Thus, the conformational ensemble of the human IAPP monomer was frequently compared to that one of the rat IAPP which is non-amyloidogenic ,35,36,37 fibrils . In contIn the same manner, the present work characterized the conformational ensemble of the non-amidated hIAPP to account for its slower rate of fibrillization than the amidated one ,23,24. AIn both amidated and non-amidated hIAPP, the helical conformations mainly occur at the N-terminal segments 5–20, whereas the  peptide  [31,35,3r et al. . The extand 75%) . OverallThe conformational ensembles of full-length hIAPP monomers were explored using all-atom MD and REMD techniques implemented in the GROMACS-4.5.5 molecular modeling package ,42. Simuormation . The nonormation  for the ormation  and SETTormation  procedurThe MD initial conformation of the amidated hIAPP was taken from the SDS micelle-bound structure (PDB ID: 2L86) resolved by NMR experiments . The iniP=2.5 ps ,50,51.The conformational ensemble of the non-amidated hIAPP monomer in solution was more exhaustively explored using REMD simulations . For thiThe whole trajectories of the classical MD simulations were used for structural analyses. However, regarding the REMD study, only the last 150 ns of the first ten trajectories generated at the lowest temperatures ranging from 290 to 313 K were analyzed (1.5  do_dssp . The “gr do_dssp  implemen do_dssp ,57. ThesIn this study, we investigated the differences between the amidated and non-amidated hIAPP conformational ensembles, by performing extensive all-atom MD and REMD simulations. The secondary structure analyses clearly showed that the non-amidation of hIAPP induces a significantly lower propensity to form  peptide ,23,24, o


Noise is pervasive in cellular biology and inevitably affects the dynamics of cellular processes. Biological systems have developed regulatory mechanisms to ensure robustness with respect to noise or to take advantage of stochasticity. We review here, through a couple of selected examples, some insights on possible robustness factors and constructive roles of noise provided by computational modeling. In particular, we focus on (1) factors that likely contribute to the robustness of oscillatory processes such as the circadian clocks and the cell cycle, (2) how reliable coding/decoding of calcium-mediated signaling could be achieved in presence of noise and, in some cases, enhanced through stochastic resonance, and (3) how embryonic cell differentiation processes can exploit stochasticity to create heterogeneity in a population of identical cells. Noise arises at all levels of biological organization and inevitably affects the dynamics of biological systems. Cellular processes, ranging from gene expression to developmental pathways, signaling, cell division, and circadian rhythms are all exposed to noise, but the way they cope with it varies from one case to another, and it is therefore not straightforward to predict the effect of noise. Noise tends to compromise the precision and the timing of molecular events, and is thus often seen as a nuisance  of the hypothalamus. The SCN receives light information from the retina and generates a robust, entrainable rhythm, transmitted to peripheral organs. Oscillations in the SCN occur at the level of single neurons while inter-cellular coupling, relying on the periodic release of neurotransmitters, ensures the synchronization between individual cells  oscillations. Similar results are obtained when individual oscillators are parameterized to yield damped oscillations (not shown). Thus, intercellular coupling can induce robust, self-sustained oscillations. Whether the noise alone is able to convert damped, sloppy circadian oscillators into self-sustained oscillations was addressed by Westermark et al. . These aBmal1 mutant in the dynamics of the SCN. Bmal1 is a core clock gene and its knock-out results in a loss of circadian rhythmicity in individual cells. Remarkably, a clear rhythm—although noisy—emerged from the SCN network even in presence of this mutation. These observations were also reproduced by a mathematical model. They suggest that the clock network can exploit noise to compensate for the loss-of-function mutation.In a combined experimental-modeling study, Ko et al.  analyzedThe cell division process plays a major role in unicellular and multicellular organisms. In the latter, it drives the development from fertilized eggs into mature organisms. In the mature organism, cell division allows the replacement of cells that die due to natural causes or external damage. Thus, the cell cycle plays a crucial role in the development of living organisms both in normal and disease conditions. The cell cycle is composed of four different phases: G1, S (DNA replication), G2, and M (mitosis) and is controlled by a network of cyclin-dependent kinases (CDK) whose activities drive the progression along the successive phases of the cell cycle Morgan, .Computational models were initially proposed for the dynamics of the cell cycle in frog embryos , a messenger that is synthesized in response to an external hormonal stimulation. When IP3 binds to specific receptors (IP3R) located in the endoplasmic reticulum (ER) membrane, these receptors release Ca2+ from the ER into the cytosol. Cytosolic Ca2+ itself regulates the activity of IP3R: low concentrations of Ca2+ stimulate the opening of the channels, while higher concentrations tend to close them. The positive feedback of cytosolic Ca2+ on its own release through IP3R, known as “Ca2+-induced Ca2+ release,” plays a crucial role in Ca2+ dynamics. It allows the occurrence of oscillations and permits the spatial coupling between IP3R, as the Ca2+ released by one IP3R stimulates the release of Ca2+ from a neighboring IP3-bound IP3R , whose activity depends on Ca2+ and on the mitochondrial membrane potential. Ca2+ efflux occurs through Na+/Ca2+ and H+/Ca2+ exchangers. At each cytosolic Ca2+ spike, mitochondria sequester some of the Ca2+ released from the ER into the cytosol through the IP3 receptors  are more variable than in the cytosol (blue curve). Moreover, this noise strongly impacts on the regularity of cytosolic Ca2+ oscillations. Simulations indicate that eliminating the mitochondrial fluxes always decreases the coefficient of variation (CV) of the interspike interval .Mitochondria are small organelles containing a limited number of Cae Figure  that mitl Figure . This de2+ signaling and mitochondrial metabolism occurs at the expense of the regularity of the stimulation-induced cytosolic Ca2+ spikes.This computational prediction thus suggests that the coupling between CaCell specification in early mammalian blastocysts is governed by interactions between transcription factors, modulated by extracellular signaling. Although cell differentiation is highly organized, both spatially and temporally, noise is involved in this process and extensive expression