Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.The yeast  SNCA gene that encodes α-syn and several point mutations  are associated with the formation of cytoplasmic protein inclusions and cytotoxicity (α-Syn misfolding and aggregation are linked to Parkinson’s disease (PD) . Duplicatoxicity . Based otoxicity .Saccharomyces cerevisiae is a well-founded model system for studying fundamental cellular processes relevant to higher eukaryotes. The high degree of conservation of protein folding and degradation, Ca2+ homeostasis and vesicle trafficking between yeast and higher eukaryotes and the fact that yeast does not express homologs of the human synuclein family allow to exploit yeast as a platform to study the molecular mechanisms underlying α-syn cytotoxicity  homeostasis by either forming membrane pores (leak channels) (2+ channels and Na+/K+ pumps)   that transports Ca2+ and Mn2+ into the Golgi complex, largely suppressed α-syn-induced Ca2+ changes. Since Ca2+ efflux across the plasma membrane in yeast may rely on vesicular exocytosis, we proposed that defects in vesicular trafficking may underlie α-syn-induced Ca2+ dysregulation.In this work, we focus specifically on the interplay between α-syn and intracellular Ca studies . In all 2+ entry . α-Syn phannels)  or affec+ pumps) . PreviouCalcium) . It was Several studies have demonstrated that α-syn overexpression strongly inhibits vesicle trafficking, thereby disrupting normal processing and transport of proteins within the secretory pathway. At physiological concentrations, the function of α-syn is closely related to SNARE complex formation and/or stabilization . Whereas2+ homeostasis and vesicular transport, we analyzed the effects of α-syn overexpression on intracellular Ca2+ handling and storage in temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1. We provide evidence that α-syn overexpression in yeast significantly affects basal Ca2+ levels and Ca2+ storage. Furthermore, we show that α-syn alters Ca2+ handling through two distinct mechanisms involving α-syn-mediated disruption of vesicle trafficking and vacuolar Ca2+ storage.To further unravel the plausible link between α-syn overexpression, Cahis3-1, leu20, met150, ura30, Euroscarf, Frankfurt, Germany). The sec4ts strain  was derived from the LRB906 strain background . PMC1 and VCX1 deletion strains in the BY4741 background were obtained from the yeast deletion collection    α-syn C-terminally tagged with yeast enhanced green fluorescent protein (α-syn-yeGFP) was expressed in the pYX212 vector (pYX212-α-syn-yeGFP) under control of the TPI promoter.Single and double deletion strains were derived from the BY4741 strain background  . The exppromoter  in the pTo visualize Pmc1 distribution in α-syn expressing cells, BY4741 cells were co-transformed with pPMC1-GFP fusion plasmid   and pGGE600  units in sample buffer containing 50 mM Tris, pH 8, 2% SDS, 0.1% bromophenol blue, 10% glycerol. Antibodies used were: Anti-α-synuclein , Anti-ADH2  and mouse Anti-rabbit complexed with HRP . Membranes were blocked in TBS-T (0.05% Tween-20) with 5% BSA. Antibodies were diluted in TBS-T (0.05% Tween-20) with 5% BSA. Primary antibodies were incubated overnight at 4°C. The secondary antibody was incubated for 1 h at room temperature.Total protein extracts were prepared from samples taken at 2 OD600 unit of cells was harvested from overnight cultures grown at the permissive temperature (25°C). Harvested cells were incubated at 37°C for 1 h in growth medium and then switched to 100 μl Y1-buffer  for 30 min at 30°C with gentle shaking in order to generate spheroplasts. Total RNA was extracted using the “RNeasy mini kit” (Qiagen). cDNA was generated using the “Transcriptor First Strand cDNA Synthesis Kit” (Roche) starting from 1 μg total RNA, using the Anchored-oligo(dT)18 Primers delivered with the kit. Unpurified cDNA was diluted 1/10 before use in quantitative-PCR using the “Lightcycler® 480 SYBR Green I Master” kit (Roche). Primer sets used with respective efficiencies and final concentrations were: (1) Pmc1 forward primer TCACCACGTTTTAGTCGG, Pmc1 reverse primer AGTTATCCACCGGAAATTTCTG, %E = 85, final concentration = 250 nM (2) Act1 forward primer AGGTTGCTGCTTTGGTTATTG, Act1 reverse primer TGACCCATACCGACCATG, %E = 91, final concentration = 250 nM. Program settings were as follows: pre-incubation at 95°C for 10 min; 40 amplification cycles 10 s at 95°C, 15 s at 53°C and 15 s at 72°C. The Pfaffl method was used to determine relative gene expression in order to compensate for varying primer efficiencies. ACT1 was used as reference gene.To determine gene expression, one OD2+ levels ([Ca2+]in) were measured in populations of yeast cells expressing apoaequorin as previously described (TPI promoter. Starting from pre-cultures (OD600 of ±2–3), two OD600 units were plated on Concanavalin A coated coverslips and incubated at 25°C for 1 h. For aequorin reconstitution, cells were washed with 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES)/ Tris pH 6.5 and incubated with 0.1 M MES/Tris, pH 6.5 supplemented with 5 μM wild-type coelenterazine (Promega) at either 37°C (non-permissive or restrictive temperature), 30 or 25°C (permissive temperatures) for 1 h. After removing excess of coelenterazine, coverslips with adherent yeast cells were placed in the perfusion chamber of a single-tube luminometer  positioned about 2 cm above the coverslip surface) and perfused with the required solutions at either 37°C (non-permissive temperature), 30 or 25°C (permissive temperatures). Adherent cells were initially perfused with 0.1 M MES/Tris, pH 6.5, followed by 0.1 M MES/Tris, pH 6.5 supplemented with 10 mM CaCl2. To estimate intracellular Ca2+ storage (2+-free medium containing (in mM): 200 KCl, 100 NaCl, 3 EGTA, 20 Hepes/KOH at pH 6.8 and then permeabilized with 0.5% Triton X-100 in the same medium. At the end of all experiments, the residual reconstituted aequorin was completely discharged by perfusing the cells with a solution containing 10 mM CaCl2 and 0.5% Triton X-100. Light impulses originating from the entire yeast cell population were discriminated, pre-scaled and integrated  with a PC-based 32-bit counter/timer board . The emitted light was calibrated offline into cytosolic Ca2+ values using the following algorithm [Ca2+]in = ((L/Lmax)1/3+[KTR(L/Lmax)1/3-1)/(KR(L/Lmax)1/3]) with KTR and KR the constants for the Ca2+-unbound and Ca2+-bound state, respectively   .2+ measurements, the same cultures were used to inoculate new cultures at a starting OD600 of 0.01. Growth was quantified by measuring the OD600 over a 72 h period at 25°C . Absorbance data were averaged for each condition and plotted as a function of time. Based on the log-transformed growth profiles, we calculated the half-times for growth (t50) of each strain, i.e., the time necessary to reach half-maximal OD600.Following Cat-test were used to determine statistical significance between datasets.Results are expressed as mean ± standard deviation (SD) or standard error of mean (SEM) as indicated. One-way analysis of variance (ANOVA) with Bonferroni posttest and unpaired Student’s 2+ homeostasis, we monitored cytosolic [Ca2+] ([Ca2+]in) after removal of extracellular Ca2+ (Rest [Ca2+]) and estimated the Ca2+ storage by monitoring the release of Ca2+ from intracellular stores when cells were permeabilized in Ca2+-free medium (Peak [Ca2+]). 2+ in wild-type LRB906 cells at 37°C  as well as the peak amplitude of the Ca2+ release transient taken as the maximum of the biphasic Ca2+ release transient (Peak [Ca2+]) are listed in To evaluate the effects of α-syn expression on intracellular Ca at 37°C . Cells w storage . For the2+ levels in 10 mM external Ca2+ (775 nM); (2) incomplete decay of [Ca2+]in after switching to Ca2+-free medium (Rest [Ca2+] 0.29 ± 0.12 μM); (3) a quasi-monophasic Ca2+ release transient with increased peak [Ca2+] (Peak [Ca2+] 2.07 ± 0.29 μM attained 1 min 15 s after membrane permeabilization) and (4) a faster decay rate  elevated cytosolic Cacay rate .2+ changes were accompanied by significant growth inhibition measured at 25°C. Specifically, LRB906 cells overexpressing α-syn reached half time for growth (t50) after 42.27 ± 5.84 h, resulting in a significant Δt50 of 12.3 h relative to wild-type LRB906 cells transformed with an empty plasmid (t50 of 29.97 ± 2.99 h). The t50 values are listed in As shown in 2+ changes are directly linked to deficits in exocytosis, we designed a series of experiments using yeast cells carrying a temperature-sensitive (ts) mutation in SEC4 (sec4ts). Sec4, an ortholog of human Rab8, is a small G-protein required for delivery and initial docking of secretory vesicles at the plasma membrane.There is compelling evidence that α-syn interferes with exocytotic pathways in both yeast and mammalian cells . To expl2+ responses in sec4ts cells are very similar to those in wild-type LRB906 cells showing complete cytosolic [Ca2+] decay after switching to Ca2+-free medium and biphasic release of stored Ca2+ following membrane permeabilization (2+ storage in sec4ts cells was significantly larger (sec4ts cells grew slower than wild-type LRB906 cells (2+]in after switching to Ca2+-free medium in sec4ts cells was somewhat slowed down (Zero [Ca2+] 0.12 ± 0.20 μM), but much more strikingly was that the Ca2+ release following membrane permeabilization became monophasic with a much higher peak value (Peak [Ca2+] 3.21 ± 0.66 μM) and a faster decay rate , the Calization . Howevery larger . In addi06 cells . At the cay rate  as compasec4ts cells , the decay of cytosolic Ca2+ upon removal of external Ca2+ was much slower (Rest [Ca2+] 0.53 ± 0.26 μM) and the Ca2+ release transient decayed faster. Peak Ca2+ release moderately increased (Peak [Ca2+] 3.71 ± 0.53 μM) but this α-syn-induced effect was clearly less pronounced than in wild-type LRB906 cells. Western blot analysis further indicated that α-syn was expressed at similar levels in LRB906 and sec4ts cells . As shown in 2+ pattern as WT cells but the peak level of the biphasic Ca2+ release was slightly higher. Also, the Ca2+ profile in LRB906-pSEC4 cells overexpressing α-syn  while the peak level of the monophasic Ca2+ release was higher (Peak [Ca2+] 2.86 ± 0.63 μM)  . Surprishibition .2+]in changes, we monitored aggregation in LRB906 and sec4ts cells expressing α-syn fused with yeast-enhanced green fluorescent protein (yeGFP). Live-cell imaging of α-syn-yeGFP under these conditions revealed a significant percentage of cells displaying foci in both LRB906 (16.7 ± 8.8%) and sec4ts cells at permissive temperatures (29.3 ± 11.8%). Upon shifting to restrictive temperatures the percentage of cells displaying foci greatly increased in both LRB906 (26.7 ± 13.1%) and sec4ts cells (64.2 ± 11.2%), which in the latter was much more pronounced . As 30°C is the optimum temperature for yeast and to minimize temperature stress, further experimental assays in the BY4741 background were performed at 30°C. Furthermore, as shown in 2+] is not an absolute requirement for the α-syn-induced shift from biphasic to quasi-monophasic Ca2+ release. When cells were not challenged