CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia  and in 8% of pediatric cytogenetically normal acute myeloid leukemia  subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins.CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile.We exposed to GANT61 AML cell lines and primary cells positive and negative for GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions.As compared to AML cells without CBFA2T3-GLIS2 fusion gene in pediatric AML.Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the The online version of this article (doi:10.1186/s13045-017-0396-0) contains supplementary material, which is available to authorized users. CBFA2T3-GLIS2 fusion gene deserves particular interest, being associated with a grim prognosis in all the reports published so far [CBFA2T3-GLIS2 is associated with upregulation of both Hedgehog (HH) and bone morphogenic protein (BMP) signaling [Pediatric acute myeloid leukemia (AML) carrying d so far –3. The id so far –3. The eignaling , 4.CBFA2T3-GLIS2 fusion gene in pediatric AML.The protein GLIS2 shares a highly homologous zinc finger domain with members of the GLI proteins, the final effectors of classic Hedgehog pathway. GANT61 is a GLI inhibitor showing a potent effect on the inhibition of transcription activity of GLI proteins, blocking their binding to DNA –8. ConsiCBFA2T3-GLIS2 fusion gene.In the present study, we investigated the in vitro effects of GANT61 on AML cell lines and primary cells from AML patients harboring the CBFA2T3-GLIS2 fusion gene is reported in Additional file The materials and methods are detailed in Additional file CBFA2T3-GLIS2 fusion gene have a higher sensitivity to GANT61 than other AML cell lines without this genetic aberration  was significantly downregulated (p < 0.05) , as for example, genes involved in cell cycle control, such as CDC25A, CDC7, CDCA2, and CCNA2. The expression of these genes is required for progression through cell cycle, and their expression is aberrant in AML, as well as in other malignancies [KIF14, MELK, MCM10, NUF2) and epigenetic regulators namely DNMT1 and DNMT3B were also present. Considering the particular interest of these DNA methyltransferase genes, we performed chromatin immunoprecipitation (ChIP) analysis using a CBFA2T3-specific antibody on WSU-AML and M07e cell lines. Our findings showed that CBFA2T3-GLIS2 fusion protein directly binds to the proximal promoter of DNMT1 and DNMT3B, positively regulating their expression  Fig. . Moreovegnancies , 10. In ion Fig. . OverexpHHIP, PTCH1, GLI1) have been described; we hypothesized that CBFA2T3-GLIS2 chimeric protein could regulate wild-type GLIS2 protein with the same feedback mechanism. Western blotting analysis confirmed the presence of chimeric protein and showed also the presence of wild-type GLIS2. These results may point to the role of the fusion protein in regulating wild-type GLIS2. Following GANT61 treatment, the expression of both proteins was decreased with respect to untreated samples, suggesting that GANT61 treatment targeted both CBFA2T3-GLIS2 fusion protein and also wild-type GLIS2 expression .Since in the classic Hedgehog signaling pathway, several target genes involved in feedback mechanisms . The combined treatment showed a higher cytotoxic effect when compared to each single drug, and the two inhibitors displayed a synergistic effect on cell growth, as indicated by the CI value  inhibitors . We ther1:10 GANT1:MK-0457CBFA2T3-GLIS2 fusion gene. Although further investigation will be required to confirm these results, our experience with GANT61 represents a preliminary background for further evaluating in vivo the inhibition of GLIS2-mediated transcription in AML harboring the CBFA2T3-GLIS2 fusion gene.This work provides preliminary data from preclinical in vitro and ex vivo studies targeting pediatric AML with


It is believed that there are key differences in the genomic profile between adult and childhood acute myeloid leukemia (AML). Relapse is the significant contributor of mortality in patients with AML and remains as the leading cause of cancer death among children, posing great challenges in the treatment of AML. The knowledge about the genomic lesions in childhood AML is still premature as most genomic events defined in children were derived from adult cohorts. However, the emerging technologies of next generation sequencing have narrowed the gap of knowledge in the biology of AML by the detection of gene mutations for each sub-type which have led to the improvement in terms of prognostication as well as the use of targeted therapies. In this review, we describe the recent understanding of the genomic landscape including the prevalence of mutation, prognostic impact, and targeted therapies that will provide an insight into the pathogenesis of AML relapse in both adult and childhood cases. Acute myeloid leukemia (AML) is a blood cancer which characterized by the infiltration of proliferative, clonal, abnormally differentiated, and occasionally poorly differentiated cells of the hematopoietic system , , or FLAG-IDA  followed by allogeneic hematopoietic stem cell transplant (HSCT) whereas low intensity therapy or best supportive care will be offered to adult patients who are physically weak and could not tolerate high dose therapies  has revealed the existence of two major patterns of clonal evolution underlying AML progression. The first pattern suggested that the founding clone gained additional mutations and evolved into the relapse clone, whereas the second pattern suggested that the subclone of the founding clone which survived initial therapy gained additional mutations and expanded at relapse   gene encodes for a phosphoprotein , isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and tet methylcytosine dioxygenase 2 (TET2) are involved in regulating the methylation of the genome  was detected in adult AML via the WGS and WES approach  as compared to childhood AML (1–7%)  encodes for a receptor tyrosine kinase which is a membrane-bound receptor with an intrinsic tyrosine kinase domain. Internal tandem duplication (ITD) of the juxtamembrane domain and point mutations of the tyrosine kinase domain (TKD) in FLT3 have been shown