heterogeneities among cells precede the emergence of lineage . Sensitivity analyses confirmed that the proposed specification mechanism is robust with respect to these factors. Indeed, the proportions of the differentiated cell types and the salt-and-pepper pattern remain unchanged in presence of a moderate amount of these extrinsic sources of noise  were identified as a potential additional source of robustness against molecular fluctuations. MicroRNAs are noncoding RNA molecules of 20–30 nucleotides. They bind to the 3′ UTR of messenger RNA which represses protein synthesis by targeting the corresponding messenger RNA for degradation and/or by inhibiting its translation Bartel, . Besides2+ oscillations is that their frequency increases with the level of external stimulation, thus leading to the transformation of an analog signal (the agonist concentration) into a digital one (the frequency of the repetitive Ca2+ spikes). It is well known that frequency-encoded signals are more resistant to noise than amplitude-coded ones  but in some cases stochasticity holds a key role in cell fate determination. Stochasticity does not mean that everything is random. A stochastic component serves as a source of heterogeneity, often required to lead cells in one or another developmental pathway. These pathways are typically associated with alternative steady states. If two or more stable steady states coexist in the same environmental conditions (bi/multi-stability), a small difference in the initial conditions of different cells can be sufficient to induce their evolution toward different steady states (fates), as exemplified in the embryonic differentiation system described above. To be reliable, the differentiation mechanism should also prevent spontaneous, noise-induced switches between cell fates and should lead to well-proportioned cells in each fate. This can be achieved by cooperative roles of multiple regulatory feedbacks (Pfeuty and Kaneko, Bacillus subtilis, a state characterized by the capability for the bacteria to uptake DNA from the environment. A model based on the gene regulatory network explains the spontaneous and transient entry into competence in term of excitability and noise-driven excursion in the phase space (Süel et al., Differentiation based on bi/multi-stable systems generates permanent, irreversible cell fates. In other cases, differentiated states are only transient. This is the case for example of the “competent” state in Finally, it should be noted that, although treated separately, the different processes discussed here are, in reality, not independent of each other. Mutual coupling between the circadian clock and the cell cycle (Feillet et al., 2+ signaling are features that would not be predicted by sheer intuition. Even less predictable is the behavior of interacting cell populations in the presence of noise. Noise-induced synchronization of circadian clocks and noise-induced cell differentiation are properties that were revealed by modeling. However many questions remain open. Various aspects such as for example, the contribution of miRNA in the robustness of oscillatory processes or the relative importance of the different sources of noise involved in cell fate determination still need to be investigated. The path, initiated by Delbrück in 1940, has beautiful days ahead.The dynamics of biological processes results from the interplay between the hard-wired architecture of regulatory circuits and the multiple sources of noise. Deterministic and stochastic modeling approaches constitute valuable tools to interpret experimental observations and to make sense of the biological complexity. The underlying bistability, dependent on positive feedback loops, and its contribution to the robustness of the cell cycle or the phenomenon of stochastic resonance revealed by models of CaDG, CG, and GD wrote the core of the paper. DG, CG, BW, AW, AT, AG, and GD contributed to ideas and revised the paper.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.


Vibrio cholerae LTTR, which regulates the expression of genes encoding cholera toxin and toxin-co-regulated pilus for inhibitor designing. Since AphB ligand is unknown, we followed a molecular fragment-based approach for ligand designing using FDA-approved drugs and subsequent screen to identify molecules that exhibited high-affinity binding to AphB ligand-binding pocket. Among the identified compounds, ribavirin, an anti-viral drug, antagonized AphB functions. Ribavirin perturbed Vibrio cholerae pathogenesis in animal models. The inhibitory effects of the drug was limited to the bacteria expressing wild type AphB, but not its constitutively active mutant (AphBN100E), which represents the ligand-bound state, suggesting that ribavirin binds to the active site of AphB to exert its inhibitory role and there exists no AphB-independent mechanism of its action. Similarly, ribavirin suppressed the functions of Salmonella Typhi LTTR Hrg, indicating its broad spectrum efficacy. Moreover, ribavirin did not affect the bacterial viability in culture. This study cites an example of drug repurposing for anti-infective therapy.Targeting bacterial virulence mechanisms without compromising bacterial growth is a promising strategy to prevent drug resistance. LysR-type transcriptional regulators (LTTRs) possess structural conservation across bacterial species and regulate virulence in numerous pathogens, making them attractive targets for antimicrobial agents. We targeted AphB, a  Antibiotic resistance of pathogenic bacteria has become a major public health threat worldwide4Anti-virulence strategies are particularly attractive, because they may be highly effective in the treatment of bacterial infections, while minimizing drug resistance. Drugs that specifically target the virulence mechanisms, such as adhesion/invasion of the host cells, biofilm formation, toxin production, virulence gene expression and secretion of virulence factors etc. will inhibit pathogenesis without compromising growth or survival of the organismsVibrio cholerae LTTR called AphB functions as a master regulator of the virulence phenotype. AphB, acting together with AphA drive transcription from the tcpPH promotertcpPH operon encodes two transmembrane regulatory proteins, TcpP and TcpH, which co-operate with ToxR and ToxS to activate the toxT gene, a direct transcriptional activator of the virulence genes, ctxAB and tcpA9. ctxAB encodes CT, an enterotoxin responsible for severe diarrhoea during cholera, while TcpA is the major sub-unit of toxin-coregulated pilus (TCP) and is essential for the attachment and colonization of the intestinal epithelium by Vibrio choleraeThe LysR family proteins are global transcriptional regulators , widely distributed in the prokaryotes. They constitute a major group of bacterial virulence determinants through the regulation of quorum sensing, motility, oxidative stress responses, toxin