with extracellular Ca2+, membrane permeabilization evoked a similar biphasic Ca2+ release as in cells challenged with 10 mM external [Ca2+]. Likewise, the response in α-syn overexpressing cells became quasi-monophasic although peak [Ca2+] was significantly reduced compared to cells previously exposed to external [Ca2+].The higher [Caand 30°C . Coolingransient  – insets1 strain  was simi2+]in values determined at the end of the Ca2+-free period before permeabilization (Rest [Ca2+]) as well as the peak amplitude of the Ca2+ release transient taken as the maximum of the biphasic Ca2+ release transient (Peak [Ca2+]) are listed in For the BY4741 strains, the [Ca2+ transporter in yeast cells that mainly controls resting [Ca2+]in  showed similar resting [Ca2+]in value as wild-type BY4741 cells  and pmc1Δ (pmc1Δ-pPMC1) cells  cells .vcx1Δ strain was also tested  but the Ca2+ release transient became nearly monophasic with a significantly higher peak value (vcx1Δ cells overexpressing PMC1 (vcx1Δ-pPMC1) showed similar resting [Ca2+]in as vcx1Δ cells but an enhanced biphasic release transient  and storage .In this work, we particularly focused on the effects of α-syn on yeast Ca2+ levels in Ca2+ free medium (increased Rest [Ca2+]). This increase may result from an enhanced Ca2+ efflux from intracellular stores and/or a reduced organellar Ca2+ uptake. Since several studies have demonstrated that α-syn may form Ca2+ permeable pores  while the second slower phase reflects the release of organellar bound Ca2+ (mainly Ca2+ bound to vacuolar polyphosphates). The finding that the deletion of Pmc1, the main vacuolar Ca2+ transporter, strongly reduces the second slower component further supports this hypothesis.In wild-type yeast cells, a biphasic Caous work , we have2+. Several studies have demonstrated that α-syn overexpression strongly inhibits vesicle trafficking  mutation in SEC4 (sec4ts). Our reigning hypothesis is that dysfunction of Sec4 disrupts exocytosis . Furthermore, while control cells showed a typical single large Pmc1-containing vacuole, cells expressing α-syn accumulated multiple small Pmc1-containing vesicles. Co-staining with endocytic marker FM4-64 revealed co-localization with vacuolar membranes. In accordance with these observations, we therefore propose that the additional pool of vesicles constitute fragmentation and vesiculation of vacuolar membranes, which display fewer fusion events as a consequence of α-syn interaction with vesicle trafficking components (2+ leakage (α-syn pore forming activity) is expected to shorten the duration of the Ca2+ release transient and to reduce the time to peak [Ca2+] release.Yeast cells overexpressing α-syn typically showed a shift from biphasic to a quasi-monophasic release of stored Cafficking . To inveocytosis  and thermponents . Additio2+ release transient was faster in α-syn expressing cells. In permeabilized cells, the rate of decay of the Ca2+ release transient reflects a balance between organellar Ca2+ release and Ca2+ leakage across the plasma membrane. In α-syn overexpressing cells, the Ca2+ release occurs at high cytosolic Ca2+ levels (peak [Ca2+] 2.51 ± 0.23 μM in BY4741 +α-syn) while this occurs in control cells at much lower levels (peak [Ca2+] 1.34 ± 0.21 μM in BY4741). Therefore, the initial Ca2+ leak across the plasma membrane will be significantly larger in the BY4741 +α-syn cells resulting in a faster decay of the Ca2+ release component. Additionally, as organellar Ca2+ leakage is likely to be increased (α-syn pore forming activity) in α-syn overexpressing cells, the total Ca2+ vacuolar storage will be lower resulting in a faster decay of the Ca2+ release component.The decay phase of the CaPMC1 in BY4741 and in pmc1Δ abolished the α-syn-related cytotoxicity on growth. As Peak [Ca2+] increased and Rest [Ca2+] decreased in parallel with PMC1 expression levels, this observation suggests that the cytotoxicity of α-syn is closer related to its propensity to form pores rather than disrupting vesicle trafficking.Finally, overexpression of PMC1 in vcx1Δ cells did not reduce α-syn-induced toxicity. In vcx1Δ cells, it was found that PMC1 expression was enhanced. As PMC1 is a calcineurin-dependent gene and calcineurin may contribute to α-syn-induced toxicity  and ADx NeuroSciences nv , but this did not influence in any way the studies reported in the manuscript.The remaining 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.


We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy‐induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high‐potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher‐potency statin (eg rosuvastatin)–based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O While the impact of O2‐controlled conditions. Drug doses chosen for the proteo‐transcriptomic studies  represent a compromise between sublethal and clinical relevance: sufficient for a detectable expected response (eg apoptosis), but still compatible with a majority of cells remaining viability. Our working model is that the surviving pool exhibits shifts in molecular programmes that are critical for surviving the first therapeutic hit. Of note, we used a conceptually similar strategy to extract information about potential synergistic therapeutic partnerships in the context of solid tumours.In the present study, we investigated the effects of two clinically relevant agents, cytarabine and quizartinib, the former representing the classic anti‐AML cytotoxic drug, while the latter being representative for the new wave of highly specific kinase inhibitors,The results of our integrated screens converged towards a key role of lipid biosynthetic programmes, and in particular cholesterol biogenesis, during AML response to therapy. The results presented herein have significant translational implications, as they provide support for the value of adding higher‐potency statins (eg rosuvastatin) to classical and emerging AML therapies.22.12. Quizartinib (AC220) and cytarabine were purchased from Selleck Chemicals, dissolved in dimethyl sulphoxide (DMSO) at stock concentrations of 10 mmol/L and stored at −80°C. Rosuvastatin was purchased from Tokyo Chemical Industry. Pravastatin, terbinafine, bempedoic acid and zoledronic acid were purchased from Selleck Chemicals. Unless otherwise indicated, all drugs were dissolved in DMSO at stock concentrations of 100 mmol/L and stored at −20°C. Fluorescein isothiocyanate‐Annexin V antibody and propidium iodide were purchased from BD Pharmingen and Sigma‐Aldrich.Human AML cell lines, Molm14, and the relatively cytarabine‐resistant cell lines OCI‐AML3 and THP‐12.2Acute myeloid leukaemia blasts derived from whole blood or the bone marrow from 10 newly diagnosed (ND), previously untreated AML patients who consecutively presented to our institution were obtained under guidelines approved by the Institutional Review Board of the Indiana University . All patients gave informed consent according to the Declaration of Helsinki. Blasts were separated by centrifugation over a layer of Ficoll‐Paque PLUS , collected as the buffy coat and then either stored in freezing medium  in liquid nitrogen or plated immediately for experiments. The clinical characteristics of the patients from whom samples were derived are listed in Table 2.34 Molm14, THP‐1 and OCI‐AML3 cells or 15 × 104 fresh primary cells suspended in RPMI 1640 culture medium with 10% FBS were seeded in 96‐well plates. Cells were treated with either drug in single agent or combined mode under ambient air  or hypoxic conditions (1% O2) for 48 hours as outlined for each experiment. Cell viability was assessed using MTT ‐2,5‐diphenyltetrazolium bromide; Roche) in accordance with the manufacturer's recommended protocols. Briefly, 20 µL MTT solution was added into each well and incubated at 37°C for 2 hours. Plates were read on at 490 nm with a plate reader. Growth inhibition percentage was calculated from the optical density ratio of treatment to DMSO control after subtraction of background under 21% O2 and 1% O2, respectively. All assays were performed in triplicate, and the results were obtained in at least three independent experiments as outlined for each experiment. Combination indexes (CIs) were determined using CompuSyn software.1 × 102.45 leukaemia cells were seeded in 6‐well plates and incubated with cytarabine and/or rosuvastatin under 21% or 1% O2 conditions as outlined for each experiment. After 48 hours, cells were harvested from the plates, washed twice with cold PBS and then re‐suspended in 100 µL binding buffer. 5 µL FITC‐Annexin V and 10 µL propidium iodide stock solution (50 µg/mL) were added to each sample and incubated at room temperature in the dark for 15 minutes. Cells were analysed on a BD Accuri (BD Biosciences).A total of 5 × 102.5HMGCS1 (F): CTCTTGGGATGGACGGTATGC and HMGCS1 (R): GCTCCAACTCCACCTGTAGG;LSS (F): GACGACCGATTCACCAAGAGCA and LSS (R): AGACATGCTCCTGGAAGGCAGT;MSMO1 (F): GCTGCCTTTGATTTGTGGAACCT and MSMO1 (R): CTGCACAACCAAAGCATCTTGCC;SQLE (F): CTCCAAGTTCAGGAAAAGCCTGG and SQLE (R): GAGAACTGGACTCGGGTTAGCT;CD36 (F): CAGGTCAACCTATTGGTCAAGCC and CD36 (R): GCCTTCTCATCACCAATGGTCCRN18S1 (F): ACCCGTTGAACCCCATTCGTGA and RN18S1 (R): GCCTCACTAAACCATCCAATCGGMolm14 and THP‐1 cells were treated with cytarabine for 48 hours. Total RNA was extracted by RNeasy Plus Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized with reverse transcription kit (Thermo Fisher Scientific) as per the manufacturer's protocols. The PCR primers used in the study were as follows:RN18S1 expression was used as a control for RNA loading. Quantitative PCRs were performed using PowerUp SYBR Green PCR Master Mix  on 7900HT Real‐Time PCR System.2.62 or 1% O2 for 48 hours. Leukaemia cells were washed twice with ice‐cold PBS and re‐suspended in 150 µL PBS. Cellular lipids were extracted using 750 µL chloroform:methanol (2:1) solution. After extraction for 30 minutes on a rocker, samples were centrifuged at 1252 g at 4°C for 10 minutes and the lower chloroform phase was collected and air‐dried in a fume hood until all liquid evaporated. The lipids were then re‐suspended in 10% Triton X‐100/isopropanol. Total cholesterol content was measured using Wako cholesterol assay kit according to the manufacturer's instructions.Cellular cholesterol was measured using Wako cholesterol assay kit . In brief, Molm14, THP‐1 or primary AML cells were incubated with cytarabine and rosuvastatin in single agent or combined mode under 21% O2.7Total RNA was evaluated for its quantity and quality using Agilent Bioanalyzer 2100. For RNA quality, an RNA integrity (RIN) number of 7 or higher was required. About 500 ng of total RNA was used for cDNA library preparation, including mRNA purification/enrichment, RNA fragmentation, cDNA synthesis, ligation of index adaptors and amplification, following the TruSeq Stranded mRNA Sample Preparation Guide, RS‐122‐9004DOC, Part# 15031047 Rev. E . Each resulting indexed library was quantified, and its quality was accessed by Qubit and Agilent Bioanalyzer, and multiple libraries were pooled in equal molarity. Five microlitres of 2 nmol/L pooled libraries per lane