to result in the constitutive activation of the receptor kinase activity  was higher compared to FLT3 TKD (10 and 7%)  and Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) belong to the RAS GTPase family that encode a membrane-associated guanosine nucleotide phosphate (GTP) binding proteins, and are involved in regulating signal transduction upon binding of ligand to a variety of membrane receptors  and childhood AML (12–13%) but KRAS mutations appeared to be more prevalent in adult AML than childhood AML (12 vs. 3–7%)  Oncogene Homolog (KIT) is involved in hematopoiesis as well as proliferation and regulation of cell survival. The frequency of KIT mutation in adult AML was reported lower as compared to childhood AML (4 vs. 12–20%)   is a transcription factor that is involved in regulating the differentiation of neutrophils. CEBPA mutations were mostly located at the N-terminal domain (NTD) and bZip domain   had favorable outcomes with prolonged survival after relapse, with a 67% 10-year overall survival rate  encodes for a core-binding factor which binds to the core element of many enhancers and promoters. RUNX1 mutations are more prevalent in AML without complex karyotype, with 5–18% in adult AML   and Additional Sex Combs Like 2 Transcriptional Regulator (ASXL2) encodes a dual-function chromatin-binding protein, which acts as a transcription activator or repressor   in childhood AML and increases with age  and childhood (26%) AML is almost similar  encodes a POZ/zinc finger transcriptional repressor , and associated with poorer prognosis, with an overall 2-year survival rate of 25.6%   or formerly known as Mixed-Lineage Leukemia (MLL) is involved in regulating gene expression during early development and hematopoiesis. MLL-PTD was found mutated in 6% of adult AML patients  was reported in childhood AML  was noted in MLL-PTD AML patients with DNMT3A mutations compared to those without DNMT3A mutations  Methyltransferase 2A (EZH2) is a histone methyltransferase which is involved in transcriptional repression by depositing histone H3 lysine 27 (H3K27) and also plays a pivotal role in hematopoiesis  and AMKL with Down Syndrome (DS-AMKL), 16 and 33% respectively , is the most well-studied tumor suppressor gene and is involved in cell cycle regulation in response to cellular stress. TP53 is frequently mutated in AML patients with complex karyotypes, with an incidence rate of 69–73%  is a transcription factor involved in urogenital development and was found overexpressed in AML with a role in promoting leukemogenesis  , the 4-year overall survival rate was only 22%, and the relapse rate was relatively high (82%)  acts as a tumor suppressor gene. PHF6 non-sense and frameshift mutations have been shown to result in loss-of-function alleles  received approval by the US Food and Drug Administration (FDA) for the treatment of adult patients with newly diagnosed L Levis, . It is aFLT3 mutated AML patients , is a relatively less selective compound compared to midostaurin, and its usage has not been able to improve the survival in FLT3/ITD patients (NCT02668653), and the QuANTUM-R trial to compare quizartinib monotherapy vs. conventional salvage therapies among relapsed/refractory FLT3-ITD mutated patients (NCT02039726), the results showed a higher composite complete remission (CRc) rate in both older and younger patients with FLT3-ITD mutated relapsed/refractory AML .Quizartinib (AC220) is a second generation FLT3 inhibitor that exhibits low nanomolar potency in biochemical and cellular assays, and has exceptional kinase selectivity   and gilteritinib (5 months)  Levis, .IDH2 mutated adult AML patients. Enasidenib (AG-221/CC-90007) is a first-in-class, oral, selective inhibitor of mutant-IDH2 enzymes and assessment outcomes in the largest relapsed or refractory mutant-IDH2 AML patient shows a 40.3% overall response rate with 9.3 months median overall survival, and 19.7 months overall survival for those who attained complete remission  and IDH305 (NCT02381886) in relapsed or refractory AML patient showed that both drugs were well-tolerated and the overall response rate was 36 and 33% respectively  is a potent and selective small-molecule inhibitor of MEK  but require further validation  inhibitor has been used in The emergence of these molecular targeted therapies has contributed to the improvement in the treatment of AML patients specifically resulting in better response rate and overall outcomes, with less toxicity than standard cytotoxic therapy. The molecular targeted therapies for the management of AML as discussed in this review are illustrated in Figure NPMI, IDH2, and CEBPA mutations predicted a favorable prognosis whilst KRAS and NRAS mutations had little clinical impact. Also, mutations in DNMT3A, IDH1, TET2, FLT3 ITD, MLL, and EZH2 are associated with poorer prognosis, whilst mutations in RUNX1, WT1, and TP53 are predictive of the worst outcomes and increased relapse risk in both adult and childhood AML. Meanwhile, the prognostic impact of KIT, ASXL1/ASXL2, and BCOR mutations between adult and childhood AML showed a contradiction. The implications of FLT3-TKD, SMC3, RAD21, STAG2, and PHF6 mutations in AML remain unclear and their usefulness as predictive biomarkers of relapse warrants further investigations. Taken together, gene mutations provide additional valuable clinical information which could help to refine the risk or prognostic classification of AML and to guide risk-adapted therapies. Discovery of gene mutations which are uniquely acquired or enriched in relapse AML may pave new ways toward the development of novel therapeutic therapies and precision medicine for AML.The breakthrough in genomic technologies, especially NGS, has enhanced our understanding about the genetic landscape of AML, and has led to the discovery of a long list of mutations which are potentially useful as prognostic markers of AML. Interestingly, the prognostic impact of the genetic events from the same functional categories are rather varied. For instance, the presence of HAz drafted this manuscript. CP, HAl, N-SA, and RJ involved in the critical evaluation of the manuscript.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.