production, attachment, secretion etcin silico screening of a library constructed from the FDA-approved drug fragments to find molecules that bind AphB at the putative ligand/ co-inducer-binding pocket and joined them to build novel molecular scaffolds. Molecular sub-structure-based screening identified a small molecule compound, ribavirin, which is a clinically approved antiviral drug. Ribavirin interacted with AphB and inhibited its functions, leading to the suppression of CT production and abrogation of V. cholerae colonization and pathogenesis in animal models. Ribavirin also inhibited Hrg, an LTTR from Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and protected mice against systemic infections due to the organism. However, the drug molecule exerted no direct bacteriostatic or bactericidal effects. Thus, ribavirin is a novel therapeutic agent for bacterial infections that functions through substrate-competitive inhibition of LTTRs.We performed To design molecules that could compete with the putative ligand/co-inducer for binding to the AphB ligand-binding pocket , we haveTo build novel molecular scaffolds, which could accommodate the AphB ligand-binding pocket out of the above three fragments, we employed LUDI De Novo Link methodhttp://pubchem.ncbi.nlm.nih.gov/compound/ribavirin#section=Top) was the only known drug molecule, which was also mentioned as ‘bioassay active’ in the PubChem Database (AID: 651758). The other six molecules we found to be potential analogs of ribavirin. The GScore for ribavirin was −7.9 kcal/mol, and it formed H-bonds with the majority of the critical residues, such as N100, N128, V144 and R262  and constitutively active mutant (rAphBN100E) proteins. This technique identifies the orientation and the mode of binding of chemical groups or residues of ligands that are in close proximity to high molecular weight receptors, where ligands undergo exchange between free and bound states1H and 13C NMR  and 2D homo-nuclear and hetero-nuclear NMR methods  (Data not shown). WT protein in the presence ribavirin  is unable to activate gene expression; whereas the constitutively active mutant AphBibavirin , whereasn (N100E . It is nn (N100E . Since tn (N100E . The difWT) and constitutively active mutant (AphBN100E) (WT protein (D ~ 300 μM). ΔH  and ΔS  that signify the thermodynamic signature of the process suggested that the binding interaction between ribavirin and AphBWT protein was an enthalpydriven (ΔH < 0) and entropically unfavorable (ΔS < 0) process. The negative value of the Gibbs free energy of binding  confirmed the spontaneous nature of the interaction. In contrast, the interaction between the mutant protein and ribavirin showed negligible and irregular heat change in ITC thermogram, which proved no binding of the drug to the protein. Overall, the ITC experiment strongly suggests binding between AphBWT and ribavirin as an enthalpy-driven spontaneous process with micromolar binding affinity.We performed ITC to study the binding interactions of ribavirin with recombinant AphB proteins, both wild type (AphBhBN100E) . The dow protein . The corV. cholerae virulence genes, we sought to investigate if ribavirin could be a functional antagonist of AphB and inhibit bacterial virulence. To this end, we studied the expression of AphB-regulated virulence genes by log phase cultures of V. cholerae El Tor N16961 strain (N16961wt). qPCR analysis showed significant suppression of sodium bicarbonate-induced expression of AphB-regulated genes ctxA, tcpA, tcpP and toxT by ribavirin, suggesting inhibition of AphB functions (mut) expressing AphBN100E, but not expressing AphBWT   assayWT strain (108) along with ribavirin or PBS. Fluid accumulation was markedly reduced by ribavirin  . Next, tibavirin , with siibavirin . That riibavirin . TogetheV. cholerae was studied using a suckling mouse model7 cfu/mouse) into the 4–5 days old Swiss Albino mice through oral gavage, followed after two hours by PBS or ribavirin. Significant decrease in the intestinal colonization of mice by N16961wt strain, but not by N16961mut strain was observed after 18 hours  at the time of the study . Out of Salmonella LTTR, is required for protection against oxidative stress and bacterial growth and survival within macrophages. Thus, a mutant S. Typhimurium strain deleted of Hrg was found to be more susceptible to H2O2D = 235 μM)  . To detenaltered .Salmonella Typhi within macrophages were studied in THP1-derived macrophage cells infected with the bacteria. Intracellular CFU counts were significantly less in the ribavirin-treated compared to untreated cells, suggesting inhibition of intracellular survival by ribavirin , PBS or ribavirin (100 mg/kg/day) was administered orally every 12 hours for 2 days. Visceral organs (liver and spleen) collected at the end of the experiment showed significantly reduced bacterial load in the mice treated with ribavirin , which encodes an adhesin (intimin) and its receptor (Tir) and a T3SS apparatus and its effectors. LTTRs namely QseA, LeuO and LrhA regulates the LEE genes and the flagellar regulon, and is involved in the microcolony formation and adherence to the epithelial cells202122ibavirin .To investigate the role of ribavirin on epithelial adhesion of EPEC, which promotes microcolony formation, we cultured HT-29 cells infected with EPEC in the presence of ribavirin. After removal of the non-adherent organisms, the numbers of bacteria attached to the cells were quantified by plating the cell lysates. We observed significant reduction in epithelial adherence of EPEC in the presence of ribavirin .9 cfu/mouse), followed after 4 hours by oral administration of PBS or ribavirin, repeated every 12 hours. On day 3, mice were sacrificed and colonization of the caecum and colon was studied by CFU counts. Oral administration of ribavirin resulted in nearly 2-log decrease in the intestinal colonization of EPEC  is the only LTTR successfully targeted for inhibitor designing till date3233in silico designing of small-molecule inhibitors for AphB, a Vibrio cholerae LTTR and a master regulator of the virulence genes, since the ligand/co-inducer of AphB is not known and currently, there is no knowledge regarding the molecular structure that could accommodate the active site of AphB. This is an established approach for structure-guided drug designing where no information about the ligand is available and delivers molecules with high specificity and selectivity and reduces off-target binding, leading to less untoward side effects of the designed drugsBacterial pathogens often develop resistance to antibiotic drugs, which target their growth or viability. In contrast, strategies that specifically target virulence pathways which are non-essential for growth would limit selection of the resistant strains, and are candidates for the development of next-generation antimicrobial therapeutics626272829in vitro. However, the