was then denatured, neutralized and applied to the cBot for flow cell deposition and cluster amplification, before loading to HiSeq 4000 for sequencing .2.8P‐values.Gene Set Enrichment Analysis was performed following GSEA User Guide (The Broad Institute). Briefly, each condition was considered as a group and gene list was ranked with GSEA default ranking metrics. Gene sets from Molecular Signature Database were used in the analysis to identify the pathways significantly enriched in each group. Gene sets were permutated 1000 times to obtain empirical FDR‐corrected 2.9In brief, cell lysate samples were serially diluted twofold for 5 dilutions  and arrayed on nitrocellulose‐coated slides in an 11 × 11 format to produce sample spots. Sample spots were then probed with antibodies by a tyramide‐based signal amplification approach and visualized by DAB colorimetric reaction to produce stained slides. Stained slides were scanned on a Huron TissueScope scanner to produce 16‐bit tiff images. Sample spots in tiff images were identified and their densities quantified by Array‐Pro Analyzer. Relative protein levels for each sample were determined by interpolating each dilution curve produced from the densities of the 5‐dilution sample spots using a ‘standard curve’ (SuperCurve) for each slide (antibody). All relative protein level data points were normalized for protein loading and transformed to linear values.2.10Cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Thermo Fisher Scientific). Proteins were separated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto PVDF membranes (Bio‐Rad). Primary antibodies used in this study included anti–stearoyl‐CoA desaturase (anti‐SCD) (sc‐58420) and anti–fatty acid synthase antibody (A‐5) (anti‐FASN) , as well as anti‐β‐tubulin . Horseradish peroxidase‐conjugated secondary antibodies (NA931V) were from GE Healthcare. Antibody detection was performed using ECL™ Western blotting reagent kit (RP2106) .2.11m/z) transitions in positive mode for rosuvastatin and simvastatin are 482.1/258.2 and 419.4/199.0, respectively. The MRM Q1/Q3 (m/z) transition in negative mode for pravastatin is 423.0/321.2. The lower limit of quantification for both rosuvastatin and pravastatin is 1 ng/mL using 200 μL media.Rosuvastatin and pravastatin were quantified per high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Briefly, rosuvastatin and pravastatin were quantified from media using simvastatin as the internal standard and HPLC‐MS/MS (ABSciex 4000). For the samples and standards, rosuvastatin, pravastatin and simvastatin were extracted from media by the addition of 0.1 mol/L citric acid buffer (pH = 3.0) followed by liquid‐liquid extraction using ethyl acetate. The analytes were separated by a gradient mobile phase  with a Zorbax 300SB‐C8 150 × 4.6 mm 5 µm column. The mass spectrometer utilized an electrospray ionization probe run in both positive and negative modes. The multiple reaction monitoring (MRM) Q1/Q3  unless stated otherwise.Statistical significance of differences was determined by Student's 33.12‐replete or O2‐depleted conditions. Differentially expressed transcripts were determined by RNAseq analysis, followed by Gene Set Enrichment Analysis (GSEA) to identify the main pathways/programmes affected by drug treatment. Using the hallmark collections, GSEA identified ‘cholesterol homeostasis’ down‐regulation as a major common feature of cytarabine and quizartinib responses under 1% O2, while at 21% O2, the suppression hallmark passed the significance threshold in quizartinib sets only, and upon further inspection revealed a similar response, albeit less robust, following cytarabine. This coordinated shift following anti‐AML agents under high and low O2 is captured by the heatmaps shown in Figure 2 in response to either drug , methylsterol monooxygenase 1 (MSMO1), lanosterol synthase (LSS) and squalene monooxygenase (SQLE). As shown in Figure 3.3A complementary dimension to the transcriptional portrait of drug response was provided by RPPA‐based comparisons Figure . RPPA is2, between cytarabine‐residual Molm14 cells and patient‐derived blasts that have become resistant to this agent in a xenograft AML model.There are notable similarities, particularly under low O3.42 conditions in the in vitro studies.Appelbaum, Penn and colleagues noted that high mevalonate pathway activity and cholesterol abundance are hallmarks of AML.Our data collectively suggest that cytarabine ‐residual cells ‘dial down’ energy‐intensive biosynthetic programmes, in particular biosynthesis of sterols and fatty acids, and switch to ‘scavenger’ mode. A central component of this switch may be up‐regulation of multifunctional translocase CD36/fatty acid translocase (FAT), as suggested by RNAseq profiles and independently confirmed by TaqMan quantitative RT‐PCR in Molm14 cells Figure A‐C. This3.52 conditions. In all cells tested and under both oxygenation conditions, rosuvastatin inhibited AML cell growth in a dose‐dependent fashion and was significantly more effective than pravastatin. Pravastatin had mild to no effects at 21% and 1% O2  and 1% O2  compared to untreated cells. While treatment with cytarabine significantly increased intracellular cholesterol levels, this response was stalled by co‐treatment with rosuvastatin  and TPH‐1 cells , and synergistic to strong synergistic effects in a diverse panel of primary AML cells (n = 10) as summarized in Table 2 abundance   Figure .42 tension in the neoplastic microenvironment is a well‐established determinant of therapy failure and disease relapse in cancer; however, the experimental evidence remains heavily biased towards solid tumours.+) AML cells to cytarabine and the highly selective FLT3 inhibitor quizartinib under O2‐controlled conditions in vitro.Decreased O2 tension. Subsequently, we revisited the concept of cholesterol‐targeted treatment approaches in the management of AML with more potent inhibitors, such as rosuvastatin. We found that rosuvastatin exerts significant anti‐leukaemic activity against a broad selection of AML cells and acts synergistically with cytarabine at different levels of O2. Intriguingly, in vitro responses were observed in AML cell lines of different subtypes and in primary cells derived from newly diagnosed AML patients. Our findings firmly support the further investigation of rosuvastatin in combination with classical and investigational therapies for the management of AML.Utilizing transcriptomic (RNAseq) and proteomic  technologies, our data indicate that the cholesterol biosynthesis pathway is transcriptionally induced under hypoxic stress conditions and attenuated by cytarabine and quizartinib. In contrast, treatment of AML cells with cytarabine led to an increase in total intracellular cholesterol levels irrespective of O2 experimental component is important, since at 21% O2, cholesterol homeostasis drops out of focus, especially in the cytarabine‐treated cells. In contrast to the reproducible increase of intracellular cholesterol levels following cytotoxic therapy, there is strikingly limited information about the response of individual pathway members, particularly under O2‐deprived conditions.Our studies provide several new insights regarding the response of cholesterol biosynthesis pathway in the AML context and the derived opportunities for combination therapy. The relevance to cholesterol biosynthesis for AML biology and therapy has been recognized for decadesA shift to a scavenger/lipid catabolic programme is the most likely scenario to reconcile this contrast. Consistently, we have confirmed the induction of CD36/FAT, a transmembrane protein that facilitates cholesterol and fatty acid uptake, previously shown to contribute to the resistance of leukaemic cells to therapy.2‐dependent and uniquely energy‐intensive , this process represents a probable liability under persistent pharmacological and environmental stress. Similar considerations may apply to the closely coordinated de novo fatty acid biosynthesis. In simplistic terms, AML cells with a lower activity window for this programme would be more likely to survive the initial wave of therapy. On the other hand, such a metabolic ‘compromise’ has important limitations since up‐regulation of scavenging transporters should only compensate the deficit of end products but not of biologically active metabolic intermediates (eg terpenoids). This ‘adaptive’ status therefore may also render residual AML cells vulnerable to a pharmacological intervention of the right magnitude at the root of this pathway.A partial suppression of lipid anabolism should provide important advantages for at least some types of AML cells. Since de novo cholesterol production is both O2‐abundant and O2‐deprived conditions. To this end, as clinical leukaemias are comprised of heterogeneous populations distributed between the O2‐abundant bloodstream and O2‐deprived tissue sanctuaries such as the bone marrow, the effectiveness of rosuvastatin alone, or combined with cytarabine, in a diverse mutational and oxygenation context is translationally significant.Our results are particularly relevant to the recent clinical studies coordinated by Applebaum and colleagues, testing the benefit of combining conventional cytotoxic agents  with high‐dose pravastatin in newly diagnosed and relapsed/refractory AML. While acceptable toxicity and promising CR/CRp rates of up to 80% and 66% were reported in a phase 1 trial of newly diagnosed and relapsed/refractory AML, respectively, a recent phase 2 trial did not meet the criteria for a positive study.2 tension, rosuvastatin more potently eliminates AML cells compared to pravastatin, which has no effects in the O2‐deprived setting and displayed underwhelming activity in AML trials. Our data suggest that the cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape and strongly support the further investigation of rosuvastatin‐based combination therapies to enhance targeting residual AML cells that reside in O2‐deprived environments, thus addressing an unmet need in AML therapy.In summary, our work demonstrates that the cholesterol biosynthesis signature is affected similarly by cytarabine and quizartinib, two unrelated pharmacological anti‐AML agents, particularly under microenvironmental stress conditions (eg severe hypoxia). We further show that, irrespective of OFC, XW, CN, MG, DP, AR, SR, BG, SG, GAC, HSB and HK do not report any conflicts of interest.FC designed and performed research, collected, analysed and interpreted data. XW designed and performed research, collected and analysed data. CN designed and performed research. MG designed and performed research. DP designed and performed research. AR performed research. SR designed and performed research. BG designed and performed research. SG contributed materials and interpreted data. GAC analysed and interpreted data. HSB contributed materials and interpreted data. HK designed research, analysed and interpreted data, and wrote the manuscript.Fig S1‐S6Click here for additional data file.Table S1AClick here for additional data file.Table S1BClick here for additional data file.


SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that  Acute myeloid leukemia (AML) is the most frequent type of acute leukemia in adults. However, there have been few significant advances in the past 40 years beyond the use of conventional chemotherapy in combination with bone marrow (BM) transplantation for AML treatment , 2. TherGenome-wide analyses have shown that genes involved in epigenetic modifications are the most recurrently mutated genes in AML, suggesting crucial roles of epigenetic regulations in AML leukemogenesis and progression , 4. ThesMLL) rearrangements. More than 79 fusion genes have been reported to translocate with MLL, sharing some correlation with disease phenotype and prognosis [MLL-r leukemia exhibits a distinct characteristic of pathology and molecular mechanism from other subtypes of AML. It has been reported that MLL-r leukemia stem cells (LSCs) exhibits aberrantly increased levels of H3K4me3 and H3K79me2 [MLL-r leukemia [MLL-r induced leukemia [+CD38– HSCs [MLL-fusion proteins to promote oncogenesis [MLL-r leukemic cells, Dot1L, via its binding to the DNA, interrupts the normal localization of Suv39h1 and Sirt1, and deletion of Suv39h1 desensitizes leukemic cells to Dot1L inhibition [MLL-r AML.Leukemia bearing translocations involving chromosome 11q23 is one of the most aggressive and drug-resistant leukemias, characterized by the presence of Mixed Lineage Leukemia   LSCs [To examine the potential clinical relevance of SUV39H1 to human AML, the expression pattern of loodSpot . SUV39H1e effect  or the cognoScan  and founned LSCs  and L-GMI+) LSCs , as comp+ cells  in c-Kit+ cells from WT controls, MA9 and MN3 AML mice. The distributions of H3K9me3 on the whole genome and around TSS  were significantly lower in AML LSCs than in normal HSPCs  that were infected with Suv39h1-overexpression lentivirus have been stably passaged for three times  for transplantation in our study. Significantly, Suv39h1 overexpression prolonged the survival of secondary murine recipients  [4 MA9 P2 AML cells, then treated with chaetocin or vehicle one day after transplantation  , 34. Sinion Fig. . Shortenion Fig. . No obviion Fig. . The samion Fig. . If chaeSuv39h1 would accelerate leukemia progression by reducing the expression of Suv39h1 using shRNA-mediated knockdown . Among these genes, 158 were down-regulated, and 526 were upregulated -induced skin carcinogenesis [It has been reported that requency , and losrequency , 44. Anoogenesis . These dogenesis  and EVI-ogenesis -mediatedMLL-fusion induced AML [Six1. Nevertheless, pathway interaction analysis of DEG-enriched KEGG pathway revealed the reduction of metabolic process as one of the most prominent downstream effects of Suv39h1. Our recent study demonstrated that Six1 was a key transcription factor involved in leukemogenesis via regulation of LSC pools and the expression of glycolytic genes [A subset of H3K9 methyltransferases including Suv39h1, G9a, GLP, and SETDB1 were reported to participate in one complex and cooperate functionally in gene silencing . In consuced AML , 49. Botic genes . In addiCcne1, Ccne2, Ccnd2 and E2F targets) as well as up-regulated expression of CDKi genes including p18, p21 family members p21 and p57 in SUV-OE cells. Thus, the decrease of S phase and the increase of G1 phase resulted by Suv39h1 overexpression were probably through coordinated regulation of G1/S transition related genes.Notably, Suv39h1 displayed a unique role in regulation cell cycle, which was not observed in Setdb1 OE AML cells. Retinoblastoma protein (Rb) is sequentially phosphorylated by CycD/Cdk4/6 and CycE/Cdk2 and full phosphorylated Rb activates the E2F transcription factor family, thus promotes the G1-S transition . PreviouHoxb13 in AML progression has not been defined. Notably, the basal expression level of Hoxb13 is very low in normal hematopoietic cells, but is upregulated in MA9 LSCs. We demonstrated here that Hoxb13 itself was epigenetically dysregulated in MA9 cells, and it was highly activated in LSCs when compared with normal HSPCs. Moreover, knockdown of Hoxb13 prolonged the survival of MA9 AML mice. Interestingly, although HoxB13 shRNA knockdown did not affect the expression of other HoxB family gene, yet we did observe the down-regulated expression levels of some HoxA family members, such as Hoxa9 and Hoxa10 . Detailed information of patient samples was described previously [Primary human AML blasts and cord blood were obtained from an experimental pathology cell bank of the State Key Laboratory of Experimental Hematology (SKLEH) from Institute of Hematology and Blood Diseases Hospital (IHBD). Sample acquisition was approved by the Institutional Review Boards at the IHBD. CD34eviously .Wild-type (WT) C57BL/6J mice were acquired from the animal facility of SKLEH. 8-week-old female mice were used for the experiments. Same batches of mice were randomly assigned to control and experiment groups. All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the IHBD, Chinese Academy of Medical Sciences.MLL translocation reported by Balgobind et al. [MLL-AF9 (MA9) and MLL-NRIP3 (MN3) AML mouse models were established as described previously , 56. MN3d et al. , and thed et al. . Serial + control mice and subjected to a limiting dilution series. The frequencies of LSCs were calculated according to Poisson statistics using ELDA software [Series doses of leukemic cells were collected from the BM of suv39h1 overexpression (SUV-OE) or experimental eGFPsoftware .+ cells from moribund SUV-OE or control mice were used. All procedures were performed as described previously [Enriched c-Kiteviously . The datt test was used for two groups’ analyses and one-way ANOVAS was for comparing more than two groups to evaluate the statistical significance. We have statistically compared the similar variance between the groups as well. Analyses were carried out using the Prism version 7.01 software .All experiments were repeated two to three time with the indicated numbers of mice. Sample size for each experiment was determined according to experience or the previously published papers. The investigator was not blinded during experiment or assessing the outcome. Distribution was tested using the modified Shapiro–Wilks method. When parameters followed Gaussian distribution, Student’s Supplemental material


In the secretory epithelial cells of the exocrine pancreas, such as acinar and ductal cells, intracellular Ca2+ elevation regulates digestive enzyme secretion in acini or fluid and ion secretion in ductal cells. Although Ca2+ is a uniquely versatile orchestrator of epithelial physiology, unregulated global elevation of the intracellular Ca2+ concentration is an early trigger for the development of acute pancreatitis (AP). Regardless of the aetiology, different forms of AP all exhibit sustained intracellular Ca2+ elevation as a common hallmark. The release of endoplasmic reticulum (ER) Ca2+ stores by toxins (such as bile acids or fatty acid ethyl esters (FAEEs)) or increased intrapancreatic pressure activates the influx of extracellular Ca2+ via the Orai1 Ca2+ channel, a process known as store-operated Ca2+ entry (SOCE). Intracellular Ca2+ overload can lead to premature activation of trypsinogen in pancreatic acinar cells and impaired fluid and HCO3- secretion in ductal cells. Increased and unbalanced reactive oxygen species (ROS) production caused by sustained Ca2+ elevation further contributes to cell dysfunction, leading to mitochondrial damage and cell death. Translational studies of AP identified several potential target molecules that can be modified to prevent intracellular Ca2+ overload. One of the most promising drugs, a selective inhibitor of the Orai1 channel that has been shown to inhibit extracellular Ca2+ influx and protect cells from injury, is currently being tested in clinical trials. In this review, we will summarise the recent advances in the field, with a special focus on the translational aspects of the basic findings.Intracellular Ca Interestingly, our results demonstrated that bile-acid-induced intracellular ROS production is remarkably different in pancreatic acinar and ductal cells. These findings provide a mechanistic explanation for the differing involvement of TRPM2 in the Ca2+ response generated by bile acid in these different cell types. The difference in ROS production may be explained by the difference in the mitochondrial mass in acinar versus ductal cells [2O2 and bile-acid-induced necrosis in pancreatic acinar cells. We also demonstrated that the rate of necrosis was significantly reduced in an experimental model of biliary pancreatitis in TRPM2 knockout mice as compared to wild type (WT) animals, consistent with in vitro observations. In agreement with our findings, Booth et al. documented that incubation of pancreatic acinar cells with TLCS in vitro leads to Ca2+-dependent necrosis, which is abolished by pre-treatment with BAPTA-AM [2+ are the major triggers of necrosis. Bile acids are known to inhibit cellular ATP production [m [m in TRPM2 KO and WT acinar cells. Genetic deletion of TRPM2 or removal of extracellular Ca2+ markedly reduced the decrease in Δψm, suggesting that TRPM2-mediated extracellular Ca2+ influx plays a crucial role in oxidative-stress-induced mitochondrial damage. In contrast, this protective effect was not detected in bile-acid-treated cells. This may be explained by the direct Ca2+-independent mitochondrial toxicity of bile acids [2+ elevation cannot completely abolish the toxic effects of bile acids. We did not observe mitochondrial fragmentation in our experiments, which has been previously linked to TRPM2 activation [In a recent study, we described the expression of TRPM2 in the exocrine pancreas and established the role of TRPM2 in biliary AP  Figure . TRPM2 wal cells ,61. As eBAPTA-AM . Using doduction  and decrction [m . Thus, wle acids . Based ole acids  and othele acids  we hypottivation . In addi2+ elevation plays a central role in the development of cellular injury in AP, regardless of the ethiology. In the polarised pancreatic acinar cells, the release of the ER Ca2+ stores in response to physiological agonist stimulation takes place at the apical granular region of the cell [3-evoked signals to global intracellular Ca2+ elevation is prevented by the mitochondria surrounding the apical region of the acinar cells [2+ ATPase (PMCA) pumps extrude the Ca2+ at the apical membrane [2+ stores activates the influx of extracellular Ca2+, which is mediated by store operated Ca2+ entry (SOCE), a process determined by the ER Ca2+ sensor Stim1 [2+ channel Orai1 [2+ store mobilisation induces a conformational change and translocation (puncta formation) of Stim1 to the ER–PM junctions [2+ entry can occur through the basal membrane of the pancreatic acinar cells [2+ entry was controlled with a Ca2+-containing pipette