VDAC1 is highly Ca2+-permeable and modulates Ca2+ access to the mitochondrial intermembrane space. Intramitochondrial Ca2+ controls energy metabolism by enhancing the rate of NADH production via modulating critical enzymes in the tricarboxylic acid cycle and fatty acid oxidation. Mitochondrial [Ca2+] is regarded as an important determinant of cell sensitivity to apoptotic stimuli and was proposed to act as a “priming signal,” sensitizing the organelle and promoting the release of pro-apoptotic proteins. However, the precise mechanism by which intracellular Ca2+ ([Ca2+]i) mediates apoptosis is not known. Here, we review the roles of VDAC1 in mitochondrial Ca2+ homeostasis and in apoptosis. Accumulated evidence shows that apoptosis-inducing agents act by increasing [Ca2+]i and that this, in turn, augments VDAC1 expression levels. Thus, a new concept of how increased [Ca2+]i activates apoptosis is postulated. Specifically, increased [Ca2+]i enhances VDAC1 expression levels, followed by VDAC1 oligomerization, cytochrome c release, and subsequently apoptosis. Evidence supporting this new model suggesting that upregulation of VDAC1 expression constitutes a major mechanism by which apoptotic stimuli induce apoptosis with VDAC1 oligomerization being a molecular focal point in apoptosis regulation is presented. A new proposed mechanism of pro-apoptotic drug action, namely Ca2+-dependent enhancement of VDAC1 expression, provides a platform for developing a new class of anticancer drugs modulating VDAC1 levels via the promoter and for overcoming the resistance of cancer cells to chemotherapy.In the outer mitochondrial membrane, the voltage-dependent anion channel 1 (VDAC1) functions in cellular Ca Moreoveartments . The majchondria . Imbalan2+ homeostasis, regulating oxidative phosphorylation (OXPHOS)  –7 and mo signals , 9, cell signals , and sec signals , 12. Encree Ca2+ .2+]i at the expense of the membrane potential across the IMM that is generated by the electron transport chain  cycle and fatty acid oxidation  homeostasis is important not only for energy production but also for regulating [Ca2+]i and activating cell apoptotic pathways   , 24 and mediator , 26. In eability –30.Three different isoforms of VDAC have been identified, VDAC1, VDAC2, and VDAC3. VDAC1 has been best studied, whereas only limited information regarding the cellular functions of VDAC2 and VDAC3 is available . Thus, w2+  into the mitochondria and the exit of newly formed molecules, such as hemes , 33  that its occupation by La3+ or Tb3+ lead to reduced channel conductance.Several lines of evidence suggest that VDAC1 possesses divalent cation-binding site(s). m uptake , essential MCU regulator, and SLC25A23 are essential for MCU activity (c oxidase assembly (2+-buffering agent (The major channel-forming subunit of the MCU complex (CCDC109A) consists of two transmembrane and the N-terminal domains and forms a complex in the IMM with many gatekeeper membrane proteins , 69–71. activity –76. MCURassembly , as a cyng agent , or in Rng agent .The functional role of MCU under physiological conditions was extensively studied using several silencing techniques –85. Inte2+ is mainly determined by the balance between influx through the MCU and efflux via NCLX  or Na+/Ca2+ exchanger (2+/H+ anti-porter in addition to being a H+/K+ anti-porter (see Other Proteins Proposed as Participating in or Mediating Ca2+ Efflux from Mitochondria) H production and matrix redox state modulation  . NCLX me the IMS , 93–95. and Ca2+ . NCLX hadulation .2+ regulates heart metabolism, where steady-state [Ca2+]m is determined by the dynamic balance between MCU-based Ca2+ influx and NCLX-based Ca2+ efflux  represents another mechanism for Caogenesis , 104. Mue matrix , 106. Hosee Ref. ]. Recente matrix .2+]m/H+ anti-porter was suggested, the leucine zipper-EF-hand-containing transmembrane protein 1 (Letm1)  , 109–111 is high .2+ transport in membranes other than in the mitochondria as ryanodine receptors (RyRs) and the transient receptor potential 3 (TRPC3) channel were reported to function in Ca2+ homeostasis [for review, see Ref.  from the IMS to the cytosol  U266 myeloma cell line to various apoptotic stimuli is well correlated with the elevated levels of VDAC1 expression that follow Ca2+ signals in response to apoptosis stimulation , VDAC levels were increased, as were [Caactivity . Downregoduction . VDAC1 hoduction . It has opathies . Thus, VVS-B wrote the review; SD and AM helped in writing.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.