effective inhibitory concentration varies considerably, depending on the virus tested and the cell lines used. The IC50 dose of ribavirin for viral replication ranges between 20 and 200 μM in most instancesS. Typhi within cultured macrophages. We and others found no cytotoxicity at this or even much higher concentrations of ribavirin37Ribavirin, a synthetic triazole compound and a guanosine analogue, inhibits replication of a wide range of DNA and RNA viruses In vivo efficacy of ribavirin was reported against several RNA viruses, although the drug is currently recommended only against severe RSV infection414243in vivo and suppressed bacterial pathogenesis.50 oral dose of ribavirin in mice is twice (2 g/kg) as that of the intraperitoneal dose (0.9–1.3 g/kg) (https://pubchem.ncbi.nlm.nih.gov/compound/ribavirin#section=Top). Intriguingly, in silico binding affinity of ribavirin and its analog ICN17261, which demonstrated significantly reduced toxicity in ratsAs opposed to injectable ribavirin used in most animal studies4143Despite considerable sequence diversity over the C-terminal domains of LTTRs, three-dimensional structural superimposition suggests that LTTRs have a conserved ligand-binding pocket. It was earlier reported that the residues S99 and A101 of TsaR and V97 and S99 of BenM, which participate in direct interactions with their respective ligands are at analogous positions to P98 and N100 of AphB46N100E). However, NMR showed a distinct peak at 8.75 ppm corresponding to the hydrogen of the 1,2,4-triazole-3-carboxamide group, but not to the other protons attached to the −OH or −NH2 group of the drug molecule. This may be explained by the fact that the experiment was conducted in D2O only due to the buffer/water solubility of AphB. Because of the labile nature, NMR signals corresponding to the 2′-, 3′- and 5′-hydroxyl or amide protons of ribavirin were absent in proton NMR and this phenomenon may be attributed to deuterium exchange. Although in ITC experiments, the KD value of the interaction was in the higher micromolar range (~300 μM), suggesting modest affinity between the molecules, specificity of the interaction is underscored by the absence of interaction between AphBN100E and ribavirin. Much higher KD values (2480 μM to undetectable) were reported for the interactions between viral RNA-dependent RNA polymerases and ribavirin, although some of these enzymes are definitive targets of the drug49Our computational analysis predicted that ribavirin strongly binds to AphB  and forms H-bonds with N100, N128, A130, D131, V144 and R262 residues of the ligand-binding pocket. This prediction was validated by NMR studies, which suggested ribavirin-binding to AphB, but not to its constitutively active mutant  registered under ‘Committee for the Purpose of Control and Supervision of Experiments on Animals’ (CPCSEA), Government of India (Registration No. 68/1999/CPCSEA dated 11-03-1999).http://ekhidna.biocenter.helsinki.fi/dali_server) and the 3D structural alignment was performed in Discovery Studio (DS 2.5) software. AphB from Vibrio cholerae (PDB ID: 3SZP) was selected for inhibitor designing. 1491 commercial fragments extracted from the FDA approved drugs as on 04-02-2014 was downloaded from e-LEA3D: Chemoinformatic Tools and Databases (http://chemoinfo.ipmc.cnrs.fr/lea.html). Fragment-based ligand designing was done in DS 2.5 using LUDI, an empirical scoring function based methodLTTR crystal structures were downloaded from Protein Data Bank (PDB). Secondary structural alignment was performed using DALI server . S.Typhi Ty2 was gifted by J. Parkhill . Plasmid pBAD24 was gifted by Dr. A.K. Mukhopadhyay . Primers (Cell lines were procured from American Type Culture Collection (ATCC). Cell culture reagents were purchased from Invitrogen and bacterial culture media from BD Difco. Ribavirin was purchased from Sigma-Aldrich. Restriction enzymes were purchased from New England Biolabs. Bacterial strains  Primers  were cusWT, AphBN100E, Hrg and QseA were transformed into E.coli strain BL21 (DE3) (Novagen). BL21(DE3) was cultured at 37 °C till the OD600 reached 0.6. Protein expression was induced with 0.1 M IPTG at 37 °C for 4 hours and purified using Ni-NTA agarose columns (Qiagen). The recombinant proteins were dialyzed against 10 mM sodium phosphate buffer containing 50 mM NaCl (pH-7.4).ORF of AphB was PCR-amplified from genomic DNA of N16961 strain, ORF of Hrg was amplified from Ty2 strain and ORF of QseA was amplified from EPEC strain, cloned into pTZ57R/T and sub-cloned into pET28a (Novagen) and pBAD24 expression vectors. Constitutively active AphB mutant (N100E) was generated by site-directed mutagenesis (Agilent technologies) as per the manufacturer’s protocol and confirmed by sequencing. Expression plasmids of AphBTMv3.1 software suiteSTD NMR experiments were performed on a Bruker AVANCE III 500 MHz NMR spectrometer, equipped with a 5 mm SMART probe at 298 K. Data acquisition and processing were carried out using TopspinITC experiments were performed on VP-ITC micro-calorimeter  at 25 °C. Ribavirin was dissolved in the same buffer as the recombinant proteinsWT or AphBN100E cloned in pBAD24.AphB deletion mutant of N16961 strain was generated using the suicide vector pCVD442 (Addgene). Gene fragments flanking upstream and downstream of AphB were amplified, ligated into pCVD442 and transformed into SM10λ-pir. The transformed SM10λ-pir was conjugated with N16961 and the conjugates were selected in TCBS agar containing ampicillin. Gene deletion was confirmed by PCR. Deletion mutants of N16961 (N16961ΔAphB) were electroporated with AphB7 cfu of bacterial cells resuspended in 50 μl of PBS4–5 days old Swiss albino mice were inoculated orally with 108 CFU) resuspended in PBS with or without ribavirin (5 mg). Rabbits were sacrificed after 18 hours and fluid accumulation per unit length of the loops was measured.The RIL assay was performed in New Zealand white rabbits (2 kg) as previously describedHuman acute monocyte leukemia cell line THP-1 was maintained in RPMI 1640 containing 10% FBS. Cells were differentiated and infected as previously describedS. Typhi infection3+ (0.32 mg/g of mice) and Desferrioxamine (Novartis) (0.025 mg/g of mice) 4 hrs prior to the bacterial challenge. Mice were orally inoculated with S. Typhi. Four hours later, ribavirin (100 mg/kg/mouse) was administered orally every 12 hours. 48 hours after infection, spleen and liver were collected, weighed and homogenized in PBS containing 0.5% Triton-X 100 and serial dilutions were plated on Hektoen enteric agar plates.An iron-overload mouse model was used for oral HT-29 cells were maintained in DMEM supplemented with 10% FBS. Cell monolayers were grown in 24-well tissue culture plates to obtain a 60% confluence. Cells were infected with bacterial suspensions in DMEM at a MOI of 100 for 3 hours in the absence of antibiotic, in presence or absence of ribavirin (100 μg/ml). Cells were washed thrice with PBS to remove the non-adherent bacteria, lysed with 0.25% triton-X 100 for 30 minutes and the lysates were diluted and plated on MacConkey agar containing ampicillin.7–8 weeks old C57Bl/6 male mice were used for the studyVibrio cholerae N16961 was cultured in AKI media supplemented with sodium bicarbonate, while Salmonella Typhi was grown in Luria Bertani (LB) broth in presence of H2O2 and EPEC in LB alone. Cultures were grown in the presence and absence of ribavirin. Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and was treated with DNase I (NEB). DNA-free RNA was used for cDNA synthesis using SuperScript II reverse transcriptase. Quantitative PCR (qPCR) was performed in a StepOnePlus system (ABI) using SYBR green master mix. Relative quantitation was done by the comparative threshold cycle (CT) method. The levels of expression of the genes of interest were normalized against 16 S rRNA using the formula 2−ΔΔCT The amount of CT in the bacterial cell supernant and fluid accumulated in rabbit ileal loop was determined by ELISA. Briefly ELISA plates were coated with 100 ng/well of GM1 ganglioside (Sigma). 100 μl of cell supernatant or intestinal fluid was used. As primary antibody rabbit polyclonal anti-CT antibody (Sigma) was used. Anti-rabbit IgG conjugated with HRP (Pierce) was used as secondary antibody. Purified CT (Sigma) was used to plot the standard curve.10 ml of culture media supplemented with or without ribavirin was inoculated with 100 μl bacterial cultures (OD600 = 1.0) and incubated at 37 °C under shaking conditions. Bacterial growth was monitored by sampling every 2 hrs followed by CFU counts after overnight growth on LB agar containing antibiotics.How to cite this article: Mandal, R. S. et al. Ribavirin suppresses bacterial virulence by targeting LysR-type transcriptional regulators. Sci. Rep.6, 39454; doi: 10.1038/srep39454 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Neurosporacrassa response to staurosporine. We highlight the importance ofthe extracellular availability of Ca2+ for this response. Limitationof the ion in the culture medium further sensitizes cells to the drug andresults in increased accumulation of reactive oxygen species (ROS). Conversely,an approximately 30-fold excess of external Ca2+ leads to increaseddrug tolerance and lower ROS generation. In line with this, distinctstaurosporine-induced cytosolic Ca2+ signaling profiles were observedin the absence or presence of excessive external Ca2+.High-throughput RNA sequencing revealed that different concentrations ofextracellular Ca2+ define distinct transcriptional programs. Ourtranscriptional profiling also pointed to two putative novelCa2+-binding proteins, encoded by the NCU08524 and NCU06607 genes,and provides a reference dataset for future investigations on the role ofCa2+ in fungal biology.Alterations in the intracellular levels of calcium are a common response to celldeath stimuli in animals and fungi and, particularly, in the  Therefore, it is not surprising thatcells commonly respond to cell death stimuli with rises in the intracellular levelsof this divalent ion. On one hand, these transient or stable modifications in thelevels of Ca2+ can operate as survival signals, leading to the activityof anti-death proteins or stimulating transcriptional defence responses. On theother hand, the loss of Ca2+ homeostasis can behave as a pro-deathsignal, since in some systems overload or a simple perturbation of the distributionof the ion within storage organelles is sufficient to trigger cell death Saccharomycescerevisiae, Candida albicans and Cryptococcusneoformans, cytosolic Ca2+ increases are a common feature ofthe cellular response to a number of antifungal stresses, including pheromone 37N. crassa, a rise in thecytosolic levels of Ca2+ was associated with cell death induced bychitosan Penicilliumchrysogenum protein PAF Calcium Ca+ is crucN. crassa to investigate the molecular mechanismsunderlying the cell death process induced by the bacterial alkaloid staurosporine.Treatment with staurosporine leads to a complex response that includes the efflux ofreduced glutathione (GSH), with a concomitant change in the intracellular redoxstate to a more oxidative environment and the consequent accumulation of reactiveoxygen species (ROS) 132+ levels with the photoreporter aequorin revealed a three-stepsignature response to the drug that involves mobilization of the ion fromintracellular stores as well as uptake from the external medium. N.crassa is a good model for the study of Ca2+ dynamics assequencing of its genome revealed a rich and versatile assortment of moleculesinvolved in Ca2+ homeostasis, including channels, pumps and signalingtransducers 17182+ machinery may exist. The best-studied Ca2+-bindingmoiety is the classical EF-hand, in which a Ca2+-interacting loop isflanked by two helices 2+-binding domains exist2+-binding Dx[DN]xDGmotifs. The distinct evolutionary origins of the motif are evident from thevariability of structural contexts flanking the Ca2+-binding region 222223Psathyrella velutina22We have been using N.crassa involves a precise sequence of Ca2+ signaling eventswhich are completely abolished by the inhibition of extracellular Ca2+uptake N. crassa cells growing in limited or excessiveamounts of the ion. The results presented here revealed that extracellular Ca2+modulates cell death and the transcriptional alterations induced bystaurosporine, and led to the identification of two novel putativeCa2+-binding proteins, encoded by the NCU08524 and NCU06607 genes.We have recently shown that staurosporine-induced cell death in 2+ dynamics play an important role duringstaurosporine-induced cell death in N. crassa, eliciting acomplex response that includes Ca2+ release from internal stores aswell as uptake from the extracellular space 2+ concentrations. We prepared a culture medium,designated ‘no Ca2+’, by removing CaCl2 from Vogel’sminimal medium (MM) 2+ as impurities from the other reagents in the solutionallowed N. crassa to grow well under such conditions . In contrasting experiments, we supplemented medium with 20 mMCaCl2 (’20 mM CaCl2’ medium), which represents anapproximately 30 fold (but not toxic) increase as compared with the 0.68 mMCaCl2 present in standard Vogel’s medium .We inoculated wild type cells on the centre of Petri dishes containing standard,no Ca2+ or 20 mM CaCl2 solid Vogel’s MM supplemented withstaurosporine and measured radial growth during a 104 hour time course. The druginhibited growth in all media . However, the staurosporine inhibitoryeffect was amplified in the absence of Ca2+ . On the other hand, growth inhibition was partiallyovercome in 20 mM CaCl2 medium . In GFS medium, which contains sorbose that promotescolonial growth 2+ was similar: theeffects of staurosporine were exacerbated in no Ca2+ medium whereasgrowth inhibition was partially suppressed in the