attached to the basal membrane in extracellular Ca2+-free media. After recharging the intracellular Ca2+ stores, repeated administration of ACh could, again, trigger an increase in intracellular Ca2+ concentration, starting at the apical secretory pole. These results suggest that the recharging of the apical ER Ca2+ stores depends on Ca2+ influx through the basal membrane. However, the cytosolic diffusion of Ca2+ from the basolateral to the apical pole could have potential unwanted effects. Therefore, the concept of Ca2+ tunneling through the lumen of the ER has been developed, which suggests that Ca2+ can diffuse more easily within the ER lumen compared to the cytosol [2+ oscillations in acinar cells.As highlighted above, sustained intracellular Cathe cell ,64  channel blocker developed by GlaxoSmithKline, which inhibits SOCE in a concentration-dependent manner and reduces the sustained Ca2+ elevation, trypsin activation, and acinar necrosis induced by FAEE. Moreover, the inhibition of Orai1 by selective inhibitors, GSK-7975A and CM-128 by CalciMedica, markedly impaired the extracellular Ca2+ influx and sustained Ca2+ overload in pancreatic acinar cells upon bile acid stimulation, significantly reducing pancreatic edema, inflammation and necrosis in experimental models of AP [2+ elevation, induced by SOCE-activated NFAT and transcription factor EB, led to the calcineurin-promoting transcription of chemokine genes and autophagy-associated genes [As highlighted, the release of the ER Caby SERCA . As a rein vitro . The autls of AP . Using Cls of AP . In addied genes . Orai1 wed genes . Orai1 p2+ elevation triggered by the bile acids and ethanol metabolites. TRPC3 deletion also prevented intracellular trypsin activation and inhibition of digestive enzyme secretion. These beneficial effects led to reduced severity of cerulein-induced AP in vivo [On the other hand, genetic deletion of TRPC3 was found to markedly reduce receptor-stimulated SOCE by about 50% and prevent sustained Ca in vivo . The sam in vivo .2+ signal development [2+ influx in acinar cells. Saraf overexpression reduced acinar Ca2+ influx and decreased the severity of AP. These results highlight the crucial role of Saraf in AP pathogenesis and identify this regulatory protein as potential target for therapy.Although Orai1 inhibition seems to be beneficial in AP, this channel is also necessary for T cell activation . Therefoelopment . In cont2+ signalling plays a central role in the pathogenesis of pressure-related pancreatic injury. They showed that incubation of mouse and human acinar cells with radiocontrast solutions triggered sustained intracellular Ca2+ elevation and activated the transcription factors NF-κB and NFAT via calcineurin. Genetic or pharmacologic inhibition of calcineurin prevented radiocontrast-induced pancreatic inflammation in mice. In addition, Wen et al. demonstrated that artificially increased intraductal pressures (100–150 mmHg for 10 min) are sufficient to trigger AP accompanied by increased serum amylase, cytokine release and tight junction integrity loss [2+ signalling, impaired mitochondrial function and calcineurin downstream activation , decreases the severity of AP [Toxin-induced mitochondrial injury is another key pathogenic step of AP development; therefore, protection of mitochondrial ATP production is another potential target . Maintenoduction . MPTP inoduction . Our groty of AP . We demo


Encephalitozoon cuniculi is an important microsporidian parasite with zoonotic potential. The present study highlights the impact of encephalitozoonosis on rabbit health in Egypt. Three rabbit farms in Giza, with a total of 16,400 rabbits were investigated due to occurrence of rabbits displaying clinical signs consistent with encephalitozoonosis.E. cuniculi were observed in the brain, retinal ganglion cells, kidneys, and liver. Transmission electron microscopy examination revealed the presence of different developmental stages of E. cuniculi in the brain and kidney. Presence of E. cuniculi was confirmed by conventional polymerase chain reaction using a universal 16S gene for Encephalitozoon spp. followed by sequencing and sequence analysis.Clinical signs observed during a 4 months observation period in 2018 included vestibular disease, paresis, limb paralysis, cataracts, phacoclastic uveitis, frequent urination, marked decrease in body weight and in some pregnant females, also repeated abortions. The total morbidity rates in adult and young rabbits were 76.7% and 81.5%, respectively. The highest mortality rate was recorded in offspring (12.3%), followed by dams (5.6%), and the lowest recorded mortality rate was in males (0.04%). Post-mortem findings included enteritis, pale enlarged kidneys, congested leptomeninges, focal brain necrosis, and endometrial congestion. Histopathological examination revealed nonsuppurative meningoencephalitis and glial nodules with central necrosis in the brain, vacuolation and necrosis of renal tubular epithelium, and corneal ulceration and ruptured lens capsule with fragmentation of lenticular fibres. E. cuniculi in rabbits was confirmed at three farms in Egypt. Nervous signs and ocular lesions were the most predominant findings in these farms.The presence of  Encephalitozoon cuniculi is an obligate intracellular spore-forming protozoan parasite belonging to the phylum Microsporidia, which contains approximately 1400 species distributed into about 200 genera. E. cuniculi can infect almost all invertebrates and vertebrates, as well as some protists [E. cuniculi, which was first identified in 1922 in a colony of research rabbits in the USA, is now recognized as a common pathogen and a significant cause of disease among pet rabbits [E. cuniculi has a zoonotic potential, especially for immunocompromised adults and for children [E. cuniculi has received increased attention [children . Therefottention , and hygttention .E. cuniculi takes 3 to 5 weeks to complete [E. cuniculi by 6 weeks [The life cycle of complete . Hosts acomplete . After icomplete . Infecte 6 weeks . The spoEncephalitozoon cuniculi infection of the central nervous system is associated with development of vestibular disease, which is characterized by torticollis, ataxia, paresis, nystagmus, seizures, and longitudinal rolling and the animal often becomes recumbent [ecumbent , 13. In ecumbent . In the ecumbent , 16. Kidecumbent . Microscecumbent .E. cuniculi genome is very small being only 2.9 Mb large and compressed. It harbours 2000 genes that are located within minute intergenic regions (the mean intergenic region is 80 bp) [E. cuniculi  [Transmission electron microscopy (TEM) can be used to confirm encephalitozoonosis by detecting the polar filament within spores and demonstrating ultrastructural features. TEM, along with newly applied molecular approaches, can contribute to the taxonomic organization of the microsporidia , 20. Pols 80 bp) . Analyse and IV) .Rabbit production recently has become well established in Egypt, but little is known about the exact rabbit numbers. Most of the rabbit population in Egypt is in the hands of smallholders, while the rest belongs to the commercial sector as a good source of protein . RabbitsE. cuniculi was not reported until 2011, when its presence was indicated by serological screening of the domestic rabbit population in northern Egypt, in Behera, Alexandria and Khafr El-Sheikh for E. cuniculi and Toxoplasma gondii [In Egypt, a gondii . HoweverE. cuniculi using pathology and molecular techniques and (3) to describe clinical signs, histopathological changes, morphology features and genetic characteristics of the parasite.The purposes of this study were (1) to investigate encephalitozoonosis in rabbit farms in middle Egypt; (2) to confirm the presence of Three commercial rabbit farms (A–C) in the Giza governorate in middle Egypt, with a total of 16,400 rabbits  founded the study population. Rabbits on these farms were investigated during a 4 months period in 2018.E. cuniculi infection  underwent a clinical examination. Of these 13,242 rabbits, 2680, 4658 and 5904 rabbits came from farms A, B and C, respectively.Rabbits that exhibited clinical signs suggestive of , were reared in a wire net cage system and fed commercial pellets. Rabbit handling procedures were performed in accordance with the applicable legislation of the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt (VetCU11112018015).Rabbits of different breeds, including New Zealand, Chinchilla, and CalifornianThe examined animals were classified into two age groups, i.e. young and adults. Young rabbits  were from 0 to 4 months old, whereas adults (n = 2072) were rabbits older than 4 months and up to 1 year old. Of the adult rabbits, 332 were males and 1740 females.The rabbits were visually assessed from a distance and any abnormal behaviour was recorded. Each visual assessment took about 5 min/cage. If abnormal behaviour was observed, the rabbit was taken out of the cage and examined according to . This cohttps://www.openepi.com/SampleSize/SSPropor.htm and a confidence level of 95%. However, the owners only allowed euthanasia of 341 rabbits. These were anaesthetized by intramuscular injection of ketamine (30 mg/kg) and xylazine (5 mg/kg) and then euthanized by exsanguination [Necropsy was performed on two subgroups of rabbits: (1) rabbits with severe clinical signs that were euthanized and (2) rabbits that had died shortly before a farm visit. The number of required euthanised rabbits was calculated to 376 by using the Open EPI free software uination . The groSamples for laboratory analyses were taken from 30 of the euthanized rabbits and from 20 of the recently dead rabbits from each farm giving a total number of 150 rabbits. These 150 rabbits were selected randomly from each farm among necropsied rabbits with prominent lesions indicating encephalitozoonosis. The following laboratory examinations were performed: (1) histopathology was performed on 10 rabbits from each of the three farms, (2) TEM was performed on seven rabbits, and (3) PCR was done on all 150 rabbits.Specimens of the brain, kidney, liver, and eye from the 30 rabbits were fixed in 10% neutral buffered formalin. The samples were then processed, paraffin-embedded, sectioned at 3 µm thickness and stained with haematoxylin and eosin and Giemsa stain .4) for 30 min at 4 °C, then washed in PBS (pH 7.3) for 1 h followed by dehydration in an ethanol gradient . The samples were then routinely processed and embedded in Epon-Araldite. Semithin sections (0.5 µm) were cut with a glass knife using a microtome  and collected on copper grids and then stained with 1% toluidine blue for 20 min, washed in distilled water and placed in Permount. The obtained slides were examined and photographed using an Olympus DP20 microscope  [Brain and kidney specimens from the seven rabbits were fixed in 