Oryctolagus cuniculus) are frequently reared for meat production in China. The aim of this study was to assess the seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi, and risk factors of infection in domestic rabbits raised in Henan province, central China. 1,213 serum samples of domestic rabbits were collected and tested for anti-T. gondii and anti-E. cuniculi antibodies using a modified agglutination test (MAT) and an enzyme-linked immunosorbent assay (ELISA), respectively. The serum positive rates of T. gondii and E. cuniculi were 128/1,213 (10.55%) and 235/1,213 (19.37%), respectively. Co-infection of T. gondii and E. cuniculi was demonstrated in 84 specimens; 44 rabbits were seropositive for T. gondii alone, while 151 rabbits were seropositive for E. cuniculi alone. The main risk factors simultaneously associated with T. gondii and E. cuniculi infection were the age of the rabbit, the type of food, and the rabbit rearing system. Serum positive rates of T. gondii and E. cuniculi among domestic rabbits were high, indicating the possibility of public health issues.Rabbits ( Toxoplasma gondii (T. gondii) is one of the most common universal zoonotic protozoan parasites, ubiquitous throughout the world, and widely prevalent in humans and animals [T. gondii [T. gondii, including rabbits [T. gondii infection is the consumption of raw or undercooked meat containing tissue cysts from intermediate hosts [ animals . It is e. gondii . Almost  rabbits . The maite hosts .T. gondii to rabbits is through water or food containing oocysts from feline excrement, or through placenta from pregnant females to offspring [The transmission mode of ffspring . Humans ffspring ,9.Encephalitozoon cuniculi (E. cuniculi) is a common pathogen of rabbits causing chronic renal and central nervous system disease characterized by granuloma formation and fibrosis [fibrosis . Howeverfibrosis ,6. Infecfibrosis . In manyfibrosis .T. gondii and E. cuniculi in humans. However, little is known about natural infection of T. gondii and E. cuniculi in domestic rabbits in China. Therefore, this study was conducted to investigate the seroprevalence and the risk factors related to seropositivity of T. gondii and E. cuniculi among domestic rabbits in Henan province, central China. The results will lay the groundwork for controlling both T. gondii and E. cuniculi infections among domestic rabbits in central China.In China, rabbits are frequently reared for meat production and are consequently considered a possible source of infection by All protocols in this study were reviewed and approved by the Ethics Committee of the Xinxiang Medical University (reference no. 2015018).2. The Yellow River flows through the middle section of Henan, which is seated within north latitude of 31°23′−36°22′ and east longitude of 110°21′−116°39′. Due to the mainland monsoon type climate, four seasons are distinct with the year-round average temperature of 12.1-15.7 °C and year-round average precipitation of 532.5-1380.6 mm. Henan province contains seventeen cities and Zhengzhou is the capital city. Seven cities which include Anyang , Sanmenxia , Luoyang , Xuchang , Zhumadian , Xinyang  and Zhoukou , located in the northern, western, central, southern and eastern parts of Henan province  [T. gondii whole cell antigen  prepared using the RH strain of T. gondii cultivated via human foreskin fibroblast cells was purchased from KeraFAST, Inc. . Briefly, serum samples were diluted by serum dilution buffer using 2-fold serial dilutions from 1:25 to 1:3,200. Fifty microlitres of diluted serum samples were used for agglutinating with 50 µl of antigen mixture  in a U-bottom 96-well microtiter plate under the temperature of 37 °C for a whole night. The formation of parasite agglutinating layers in wells which contained diluted serum specimens with a ratio of 1:25 or higher indicated positive results. Each assay contained negative and positive control groups.Based on previous studies, the anti-st (MAT) ,13,21. TE. cuniculi antibody levels of serum samples from domestic rabbits were verified and detected using a commercial Encephalitozoon cuniculi (EC) ELISA kit , following the manufacturer’s instructions.The anti-T. gondii and E. cuniculi resulting from variates including species, gender, ages, the type of food, and the rearing system. Windows version SPSS 20  was used to perform all statistical analyses. A p-value lower than 0.05 was regarded as the threshold for statistically significant differences.Chi square tests were used to analyse the variation of serum positive rates for T. gondii among rabbits from Henan province, central China was 10.55% (128/1213), with titers of 1:25 in 69, 1:50 in 36, 1:100 in 14, 1:200 in 5, 1:400 in 3, and 1:800 in 1. The serum positive rates of 7 sampling sites were within the range of 7.64% in Sanmenxia city to 14.06% in Luoyang city  and in female rabbits 9.87% (67/679) (p>0.05). The prevalence of T. gondii infection in rabbits increased significantly (p < 0.01) with age. Rabbits no younger than twelve months exhibited