presence of 20 mMCaCl2, . Enhancement and suppression of inhibition ofgrowth by absence of Ca2+ and 20 mM CaCl2, respectively,was not reproduced when cells are treated with phytosphingosine (data notshown), another cell death inducer in N. crassa, supportingprevious observations that staurosporine and phytosphingosine act by distinctmechanisms 1429Intracellular Ca2+medium, these levels were augmented to ~40%  and ~34% , respectively . In contrast, the percentage of YOPRO-1 andPI-positive cells was reduced to ~15%  and ~6% , respectively, in 20 mM CaCl2 medium.In order to check cell death, we analysed cell staining with YOPRO-1 (earlyapoptosis marker) or PI (late apoptosis/necrosis marker) by flow cytometry. A2-hour treatment with staurosporine in standard MM resulted in ~22% and ~17% ofYOPRO-1 and PI positive cells, respectively . In no CaN.crassa to staurosporine, as the addition of antioxidants blockscell death 2+ in the culture medium influenced cellular ROSproduction induced by staurosporine. While in standard MM cells stressed for 30minutes with staurosporine displayed a ~4.2 fold-increase in ROS accumulation,the equivalent accumulation was ~10.4  and ~1.3  in no Ca2+ and 20 mM CaCl2 media, respectively. Given the importance of ROS formation for cell death provoked bystaurosporine, this Ca2+-ROS dependence is likely associated with thedistinct cell death phenotypes in the different culture media.The production of ROS is an essential event during the response of 2+in staurosporine-induced cell death, we combined the drug with theCa2+ chelator BAPTA and the Ca2+ ionophore A23187 in GFS plates with standard MM. As expected, extracellularCa2+  blockage with BAPTA synergized whereas elevation ofintracellular Ca2+ with A23187 antagonized the effects ofstaurosporine . In agreement, pre-incubation with BAPTA significantlyenhanced apoptotic levels induced by staurosporine, whereas they were partiallyinhibited by A23187, as measured by YOPRO-1 staining . Altogether,these data consistently indicate that the absence of Ca2+ enhancesstaurosporine-induced cell death, whereas excess (but not toxic) concentrationsof Ca2+ partially prevent it.To further stress the modulatory effect of altering the levels of Ca2+ reporter gene aequorin, wedescribed recently that staurosporine induces a three-step Ca2+response comprising three main signals called "A", "B" and"C". Peak "A" occurs immediately after the addition of thedrug and lasts approximately 20 minutes; peak "B" appears after 35-40minutes and lasts approximately 80 minutes; phase "C" is a prolongedand continuous elevation of cytosolic Ca2+2+, the Ca2+ response to staurosporine wascompromised, with peaks "A" and "B" showing substantialreduction . In medium with 20 mM CaCl2 there was a strongboost in peak "A", while peak "B" remained similar tostandard MM. These alterations in intracellular Ca2+ dynamics are inagreement with our findings that extracellular Ca2+ uptake isimportant for the Ca2+ signaling response to staurosporine Using cells expressing the cytosolic CaN. crassa genes (1921 genes) 2+ (~36% of genes (2749genes) were altered by the drug), and markedly suppressed by 20 mM ofCaCl2 (~6% genes (454 genes) showed altered profiles) . In standard medium, approximately half of the genes were induced andapproximately half were repressed by staurosporine 2+ medium, 43.1% of the alteredgenes were upregulated, whereas 56.9% where downregulated. Remarkably, in thepresence of excessive Ca2+ most of the altered genes were repressed(~68%). The complete dataset obtained from these RNA-seq experiments ispresented as File S1.Treatment with 20 μM staurosporine led to significant alterations in theexpression of ~28% of the 2+,792 (66.8%) of the induced genes were specific to this condition while in 20 mMCaCl2 this corresponded to 67 (46.2%) genes . Among repressed genes, 875 (56.0%) and 157 (50.8%) genes were specific tono Ca2+ and 20 mM CaCl2 media, respectively . Only minor fractions of the altered genes  were common to all conditions.We analysed the distribution of the individual genes induced and repressed bystaurosporine in the different culture media. In the absence of Ca2+concentration-specific differences were found among the downregulated genes. Inthe absence of Ca2+, staurosporine led to the downregulation of genesincluded in numerous categories. These encompass various groups within the‘Metabolism’ super-category such as ‘metabolism of the aspartate family’,‘purine nucleotide/nucleoside/nucleobase metabolism’, ‘phosphate metabolism’ and‘tetracyclic and pentacyclic triterpenes metabolism’. Also, there was a specificrepression of genes involved in ‘Cell cycle and DNA processing’ and‘Transcription’, namely ‘mRNA synthesis’ and ‘mRNA processing’. Categoriesrelated to stress responses like ‘Protein fate’, ‘unfolded protein response’,‘cellular sensing and response to external stimulus’, ‘cell growth’,‘anti-apoptosis’ were also repressed. Finally, while in standard MM there was adownregulation of ‘aerobic respiration’ genes, in no Ca2+ medium,other bioenergetic pathways were repressed: ‘glycolysis and gluconeogenesis’ and‘pentose-phosphate pathway’. These data indicates that a highly complextranscriptional program is established by staurosporine-treated cells growing inthe absence of Ca2+ resulting in the repression of several genesrelated with an anti-stress response. It seems that, when Ca2+ islimited, staurosporine causes the downregulation of cellular defences and thismay be related to the high susceptibility of these cells to the drug.FunCat was used to examine the enrichment of functional categories in thedifferent gene sets . The most interesting Ca2+ wasanalysed by comparing basal gene expression in no Ca2+, 20 mMCaCl2 and standard MM (File S3). The fraction of genes withaltered expression in no Ca2+ and 20 mM CaCl2 comparedwith standard MM was 3.9% and 1.8%, respectively , corresponding to281 and 129 genes, respectively. While half of the genes altered in noCa2+ were induced and half were repressed, in 20 mMCaCl2 most of the altered genes were upregulated (71.3%). Most ofthe genes were specifically induced or repressed depending on the particularculture medium: 97.2% and 97.1%, respectively, for no Ca2+, and 95.7%and 89.2%, respectively, for 20 mM CaCl2 . Notsurprisingly, categories related to ‘ion transport’, ‘homeostasis of cations’and ‘homeostasis of anions’ were enriched in the set of genes induced in theabsence of Ca2+ , which is perhaps linked to acellular attempt to recover proper conditions of osmolarity. Consistently, thesecategories were also significantly enriched among the genes repressed in thepresence of 20 mM CaCl2 . Lack of Ca2+ also seemsto be associated to a metabolic adaptation of the cells, since there wasenrichment in the induction of genes falling on the ‘metabolism’ super-category,like ‘amino acid metabolism’, ‘nitrogen, sulfur and selenium metabolism’ and‘anaerobic