2.5% glutaraldehyde, kept for 4 h at 4 °C, thoroughly rinsed overnight at 4 °C in 0.1 M phosphate buffer, fixed in 1% osmium tetraoxide  . UltrathDNA was extracted from brain, eyeball, liver, and kidney of the 150 rabbits using the DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions.E. bieneusi, E. intestinalis, E. cuniculi, E. hellem, Vittaforma corneae, and Pleistophora spp. using the forward primer F (5′-CAC CAG GTT GAT TCT GCC TGAC-3′) and reverse primer R (5′-CCT CTC CGG AAC CAA ACC CTG-3′) [PCR was performed for the target regions of the small subunit of 16S rRNA of zoonotic microsporidia, including  CTG-3′) . These p CTG-3′) .E. cuniculi.Among the PCR positive products, 10 samples representing three from each of farm A and B and four from farm C were purified using the QIAquick purification kit (Qiagen) according to the manufacturer’s instructions and sequencing was conducted using the Big Dye Terminator V3.1 Sequencing kit  with the forward and reverse primer for 16S rRNA. The obtained 10 nucleotide sequences were aligned with the sequences in GenBank using the NCBI BLAST server to confirm the identity with E. cuniculi 16S ribosomal RNA gene was deposited in GenBank, Accession number MK615616.The sequence (177 bp) of the E. cuniculi were downloaded from the NCBI GenBank and imported into BioEdit version 7.0.1.4 for multiple alignments using the ClustalW program in BioEdit. A similarity matrix was done using the DNASTAR program . The genetic distance values of species variations of E. cuniculi were analyzed with MegAlign project of DNSTAR software.The submitted gene sequence was compared with the aligned sequences available in the NCBI GenBank database. Publicly available 16S rRNA gene sequences for Signs of central vestibular disease were observed in 81.5% of the 13,242 rabbits and consisted of ataxia, circling or rotation around the longitudinal axis, head tilt  were present inside tubular epithelial cells and were released in the tubular lumen with little or no associated inflammatory reaction Fig. b. In semE. cuniculi were all found in brain sections. The meront stage was ovoid with a size of 2.21–3.1 × 3.175–3.8 μm, had an electron-lucent endospore layer and was surrounded by a dense outer coat. The sporont and sporoblast stages were also ovoid but measured only 1.91 × 2.3 μm and 1.85 × 2.2 μm, respectively . The sequence analyses from the ten selected positive samples were identical and revealed a 100% similarity to E. cuniculi were performed.  with the Donovan sequence of E. cuniculi (Accession number X98470) isolated from a human in the UK . However, the Donovan sequence of E. cuniculi was genetically more distant (GD 0.6) from E. cuniculi isolated from humans from the UK and USA .Inter- and intra-species analyses of genetic distance among the obtained sequence and other 11 aligned sequences available in the NCBI GenBank database of ed. Fig. . The gen UK Fig. . InterspE. cuniculi to rabbit farms threatens the fate of this industry due to high morbidity and a potential zoonotic risk [E. cuniculi spores further spread through the urine of infected rabbits [The rabbit industry in Egypt is at present flourishing and rabbits have become an alternative source for meat . The tratic risk . The hig rabbits . The dif rabbits . The hig rabbits .E. cuniculi infection in rabbits [E. cuniculi [The diagnosis of diseases causing nervous signs in rabbits are challenging as they may be caused by many conditions. In the present study, the association of nervous signs with ocular lesions suggested encephalitozoonosis. Nervous signs including central vestibular disease and unilateral ocular lesions are the main signs associated with  rabbits –35 and lcuniculi  as also cuniculi . Brain lcuniculi , 37, 38.Ocular lesions due to parasitic colonisation of the lens are very common in rabbits having encephalitozoonosis , 39, 40.E. cuniculi infection results in renal interstitial lymphocytic proliferation, necrosis of tubular epithelium, and interstitial fibrosis associated with presence of intact or ruptured parasitic cysts [Some rabbits showed symptoms of polyuria and urinary incontinence. ic cysts . In the Hepatic lesions were restricted to mild periportal mononuclear inflammatory cell infiltration and mild vacuolar degeneration but the parasite was observed by Giemsa stained sections. In contrast, other studies have reported moderate to severe lesions in the liver, including periportal nonsuppurative interstitial infiltration, fibrosis and necrosis of the periportal area .E. cuniculi stages in the brain and kidneys of rabbits has not been reported previously. The different intracellular stages of E. cuniculi can be discriminated from each other because the parasite undergoes morphological changes [E. cuniculi were found to have a nucleus, lamellar polaroplast, a single row of coiled polar tubes and surrounding spore wall similar to the findings of Vavra and Larsson [E. cuniculi ranged from 1.75 × 3.3 μm, whereas the spore of Encephalitozoon spp. in another study was 1.0–1.5 × 2.0–2.5 μm [E. cuniculi from all other Encephalitozoon spp. is the parasitophorous vacuole (PV). E. cuniculi is the only species of microsporidia known to exist in a septated PV that separates each developing spore [To the best of our knowledge, the ultrastructure of  changes . The mat Larsson , and Hab Larsson . The mat0–2.5 μm . The var0–2.5 μm . The maing spore .Encephalitozoon spp. was done by amplification of the specific 16S gene [. The results confirmed E. cuniculi infection in all examined brain, kidney, eye and liver tissues. This result is in accordance with other studies that have suggested that encephalitozoonosis is a systemic disease affecting many organs [E. cuniculi presented 99.4% similarity with E. cuniculi (Donovan sequence) isolated from human thus indicating a potential zoonotic transmission of E. cuniculi circulating in rabbits farms in Egypt.Molecular detection of the 16S gene . The resy organs . The ideEncephalitozoonosis in farmed rabbits was very prevalent in three farms investigated in middle Egypt and associated with a high mortality/culling rate. Sequence analyses revealed a potential zoonotic risk of this infection in Egypt.


Moreover, NOM adhesion on the UF membrane was investigated using atomic force microscopy (AFM) with colloidal probes and a quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicated that the adhesion forces at the NOM-membrane interface increased in the presence of background cations, particularly Ca2+, and that the amount of adhered NOM increased due to reduced electrostatic repulsion. However, the membrane permeability was almost not affected by background cations in the pore blocking-dominated phase but was aggravated to some extent in the cake filtration-governed phase. More importantly, the irreversible NOM fouling was not correlated with the amount of adhered NOM. The assumption for membrane autopsies is doubtful that retained or adsorbed organic materials are necessarily a primary cause of membrane fouling, particularly the irreversible fouling.To understand impacts of organic adhesion on membrane fouling, ultrafiltration (UF) membrane fouling by dissolved natural organic matter (NOM) was investigated in the presence of background cations (Na Because of outstanding advantages such as low-pressure running, small footprint, and high compatibility with other water purification technologies, ultrafiltration (UF) is increasingly applied in potable water treatment to meet stringent requirements for water quality. In particular, UF can totally retain particles, bacteria, and pathogens, therefore substantially improving water security . HoweverNOM fouling of UF membranes is very complicated due to the heterogeneous composition of NOM which is specific to water quality, type of membrane material, running conditions, and solution chemistry ,4. On th+ and Ca2+, in surface water play a significant role in the formation of NOM fouling. Essentially, the presence of cations can weaken electrostatic repulsion at interfaces via charge screening, double layer compression, bridging effects, or complexation according to the classical Derjaguin–Landau–Verwey–Overbeek (DLVO) theory [2+ and Mg2+) at different concentrations, a linear relationship was observed between the irreversible fouling rate and concentration of cations [2+ concentration in a range of 0–10 mM [In addition to NOM, coexisting cations, particularly Na) theory . On the ) theory ,11,12. F cations ,14. On t cations . In a st 0–10 mM . Hence, +, Ca2+, and Mg2+. Progress has been made in understanding the impact of cations on the migration of foulants and fouling behaviors during filtration, particularly for polysaccharides. Biphasic fouling behavior has been demonstrated with increasing concentrations of cations. At low concentrations, Ca2+ ions preferentially interact strongly with guluronate functional groups, forming a highly interconnected gel assemblage with egg-box structure; despite the highly porous structure, the fouling layer shows very high resistance to water flow due to the accelerated crosslinking of the alginate gel layers [Recently, further investigations on the impact of cations on UF membrane fouling by NOM have been performed in a broad concentration range for cations such as Nal layers . With anl layers ,19. Morel layers . Miao etl layers  reportedl layers  compared+ and Ca2+ in surface water.In this work, NOM in surface water was extracted by reverse osmosis concentration and dialysis desalination was used to remove background ions. Filtration tests were performed with the reconstituted NOM solutions; the removal of NOM and fouling extent during filtration were assessed. Moreover, the membrane-NOM interaction and the adhesion of NOM onto the membrane were investigated using AFM coupled with a probe and a quartz crystal microbalance with dissipation monitoring (QCM-D), respectively, to obtain more insight into the fouling behavior of NOM at typical concentrations of Na−1 and values of UV absorbance at 254 nm (UV254) from 0.172 to 0.218 cm−1. Other water quality indexes for the raw water are found in Natural water was sampled from the Songhuajiang River in Harbin, China. The raw water was immediately filtered through a 0.45 μm mixed cellulose filter  to remove solid particles and NOM colloids with large size. The raw water quality is summarized in Flat (PES) UF membranes (OM100076) were purchased from Pall Co. . The membranes were made of polyethersulfone and had a nominal molecular weight cutoff of 100 kDa. A bench-scale filtration system, which was introduced elsewhere , was use2 to understand the effects of background ions in surface water on membrane fouling. The concentrations of Na+ and Ca2+ were 15 and 18 mg/L, respectively, which were identical to the concentrations in the surface water. The membrane fouling was assessed by three types of resistance, i.e., intrinsic membrane resistance, reversible resistance, and irreversible resistance, which were related to the pore characteristics, cake layer formation due to foulant deposition, and pore blocking due to foulant adhesion inside membrane pores, respectively. The fouling resistance was evaluated via the resistance–in-series model.The feedwater was prepared by diluting the concentrated natural water to a DOC concentration of 6 mg/L. The feedwater and permeate were sampled to investigate the rejection of NOM by UF. In addition, the feedwater was spiked with NaCl or CaClP is the operating pressure (Pa), μ is the dynamic viscosity (Pa s), J0 is the initial flux for the UF membrane, eJ is the flux at the end of filtration, and wJ is the clean water flux of the backwashed membrane.Briefly, filtration of 300 mL milli-Q water under 60 kPa was performed in each filtration test to determine the initial membrane flux. Subsequently, 400 mL water was fed into the UF cell and 350 mL was filtered at a constant pressure of 60 kPa. The flux was monitored to investigate the membrane fouling. When filtration was terminated, the fouled membranes were turned over and flushed with Milli-Q water in the cell for 2 min under 60 kPa. Finally, filtration of milli-Q water was performed again with the backwashed membranes to determine fouling reversibility. Each filtration test was performed in triplicate. The intrinsic membrane resistance, reversible resistance, and irreversible resistance during filtration were obtained via Equations (1)–(3) .