the highest seroprevalence (17.07%), followed by rabbits of 6-12 months (10.40%), and then rabbits ≤ 6 months (6.65%) (The rate of antibodies to (67/679) . Althoug (6.65%) .T. gondii infection (15.99%) was found in rabbits fed with a mixture of fruits, vegetables or grains. The probability of rabbits being infected by T. gondii was increased by the presence of cats at the feeding farm compared to farms without cats . In terms of rearing systems, the T. gondii seroprevalence in rabbits raised on commercial farms (6.01%) was significantly lower than that of animals raised on household farms .The highest seroprevalence of T. gondii and possible risk factors are shown in T. gondii and food type, rabbit ages, cat presence on feeding farms, as well as the rearing system.The univariate analyses on the correlation between serum positive rates of E. cuniculi antibodies. Rabbits with positive antibodies in serum were found at all tested sites, varying from 11.83% in Xuchang city to 29.55% in Zhoukou city (p < 0.05).As shown in kou city . Rex rabE. cuniculi seropositive animals (12.34%) were young (≤ 6 months), 135/692 (19.51%) were adults (6∼12 months), and 61/205 (29.76%) were in older age groups (≥ 12 months). The E. cuniculi seropositivity of rabbits increased significantly (p < 0.01) with the increase of rabbit’s age.Moreover, it was revealed by comparing infection rates by ages that 39/316 T. gondii infection, the highest E. cuniculi seroprevalence (27.89%) was found in rabbits fed with a mixture of fruits, vegetables or grains. Moreover, E. cuniculi seroprevalence in rabbits raised on commercial farms was also significantly lower than that of animals raised on household farms .Like in E. cuniculi and T. gondii was demonstrated in 84 specimens (30.11%). Forty-four rabbits (15.77%) were seropositive for T. gondii alone, while 151 rabbits (54.12%) were seropositive for E. cuniculi alone  [E. cuniculi was 19.37% in domestic rabbits in Henan. In comparison to other provinces within China mainland, the serum positive rate of 19.37% was lower than the values of 22.2% among rabbits investigated previously in Inner Mongolia, 30.9% in Liaoning [T. gondii and anti-E. cuniculi antibodies among different regions are probably attributable to different rabbit breeds, sample capacities, time of investigations, and testing methods, as well as geographical and ecological factors.The current investigation showed that the total serum positive rate of  (23.4%) , but hig (23.4%)  of ChinaLiaoning , and 41.Liaoning , but higLiaoning , and HeiLiaoning . The varT. gondii in the current work was lower than that of E. cuniculi, which was consistent with previous literature reports [E. cuniculi among herds was remarkably promoted by directly transmitting spores from urine of affected animals [The serum positive rate for  reports ,14. The T. gondii was found in New Zealand rabbits, while Rex rabbits had the highest seroprevalence of E. cuniculi. Regarding the breed of rabbits, de Lima et al. [et al. [T. gondii in New Zealand rabbits than in other breeds. Pan et al. also revealed higher serum positive rates for E. cuniculi among Rex rabbits than those among New Zealand and Japanese White Rabbits (p < 0.01) [T. gondii and E. cuniculi. Most epidemiological surveys indicated no association between T. gondii and E. cuniculi infection and gender of the animals [T. gondii, which was in agreement with many previous studies [E. cuniculi in comparison to male animals of our study. The role of gender in the epidemiology of rabbit encephalitozoonosis requires further research.In the present study, the highest seroprevalence of a et al.  and Alva [et al.  reported < 0.01) . These r animals ,18,19. I studies ,18. HoweT. gondii and E. cuniculi infection. The observed higher seroprevalence for both protists in older rabbits suggests that these infections are mainly maintained by horizontal rather than vertical transmission. This finding is in accordance with other similar surveys [Increasing age was confirmed as a risk factor for both  surveys ,17.T. gondii and E. cuniculi according to the current study, which was consistent with other reports [T. gondii oocysts or E. cuniculi spores than feed or mixed food. Cats play an essential role in T. gondii spreading since they are the final hosts and shed oocysts into the environment [T. gondii infection among rabbits, which accorded with findings in previous literature reports [Rabbits fed with fruits, vegetables and grains had the highest serum positive rates of  reports . This miironment ,20. In t reports ,9.T. gondii and E. cuniculi seroprevalence rates were significantly higher in rabbits raised on household farms than in those raised on commercial farms, which is probably due to lower hygiene standards on household farms. Our findings are similar to those of previous reports [T. gondii infection in rabbits as well as E. cuniculi infection.In terms of rearing systems, both  reports ,14. ThesThe authors declare that they have no conflicts of interest in relation to this article.


NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin ( Acute myeloid leukemia (AML), which results from hematopoietic stem cell disorders, is a heterogeneous disease. Indeed, AML is associated with a high diversity of molecular alterations promoting blast transformation ,2. Consepol I) associated with an increase in protein synthesis [In neoplastic cells, the high proliferation rate of tumor cells is sustained by an increased ribosome biogenesis due to hyper-activation of RNA polymerase I  and fibrillarin (FBL), or ribosome composition—such as FBL, NOP56, NOP58 and NHP2L1. NCL and FBL are two of the most abundant nucleolar proteins playing a central role in rDNA transcription by regulating activity of RNA pol I [NOP56, NOP58 and NHP2L1 constitute the rRNA 2’-O-ribose methylation complex whose pattern alterations have been shown to modulate gene expression [Here, we investigated for the first time the clinical significance of a panel of factors involved in ribosome biogenesis. We focused our study on genes encoding factors regulating either ribosome production, such as nucleolin . The TCGA series was extracted from a public database hosting datasets issued from the Cancer Genome Atlas project . All samples were obtained at time of diagnosis with written informed consent at corresponding hospitals with approval of local ethics committees (Comité d’Ethique de Lyon) that approved this study. Characterization of the classical cytogenetic markers was determined by the corresponding hospitals. Clinical annotations were available for the series 3 and TCGA  and AML patients (n = 6) at initial diagnosis to analyze number of nucleoli per cell and nucleoli morphology by immunofluorescence. In addition, RNA samples of five series  issued from bone marrow or blood samples were used to investigate clinical value of ribosome biogenesis factors (see characteristics in and TCGA .®/AlexaFluor 555® conjugated secondary antibody (Molecular Probes). Following immunostaining, nuclei were stained with Hoechst 33342 (Molecular Probes), and smears were mounted with Fluoromount G mounting medium (Electron Microscopy Sciences). Images were collected with a x63 NA 1.4 Plan-Apochromat objective on a Carl Zeiss LSM 780 confocal microscope. Image analysis and quantification were performed on wide-field images collected with a x40 NA 1.30 Plan Fluor objective on a Nikon NiE microscope equipped with an Orca Flash 4 camera (Hamamatsu), and using the NIS Elements software (Nikon). Briefly, nuclei were segmented using intensity thresholding of Hoechst signal, and saved as individual ROI (Region Of Interest). Nucleoli were segmented using intensity thresholding of FBL signal within the ROIs and their number and size were extracted.Air dried bone marrow smears were hydrated with 1X PBS, fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining was performed using a rabbit anti-FBL antibody at 1/500 dilution , a mouse anti-NCL antibody at 1/2000  and AlexaFluor 488-ΔΔCT method using GAPDH gene expression levels as normalizer . Gene expression was quantified by real-time qPCR using Sybr Green chemistry and specific primer sets . Clinical and pathological information from only 113 patients with AML were downloaded. Indeed, some AML cases lack information about patients’ survival and AML cases with history of neoadjuvant treatment were excluded from the analysis since chemotherapeutic drugs have been shown to inhibit ribosome biogenesis that may thus affect expression of factors involved in ribosome biogenesis [The Cancer Genome Atlas (TCGA) was queried (last query in July 2014) . Median comparison between two unpaired groups was performed using the non-parametric Mann-Whitney test. Correlations between different methods or primers sets were tested using the non-parametric Spearman r test. Overall survival was measured from the date of diagnosis to death or censored at the last follow-up. Prognostic value of the different genes was estimated using the Kaplan-Meier method and the Log-Rank Mantel-Cox model was used to assess statistical significance. A cut-off based on data dispersion was used instead of an absolute cut-off since quantification of mRNA levels was measured using two different methods (RT-qPCR and RNA-seq). In particular, the 75In a first instance, we investigated whether nucleoli number and organization are modified in AML patients. The morphological aspect of nucleoli reflects ribosome biogenesis activity and correlates with the neoplastic status of cells . To inveC-Myc and NPM1 genes were significantly over-expressed in AML samples compared to controls  . Gene excontrols . These r18S rRNA together with that of NCL and FBL, these three genes reflecting ribosome synthesis, as well as the expression of the components of the rRNA 2’-O-ribose methylation complex . Analysis of series 1 showed that only NCL, NOP58 and NOP56 genes are significantly over-expressed in AML samples compared to controls  the highest levels of NCL expression in healthy donors correspond to about the NCL median levels in AML patients  NCLy donors , and (3)spersion . No absospersion . We firspatients . Similar outcome .NCL and NOP56 expression in predicting AML outcome, an additional, independent series was studied  determined by RNA-seq of AML samples from the TCGA dataset . Filters