respiration’.The transcriptional response to the lack or excess of Ca2+ channels,Ca2+-ATPases, Ca2+-exchangers,Ca2+-dependent signaling molecules and other Ca2+ bindingproteins 1819312+-ATPase NCA-2 and the Ca2+/H+ antiporterCAX, respectively, were repressed in the absence of Ca2+. NCU04265,encoding the invertase enzyme (INV) was induced in the absence ofCa2+. The induction of INV, an enzyme that hydrolysesextracellular sucrose into glucose and fructose, is a marker of the process ofcarbon catabolite repression 332+-ATPase ENA-2 2. NCU05046 and NCU07966,encoding ENA-1 and TRM-1, respectively, were induced by no Ca2+ andrepressed in 20 mM CaCl2 medium. These results further suggest anintracellular metabolic remodeling in response to Ca2+availability.We looked for alterations in the expression of known Ca2+ or in 20 mM CaCl2medium. We screened the respective protein sequences for the presence ofCa2+-binding patterns using CaPS 2+-binding motif. NCU08524 was repressedin Ca2+-free medium whereas NCU06607 was induced by 20 mMCaCl2 . Fig. 6B shows the alignment of the predictedCa2+-binding motifs of NCU08524 and NCU06607 with those ofproteins containing the Dx[DN]xDG motif 22Bacillus subtilis. YesW exemplifies β-propeller structuresin which the Dx[DN]xDG motif can be embedded in one of the propeller blades,forming a ‘calcium blade’ structure Psathyrella velutinaMacrophomina phaseolina (Table S3)and matches from HHpred suggest that it contains a 6-bladed propeller structurein which β-strands flank the putative Ca2+-binding motif. Takingadvantage of the similarity between fucose-specific lectins and NCU08524,molecular models were built for the latter in a situation of Ca2+binding . In proteins containing the Dx[DN]xDG motif, interactionswith bound Ca2+ ions are supplemented by the interaction of sidechains from at least one downstream acidic residue 502, Glu503 or Asp504 as thepossible additional acidic residue. The models show that NCU08524 canaccommodate a Ca2+-binding geometry by combining the Dx[DN]xDG motifwith any of these downstream residues .We found that 188 genes with unknown function had altered expression in no Ca2+-binding motif of NCU06607  is comparableto the Dx[DN]xDG motif of a calmodulin from ParameciumtetraureliaThe predicted Ca2+ availability. The growth of bothmutants was significantly reduced in no Ca2+ medium and in mediumcontaining the Ca2+ chelator BAPTA . ΔNCU06607 also showed asignificant reduction in growth in the presence of 20 mM CaCl2 ascompared with wild type. The observed differences in growth were not merely dueto non-specific osmotic stress, since all strains were similarly affected by theaddition of 4% NaCl. ΔNCU06607 was slightly more sensitive to staurosporine thanwild type, suggesting that the respective protein may be involved in theresponse to the drug . In summary, the RNA-seq dataset and furtheranalysis revealed two putative novel components of the Ca2+-handlingmachinery in N. crassa.The growth of ΔNCU08524 and ΔNCU06607 was compared with the wild type strainunder different conditions of Ca2+ during the N.crassa response to staurosporine and to identify mediators of theprocess, we assayed the drug sensitivity profile of approximately 50 deletionstrains encoding proteins involved in Ca2+ homeostasis. We showedrecently that deletion of genes encoding phospholipase C enzymes, especiallyPLC-2, results in mutants resistant to staurosporine, while strains lacking theCa2+ channels CCH-1 and MID-1 are hypersensitive 2+-permeable channels, Ca2+-ATPases or otherCa2+ binding molecules involved in cellular signaling showed aresistance phenotype to staurosporine that was dissimilar from the wild typestrain . A resistance phenotype was particularly strong in deletionmutants for genes encoding the Ca2+-ATPases NCA-2 and PMR-1, anddeletion of other Ca2+-ATPases, namely ENA-1, ENA-2 and NCU10143 alsoled to increased resistance. The disruption of the signaling moleculescalmodulin, CAMK-4, and CSE-1 augmented resistance, whereas strains carryingdeletions in genes encoding STK-16 and PRD-4 resulted in increasedsusceptibility to staurosporine. Table 1 lists strains with altered sensitivityto staurosporine and Table S4 lists all strains tested. The deletion strains forNCU05360, NCU03804, NCU03833, NCU09871, NCU06347, NCU06617, NCU02411 andNCU09265 were not tested due to strain unavailability or their heterokaryoticnature. This data further stresses the importance and complexity of the fungalresponse to staurosporine, implicating numerous Ca2+-related proteinsof different function and subcellular localization in the process.To further substantiate the crucial role of Ca2+ available in theextracellular milieu is enough for a robust modulation of the effects ofstaurosporine in N. crassa. Cell death is strongly enhanced byCa2+ limitation whereas it is partially suppressed by a ~30-foldexcess of external Ca2+. A similar behavior was observed in S.cerevisiae cells treated with miconazole or terbinafine, inhibitors ofergosterol biosynthesis 2+concentrations  protected N. crassacells from toxicity induced by PAF, an antifungal protein fromPenicillium chrysogenum that also disturbs intracellularCa2+ homeostasis 2+ is an important player duringcell death.Here we show that controlling the amount of Ca2+are remarkably paralleled by distinct cytosolic Ca2+ dynamics, in linewith our previous findings that extracellular Ca2+ uptake is required forthe development of the cytosolic Ca2+ signature induced by staurosporine2+ signature, peaks "A" and"B" precede cell death, suggesting that they represent signaling events.In the presence of 20 mM CaCl2 the peak "A" is enhanced,whereas in medium lacking Ca2+ peaks "A" and "B" ofthe staurosporine-induced cytosolic Ca2+ response are abolished. Thus,intracellular Ca2+ signaling that is triggered by staurosporine ismarkedly modulated by the concentration of Ca2+ in the external mediumand might have a role in determining cell survival. However, the intracellularresponse to staurosporine is very complex and direct conclusions on thesusceptibility of the cells based solely on the cytosolic Ca2+ profile,although tempting, are not possible. For instance, a Δplc-2 mutantshows increased resistance to the drug despite that its cytosolic Ca2+response to staurosporine is nearly obliterated 2+ levels ultimately define cell fate in response tostaurosporine.The effects of staurosporine in media containing different amounts of Ca2+-handling mutantstrains revealed a number of proteins likely involved in the cell death response tothe drug. Further studies are needed to contextualize them functionally in themechanism of action of the drug. NCA-2 is a Ca2+-ATPase which pumpsexcess of cytosolic Ca2+ to the vacuoles or extracellular space 382+-ATPase PMR1 knockout cells accumulate much less Ca2+ inthe ER nca-2 and Δpmr-1 cells is likely related tothe phenotype of the strains upon treatment with