(1)Ri=ΔMembrane-based size fractionation and nonionic resin isolation were used to characterize the NOM in the surface water. The details for the methods were induced elsewhere . Briefly−1 were adopted. Measurements were taken at room temperature (21 ± 1 °C). The absorbance at 254 nm of each sample was measured using a UV/Vis spectrophotometer  beforehand. Samples with UV254 values exceeding 0.05 cm−1 were diluted with Milli-Q water to minimize the inner filter effect, and the dilution factor of each sample was recorded [A fluorescence spectrophotometer  was used to obtain three-dimensional (3D) fluorescent excitation-emission matrix (EEM) fluorescence spectra of the water samples. The FEEM measurements were performed at excitation wavelengths of 220 to 450 nm in 5 nm increments and at emission wavelengths of 250 to 550 nm with 1 nm increments. A photomultiplier tube (PMT) voltage of 700 V and a scanning speed of 2400 nm minrecorded . The flurecorded .−1) was used to access interfacial adhesion force between NOM and the UF membrane according to Miao et al. [An atomic force microscope  in conjunction with a SiN cantilever   combined with a PES-coated sensor crystal. Specifically, a small amount of epoxy resin was coated on a clean sensor crystal via a spin coater . The coating was performed at a speed of 3000 rpm for 60 s. A clean membrane was cut and pasted onto the coated sensor crystal. Then, the membrane-coated sensor crystal was rinsed with milli-Q water and dried under pure nitrogen gas for approximately 2 min. For adhesion tests, the sensor crystal was installed in the QCM-D, and milli-Q water flowed through the measurement chamber until a stable baseline was achieved. The temperature was maintained at 23 °C and the flow rate was set at 0.1 mL/min with a peristaltic pump . After that, the testing solution was shifted to the reconstituted NOM solutions. The measurement consisted of two parts, i.e., an adsorption process and desorption process. The NOM solutions and milli-Q water were used as the testing solutions in the adsorption process and a desorption process, respectively. During measurements, both the frequency and dissipation were monitored and recorded to assess the amount of adsorbed foulants and the structure of the adsorption layer, respectively. According to the Sauerbrey relationship (Equation (4)), the crystal sensor frequency is negatively correlated with the adsorbed mass; the dissipation reflects the energy dissipation of adsorbed foulants during deposition and provides insight into the structure of deposit layers  as:(4)Δm−1) was obtained with an R2 value of 0.999. An ultraviolet (UV) spectrometer  was used to measure the UV254 values of the water samples. The water samples were prefiltered by a 0.45 μm filter to remove suspended particles prior to measurements. The filters were cleaned in boiled water for 20 min to minimize leached organics, and the first 10 mL filtrate was discarded. For SEM observation, membrane samples were coated with an approximately 20 Å Au/Pd layer using a precision etching coating system , and then observed under SEM . The membrane samples were fractured in liquid nitrogen and naturally dried under ambient conditions.A total organic carbon (TOC) analyzer  was used to determine the concentrations of DOC in the water samples. Calibration was performed with potassium hydrogen phthalate and a linear correlation  of the quartz crystal and the energy dissipation (ΔD) describe the amount of adsorbed organics and the structural properties of the fouling layer, respectively [F substantially increased as the reconstituted NOM solution was fed and gradually reached a plateau with a ΔF value of 4.35 Hz, implying that the adhesion of NOM on the PES membrane was saturated. In the presence of Na+ and Ca2+, the ΔF values increased to 5.34 and 7.30 Hz, respectively. This is consistent with the adhesion force results, in which the maximum adhesion force was observed in the presence of Ca2+. It can be inferred that background cations, particularly Ca2+, substantially enhanced the adhesion of NOM on the PES membrane due to reduced electrostatic repulsion. Wang et al. [2+ and Mg2+ promoted the assembly and deposition of humic acids on the PES surface, which resulted in accelerated foulant accumulation. When the test solution shifted to milli-Q water, the ΔF values in all three cases started to decrease, indicating the desorption of organics from the membrane surface. However, the increased ΔF values could not be totally restored. The residual ΔF values were 0.43 Hz in the absence of background cations, implying that some NOM adhesion was irreversible. In the presence of Na+ and Ca2+, the residual ΔF values were 1.11 and 2.46 Hz, respectively. The irreversible adhesion of NOM was aggravated in the presence of background ions.The adhesion of the reconstituted NOM on the PES membrane was assessed by QCM-D; the changes in the oscillating frequency . This is attributed to reduced electrostatic repulsion and increased organic adhesion on the membrane surface in the presence of cations, which is generally consistent with the QCM-D results.To gain more insight into NOM removal, FEEM was used to characterize fluorescent components in the feed and UF permeate. The FEEM spectra for the feedwater and permeate samples are shown in + and Ca2+, the flux decline in the initial phase was almost identical to that with NOM alone, regardless of the increased NOM adhesion  [adhesion . The prong layer  in the png layer , and thu (<1 mM) . Overall11 m−1, respectively, during filtration of the reconstituted NOM solution. The reversible resistance increased to 14.91 × 1011 m−1 and 18.42 × 1011 m−1 in the presence of Na+ and Ca2+, respectively, which is consistent with the variations in membrane flux. It is known that Ca2+ can preferentially reduce foulant–foulant rather than membrane–foulant electrostatic repulsion, resulting in enhanced aggregation of foulants, and thus formation of a more porous cake layer [2+. Moreover, as shown in 2+ concentration, because the initial increase in Ca2+ concentration facilitates intermolecular rather than intramolecular binding, resulting in a more homogeneous gel layer and higher resistance, whereas excess Ca2+ induced the formation of alginate calcium clusters and less filtration resistance due to the macrovoid pores in the interclusters [2+ concentration in the surface water was not sufficient to cause intramolecular binding for organics, therefore, NOM fouling was aggravated in the presence of Ca2+ in this work. In another similar work, reversible membrane fouling by humic acids was also aggravated in the presence of divalent cations (Ca2+ and Mg2+) at concentrations lower than 10 mg Ca2+ mg−1 DOC [11 m−1 in the presence of Na+ and Ca2+, respectively, which was inconsistent with the increased adhesion force (As shown in ke layer . Howeverclusters ,20. The mg−1 DOC . Regardion force  and incron force . This re+ and Ca2+, thick and dense fouling layers were observed on the membrane surface, which was consistent with the increased reversible fouling resistance in the presence of background cations. This result further verified the unfavorable impact of background cations at low concentrations on membrane permeability. Moreover, the fouled membranes were characterized by FTIR, and the results are shown in −1. Beyond this range, typical peaks at approximately 3300 cm−1, which refer to the bending vibration of adsorbed molecular water and the stretching vibration of hydroxyl groups [l groups , were ob+ and Ca2+ at typical concentrations in surface water, and the amount of adhered NOM increased due to reduced electrostatic repulsion, resulting in improved NOM retention. The membrane permeability was minimally affected by background cations in the pore blocking-dominated phase but aggravated to some extent in the cake filtration-governed phase. The presence of Na and Ca2+ increased the adhesion of NOM on the membrane, but irreversible NOM fouling was not correlated with the amount of adhered NOM.In this work, membrane fouling was systematically assessed in the presence and absence of background cations at typical concentrations in surface water. The NOM-membrane adhesion forces were increased in the presence of Na