were used to restrict the analysis to primary AML samples thus allowing the collection of 113 samples to build the validation series. Dichotomization of expression levels was performed using the 75th percentile as a cut-off to conserve similar strategy within the study  showed association with AML patient outcome, supporting the specificity of NCL as prognostic marker in AML   . NCL expression levels could therefore be used as a prognostic factor for survival in elderly AML patients.The lack of strict concordance in series 3 and TCGA in terms of prognostic impact of  0.023*) . Indeed,e marrow . In contars old) . ElderlyNCL expression in improving cytogenetic risk classification in AML patients of the TCGA series using Kaplan-Meier and Cox regression analyses. While NCL expression had no significant impact on patient outcome in the favorable and poor risk groups, enhanced NCL expression was associated with shorter overall survival in the intermediate group  on all AML samples issued from the TCGA dataset (n = 113). The best model associated with poor overall survival contained three independent markers: age at diagnosis (P = 0.00012***); cytogenetic alterations (P = 0.0170*); and NCL mRNA levels (P = 0.0230*). These analyses demonstrated that NCL expression is associated with poor overall survival independently of these other common prognostic factors. Overall, our data indicate that NCL expression is a novel prognostic marker of survival in AML.Finally, we investigated whether NCL is an independent marker of prognosis in AML patients using the TCGA series. We performed a multivariate analysis to adjust possible confounding variables  are not significantly over-expressed. This observation supports the specificity of over-expression of NCL and NOP56 ribosome biogenesis factors in AML disease and suggests that NCL and NOP56 may have important and additional functions in ribosome biogenesis that are necessary to promote acute myeloid leukemogenesis.Because of the need for improving the current risk classification in order to improve AML patient management, and given the emerging role of ribosomes in controlling HSC differentiation and HSC-derived diseases ,15,17,37NOP56 as being over-expressed in AML samples, the prognostic significance of NOP56 remains to be clarified. Indeed, we observed a significant association between high NOP56 expression levels and poor outcome of AML patients in the test series 3 that was not confirmed in the validation series TCGA. Few studies investigated either the clinical significance or the biological role of NOP56 in hematological disorders. NOP56 expression is increased in Burkitt’s lymphoma-associated C-Myc mutants and its expression promotes wild-type C-Myc-induced cell transformation and increases tumor size, suggesting a link between B-cell lymphoma and NOP56 [NOP56 in hematopoietic-related disorders, use of larger data sets will be required to clearly conclude concerning the clinical significance of NOP56 in AML.Although we clearly identified nd NOP56 ,7,8,39. NOP56, our data clearly demonstrated that NCL expression has a prognostic value in AML outcome. We first demonstrated that NCL is over-expressed in AML patients compared to controls. Shen and collaborators recently showed using analysis of previously published GEO data sets (2 series of 54 AML younger than 15 year-old and 26 AML patients) that NCL mRNA expression levels is statistically higher in AML patients than in healthy donors [NCL in AML is supported by our observation that NCL staining is higher in AML bone marrow smears than in controls  (P = 0.0137**) (data not shown), however such observation cannot explain the association between high expression of NCL and poor prognosis of AML patients. Interestingly, none of the five additional genes of interest showed association with AML patient outcome, supporting the specificity of NCL as prognostic marker in AML. Using a GEO data set of 86 AML patients, Shen et al. also reported a significant association between high NCL mRNA levels and poor prognosis in AML patients [NCL is a novel prognostic factor in AML. The stratification using NCL expression levels allows to discriminate two groups in elderly AML patients (> 60 years)–low and high expression-related groups of NCL expression associated in TCGA series with median survivals of approximately 15 or 6 months, respectively. However, these results cannot be validated using the series 3 due to the small sample number or using additional data sets that are currently unavailable. Ederly AML patients are more often treated with “curative intent” when the cytogenetic profile is favourable and receive palliative or experimental therapy when the cytogenetic profile is unfavourable [NCL could thus be useful to guide therapy choices in these particular patient subgroups. Establishment of prospective series enriched in elderly AML patients with information regarding their current treatment regimen will be required to firmly conclude about the utility of NCL to improve current risk classifications.In contrast to y donors –12. The controls . In addipatients . Howevervourable –43. StraThe best characterized activity of NCL remains its role in regulating ribosomal RNA synthesis –18. IndeNOP56 and NCL. More importantly, expression of one of them, NCL, predicts