staurosporine, as both the ER andthe vacuoles play an important role during the fungal response to staurosporine2+ in thecytosol and this is consistent with the observations that high levels ofCa2+ (below a toxicity threshold) protect against the effects ofstaurosporine. The lack of NCA-2 has been recently associated with increasedsensitivity to UV exposure, further implicating the molecule in cell survival 2+-related deletionstrains present altered resistance to staurosporine, includingΔcse-1, Δcamk-4 and Δstk-16.Knockout of cse-1 has been shown to lead to impaired germlingcommunication and fusion 2+camk-4 results in benomyl resistance, whiledisruption of stk-16 leads to increased sensitivity to fludioxonil,sodium chloride and benomyl The screening for staurosporine sensitivity of CaN. crassa transcriptional response to staurosporine is robustlyreduced in the presence of 20 mM CaCl2 when compared with standard MM,whereas limited extracellular Ca2+ causes alterations in an increasednumber of genes. Thus, different concentrations of extracellular Ca2+ notonly lead to distinct intracellular Ca2+ dynamics, ROS accumulation andcell death levels, but also seem to be coupled to unique transcriptional profiles.It appears that, in the absence of Ca2+, cells treated with staurosporineturn off a diverse array of biological processes. We discovered several enrichedfunctional categories in the set of no Ca2+ medium-specific repressedgenes, including the repression of genes involved in cell cycle, signaltransduction, cell growth, anti-apoptosis, cellular polarization, protein fate andthe unfolded protein response. It is possible that the decreased activity of variousintracellular pathways explains the inability of cells to cope with thestaurosporine insult and the consequent increased cell death.The N. crassa clearly translates changes in extracellularCa2+ into a transcriptional response, since alterations in geneexpression are caused alone by extracellular Ca2+ limitation or overload.This likely results from the homeostatic adaptation to the abnormal Ca2+environment, i.e., the observed differences in gene expression might be triggeredafter the uptake or release of Ca2+ to balance the intracellular levelsof the ion. Alternatively, these alterations may be secondary to the activity of asurface-localized Ca2+ sensor, although a sequence homologue of theextracellular Ca2+-sensing receptor of animals and plants 46N. crassa. Given the predicted cellsurface localization of the two putative novel Ca2+-binding proteins,NCU08524 and NCU06607, and the growth phenotype of the respective deletion strainsunder Ca2+ stress, it is tempting to speculate that they could play arole in Ca2+ sensing. The chemotropic interaction during conidialanastomosis tubes during cell fusion is Ca2+-dependent 482+ in the culture medium. The amount ofextracellular Ca2+ affects staurosporine-triggered intracellular eventslike Ca2+ signaling, the production of ROS and cell death. Our resultsindicate that these differences are linked to the stimulation of uniquetranscriptional programs and further highlight the importance of Ca2+during fungal cell death. In addition, our RNA-seq dataset will be a useful resourcefor investigations on the role of Ca2+ on different aspects of fungalbiology.In summary, we show that the effects of the antifungal 131415162PO4 in the 50x Vogel’s stock solutionwas limited to 10 mM to avoid precipitation with supplemental calcium Wild type 74-OR23-1VA strain and deletion mutants were obtained from the FungalGenetics Stock Center 4 conidia on the centreof large Petri dishes (ϕ 14.2 cm). Growth in liquid medium was examined byincubating 1x104 conidia/ml at 26°C, 100 rpm, under constant light in96-well plates (200 μl/well) and following absorbance at 450 nm 4 cells/ml wereprepared for each strain. 5 μl from each dilution were spotted on platescontaining glucose-fructose-sorbose (GFS) Hyphal growth in solid medium at 26°C was obtained by measuring colony elongationafter the inoculation of 20 μl containing 5x106 conidia/ml in the appropriate medium was incubatedfor 4 hours at 26°C . Staurosporine (20 μM) was addedand growth resumed for further 2 hours. The cells were harvested bycentrifugation, washed twice with PBS and incubated either with 0.1 μM YOPRO-1(samples were kept 20 min on ice before reading) or 2 μg/ml PI (samples wereimmediately ready for cytometry). The production of ROS was measured using thefluorescent probe dihydrorhodamine 123 (Sigma-Aldrich). After growing106 conidia/ml for 4 hours in the indicated medium at 26°C, 20μg/ml dihydrorhodamine 123 and staurosporine were added for further 30 minutes.Samples were harvested by centrifugation and washed twice with PBS before beingresuspended in PBS and read in the cytometer.For each experiment and strain, a control without staining was prepared to defineautofluorescence. Samples were read in a BD FACS Calibur and data analyzed withFlowJo (Tree Star). The levels of cell death were determined by staining cellswith YOPRO-1 (Life Technologies) or propidium iodide . Aninoculum of 102+ withthe genetic probe aequorin in wild type cells was employed as previouslydescribed 6 cells/ml, 5 μM coelenterazine (Santa CruzBiotechnology) and minimal medium were added to white opaque 96-well plates andincubated for 6 hours at 26°C, in the dark, without agitation. Luminescence  was captured over time on a Bio-Tek Synergy HTmicroplate reader. Maximum levels of luminescence, determined in extra wells bymeasuring luminescence during 3 mins after the injection of 100 μl of 3MCaCl2 in 20% ethanol, were used to normalize each experiment(cytosolic Ca2+ levels (arbitrary units) = experimental RLU values /total emitted luminescence). Quantification was performed by summing thenormalized values and data are expressed as mean ± SEM. The values from controlDMSO-treated samples were subtracted from staurosporine-treated samples toobtain the "staurosporine-induced amplitude of response".A bioluminescent method based on the reaction of cytosolic Ca2+-binding motifs with CaPS The non-parametric Mann-Whitney test was used for comparisons between two groupsusing SPSS 20 (SPSS Inc.) and p-values <= 0.05 were considered statisticallysignificant. ClustalW2 6 cells/ml were incubated in culturemedium with the indicated concentration of Ca2+ for 6 hours , staurosporine  was added and growthresumed for 1 more hour. Cells were harvested and immediately frozen in liquidnitrogen. RNA isolation, mRNA purification and fragmentation and cDNA synthesiswas performed as previously described http://www.ncbi.nlm.nih.gov/geo; series record: GSE53013).Conidial suspensions of 1x10Click here for supplemental data file.http://microbialcell.com/researcharticles/extracellular-calcium-triggers-unique-transcriptional-programs-and-modulates-staurosporine-induced-cell-death-in-Neurospora-crassa/.All supplemental data for this article are also available online at