Compelling evidence indicate that chloroplasts can generate specific stromal Ca2+ signals and contribute to the fine tuning of cytoplasmic Ca2+ signaling in response to different environmental stimuli. The recent set up of a toolkit of genetically encoded Ca2+ indicators, targeted to different chloroplast subcompartments  has helped to unravel the participation of chloroplasts in intracellular Ca2+ handling in resting conditions and during signal transduction. Intra-chloroplast Ca2+ signals have been demonstrated to occur in response to specific environmental stimuli, suggesting a role for these plant-unique organelles in transducing Ca2+-mediated stress signals. In this mini-review we present current knowledge of stimulus-specific intra-chloroplast Ca2+ transients, as well as recent advances in the identification and characterization of Ca2+-permeable channels/transporters localized at chloroplast membranes. In particular, the potential role played by cMCU, a chloroplast-localized member of the mitochondrial calcium uniporter (MCU) family, as component of plant environmental sensing is discussed in detail, taking into account some specific structural features of cMCU. In summary, the recent molecular identification of some players of chloroplast Ca2+ signaling has opened new avenues in this rapidly developing field and will hopefully allow a deeper understanding of the role of chloroplasts in shaping physiological responses in plants.Calcium has long been known to regulate the metabolism of chloroplasts, concerning both light and carbon reactions of photosynthesis, as well as additional non photosynthesis-related processes. In addition to undergo Ca However, in the last few years the increasing availability of specifically targeted Ca2+ reporters has rapidly expanded the possibility of accurately monitoring organellar Ca2+ dynamics. The set up of a toolkit of aequorin-based probes targeted to the different subcompartments of chloroplasts  has allowed for the elucidation of stimulus-specific intra-organellar Ca2+ signals and their contribution to fine-tuning cytosolic Ca2+ signatures , as well as their contribution to the modulation of intra-chloroplast Ca2+ signals  of free Ca2+ elevation in the cytosol ([Ca2+]cyt) acts as a signal to be implemented in the transducing machinery of the cell. Different stimuli are followed by different Ca2+ signatures, leading in turn to different specific responses, in terms of gene expression, protein activity and localization. The Ca2+ signature is shaped by the activity of Ca2+-permeable channels and transporters regulating the ion entry into and exit from the cytosol, respectively. Ca2+-permeable channels are grouped in five families: cyclic nucleotide-gated channels (CNGCs), glutamate receptors-like channels (GLRs), two-pore channels (TPCs), mechanosensitive channels (MCAs), hyperosmolality gated channels (OSCAs) , P2A-type calcium cation-transporting ATPase (ECAs) and P2B-type calcium cation-transporting ATPase (ACAs)   . Ca2+ tre (ACAs) . Other Cchanger)  and CaCA2+ signal is mediated by Ca2+ -dependent/binding proteins. The Arabidopsis genome encodes for 250 proteins harbouring at least one Ca2+ binding domain (EF-hand), hence acting as putative Ca2+ sensors [e.g.2+-dependent protein kinases (CPKs) all harbour EF hand motifs. Ca2+ sensors directly (CPKs) or indirectly  [e.g.  and/or protein subcellular localization (e.g. transcription factors). The redundancy of sensor isoforms allows the discrimination between different signals and carry the specificity of the message brought by the Ca2+ signature.The transduction of the Cas) [e.g. ] modulat2+-binding proteins acting as buffers in the chloroplast have not yet been identified. Nevertheless, organellar Ca2+ buffering mechanisms are likely to play an essential role, generating heterogeneity in local Ca2+ concentrations inside chloroplasts. How Ca2+ is stored in the chloroplast remains an open question for future investigations, aimed to unravel whether Ca2+ interacts with specific Ca2+ binding proteins or with the thylakoid surface, which harbours a significant amount of phosphorylated proteins that have been suggested to bind calcium ions  have been found to mediate Cay AtMCU1  and AtMCy AtMCU1 . In animy AtMCU1  is respo as well . In partolerance .2+ handling .Querying the protein databases Uniprot , SUBA4  and Aram2+ channels/transporters: 6 are confirmed to be located in plastid membranes either by biochemical and cell biology methods or by mass spectrometry. Among them, for AtcMCU, AtGLR3.4 and AtGLR3.5 a role in stress response was suggested. Altogether, these channels/transporters can be involved in the formation of the plastidial Ca2+ transients, along with the putative calcium-transporting protein PAM71/BICAT  no proteins with unique localization to chloroplasts are present. Thus, the mechanism by which dual localization occurs awaits clarification.As mentioned above, AtcMCU is one of the very few molecular entities among the plastidial Ca isoform , Arabido isoform , AtMCU2/ isoform , AtMCU3/ isoform . For AtM isoform . Thus, e2+ uptake by cMCU, as demonstrated by mutagenesis studies (D107A/E118K mutant) and Ca2+ uptake assays in an aequorin-based E. coli system  of AtcMCU harbours motifs rich in acidic residues, one of which (107-118) playing a role in Cai system . Two groaequorin  or the fr GCaMP2 . This va2+-permeable channels on their own in contrast to vertebrates, where the uniporter is a complex (MCUC) consisting of multiple subunits, including: 1) the channel forming unit (MCU) with two transmembrane segments and a conserved DXXE sequence forming the Ca2+ selectivity filter   froma crassa ; 3) fromfischeri ; and froeuropaea . All theuman MCU . This si2+ flux, without the need of EMRE, as confirmed by different groups  to LN.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.


Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy. Protein phosphorylation is an essential post-translational modification in most cellular processes, making of protein kinases promising therapeutic targets for a wide variety of disorders, including cancer ,2. AmongProtein kinase CK2 has been linked to basically all the hallmarks of malignant diseases ,8. AccorAcute myeloid leukemia (AML) is one of the most frequent hematologic malignancies and high-expression of CK2α subunit has been connected to a worse prognosis in AML patients with normal karyotype ,17. ActuIn recent years, quantitative phosphoproteomic approaches have been useful to explore the cellular response to kinase inhibition in different types of cancer cells . In factv/v) fetal bovine serum  and 50 µg/mL gentamicin . Cells were maintained under standard cell culture conditions.Human AML cell lines HL-60 and OCI-AML3 were originally obtained from the American Type Culture Collection  and the German Collection of Microorganisms and Cell Cultures , respectively. Both cell lines were cultured in RPMI 1640 medium  supplemented with 10%  were treated or not with 5 µM CX-4945  for 8 h. After collected by centrifugation and washed with PBS, cells were resuspended in lysis buffer containing 2% SDS and 50 mM DTT. Samples were boiled at 95 °C for 10 min and proteins were extracted by multienzyme digestion filter-aided sample preparation (MED-FASP) with overnight lys-C and tryptic digestions . Th. Th43]. CK2 has direct interactions with 13 and 27 kinases related to the phosphoproteomic profile identified in HL-60 and OCI-AML3 cells, respectively . Such kiHighly connected regions in the kinome networks associated to HL-60 and OCI-AML3 phosphoproteomic profiles were identified using MCODE algorithm . ClusterImportantly, previous phosphoproteomic results from primary AML cells have indicated that at the diagnosis time, patients that relapse after chemotherapy had a higher CK2, MAPK and CDK activity in comparison with patients which have free-relapse evolution . HoweverBesides the bona fide CK2 substrates annotated in iPTMnet and KEA databases ,42, addiFirst, differentially phosphorylated proteins identified in AML cells were searched as candidate CK2 substrates using NetworKIN database . Such daTaking into account the four levels of predictions  are reported for the first time. Coilin protein is an integral component of Cajal bodies-subnuclear compartments, whereas IMPDH2 catalyzes the first and rate-limiting step for de novo guanine nucleotide biosynthesis pathway . InteresPhosphoproteins identified as candidate CK2 substrates are related to transcription, mRNA splicing, rRNA processing, translation, DNA repair and cell cycle in both AML cells lines . HoweverCK2 inhibition in AML cells could impact the transcriptional machinery by modulating the phosphorylation of several candidate substrates. Such CK2 candidate substrates in OCI-AML3 phosphoproteomic profile are centered around the RNA polymerase II subunit A (POLR2A) according to protein-protein interactions gathered from STRING database  47]. Th. Th47]. Substrates related to transcription include bona fide CK2 targets such as the non-histone chromosomal protein HMG-14 (HMGN1) and the high mobility group protein HMG-I/HMG-Y (HMGA1) ,78,79. TThe most densely down-phosphorylated protein among the candidate CK2 substrates is the protein IWS1 homolog (IWS1) which was identified with eight phosphopeptides in OCI-AML3 cells . This prCK2 phosphorylation of proteins related to rRNA processing and translation has been well documented . Among tAnother function attributed to CK2 is the regulation of the cellular DNA damage response . After CWorthy of note, we identified members of the heat shock protein 90 (HSP90) chaperone proteins differentially modulated in OCI-AML3 phosphoproteomic profile. CK2 mediated phosphorylation of HSP90 is required for its chaperone activity toward client kinases, some of them involved in human cancers . PhosphoIn summary we found that the phosphoproteomic profiles modulated after CK2 inhibition with CX-4945 in AML cell lines, contain protein mediators of signaling pathways and biological processes previously described in primary AML cells  22,23,5,68. TherOur study provides the first quantitative phosphoproteomic analysis exploring the molecular impact of the ATP-competitive CK2 inhibitor CX-4945 in human cell lines representing two differentiation stages and major AML subtypes. Here, we identified a total of 273 and 1310 unique phosphopeptides as significantly modulated in HL-60 and OCI-AML3 cells, respectively. Modulated phosphopeptides are mainly related to mRNA processing and splicing, response to DNA damage stimulus, protein sumoylation and regulation of viral processes. In addition, the network analysis illustrated how the relationship of CK2 with other kinases could orchestrate the perturbation of AML cells phosphoproteome. In this complex cellular response, phosphorylation mediated by other kinases besides CK2 could be interpreted as a consequence of signal propagation downstream of CK2 inhibition, rather than off-targets effects. Additionally, using database mining and prediction tools, in HL-60 cells we identified 64 phosphosites on 53 proteins as high confidence CK2 substrates responsive to CX-4945, whereas 168 phosphosites on 120 proteins were identified in OCI-AML3 cells. Such substrates not only explain the variety of cellular effects exerted by CX-4945, but also reinforce the instrumental role of protein kinase CK2 in AML biology. Besides, selected cells lines not only represent two major AML subtypes, but also different niches that can be found in the clinical practice if we consider the differential sensitivity to CK2 inhibition with CX-4945 displayed by these cell lines. Finally, our results, in conjunction with previous findings in primary AML cells, support the suitability of using CK2 inhibitors for AML targeted therapy, a pharmacologic approach that could significantly improve the outcome in AML patients.