AML outcome, independently of the current risk factors—age at diagnosis and cytogenetic alterations. Altogether, our study supports the usage of this ribosome biogenesis factor as clinical markers. It opens up an original and yet unexplored pathway, the ribosome biogenesis pathway, in clinic. In particular, NCL could represent a novel opportunity to improve management of elderly AML patients, not only as an original and independent prognostic factor useful to guide therapy choices but also as novel therapeutic target to develop innovative strategy dedicated to a particular elderly patients subgroup.Several data suggest that ribosome biogenesis contributes to hematopoietic stem cell disorders (HSCs), such as AML –17,33–37S1 Dataset(XLS)Click here for additional data file.S1 FigGAPDH (A), ACTB (B) and 18S (C), were compared to identify the most suitable housekeeping gene in our experimental conditions. Three major criteria were required: first, the absence of significant difference between controls and AML patients; second, the smallest coefficient of variation; and third, a mean CT of about 20 that corresponds to the mean CT of most of our genes of interest. Only GAPDH gene matched these criteria and was thus used as normalizer.Ct values of three internal reference genes, (TIFF)Click here for additional data file.S2 FigImmunofluorescence staining of nucleolar FBL and NCL on control (top panel) and AML (bottom panel) patient's bone marrow smears. FBL (green) and NCL (red) pattern are shown individually in top and middle images, and merged image with nuclei staining (blue) is shown in the bottom image. Images were collected using confocal microscopy .(TIFF)Click here for additional data file.S3 FigA) Quantification and analysis of nucleoli number by image analysis from 4 control and 6 AML bone marrow smears. The number of nucleoli per cell was measured using FBL signal in each samples and ranged from 0 to 4. The median number of nucleoli per cell was represented for each individual samples. No nucleolus was observed in the control Ind4. (B) Quantification and analysis of nucleoli size by image analysis from 4 control and 6 AML bone marrow smears. When nucleoli are detected within a sample, the nucleolus size was measured using FBL signal in each samples. The median size of nucleoli per cell was represented for each individual samples. No nucleolus was observed in the control Ind4. Box and whisker plots represent median (middle bar in the box), interquartile range (bottom and top of box) and minimal/maximal values (bottom and top whisker). The number of cells or nucleoli analyzed for each samples are indicated in bracket. Representative panels of images used to perform these image analyses are shown in ((TIFF)Click here for additional data file.S4 FigA) Validation of high-throughput qPCR method using microfluidic system device. Correlation between fold-changes calculated from Ct values determined by classical RT-qPCR and high-throughput qPCR was investigated on few samples for C-Myc and NPM1 gene using Spearman test. (B-C) Expression of C-Myc and NPM1 genes in AML patients. Mean comparison of C-Myc (B) and NPM1 (C) gene expression between controls and AML patients was investigated using Mann-Whitney test in serie 1 analyzed by classical RT-qPCR. Graphs represents median  and interquartile range (bottom and top bars) calculated on the Log2(Relative mRNA levels) of each individual samples (grey dot). n: number of samples; *: P<0.05; **: P<0.01; ***: P<0.001.((TIFF)Click here for additional data file.S5 FigA-B) Correlation between log2 (Relative mRNA levels) issued from two different sets of primers to analyze expression of NCL (A) and Nop56 (B). Correlation was determined using Spearman test. (C-H) Mean comparison of NCL and Nop56 gene expression between controls and AML patients using Mann-Whitey test. Relative RNA levels of NCL  and Nop56  were determined using high-throughput qPCR in series 1 (C-F) and series 2 (G-H) with two different sets of primers . Graphs represents median  and interquartile range (bottom and top bars) calculated on the log2 (Relative mRNA levels) of each individual samples (grey dot). n: number of samples; *: P<0.05; ***: P<0.0001.((TIFF)Click here for additional data file.S6 FigA) Expression distribution of the different genes analyzed by high-throughput qPCR in series 3. (B) Expression distribution of the different genes analyzed by RNA-seq in series TCGA. Box and whisker plots represent median (middle bar in the box), interquartile range (bottom and top of box) and minimal/maximal values (bottom and top whisker). Grey: gene usually over-expressed in AML samples; green: genes coding proteins regulating rRNA synthesis; red: genes coding proteins of the rRNA methylation complex.((TIFF)Click here for additional data file.S7 FigA-B) Kaplan-Meier analysis of overall survival rates (event = death related to AML disease) according to cytogenetic risk classification (A) and to NCL in intermediate group of cytogenetic risk classification in AML patients (B). The data are dichotomized at the 75% percentile value into high and low mRNA level groups. n: number of samples.((TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Saccharomyces cerevisiae and Candida albicans. Whether and how Fig1 regulates Ca2+ homeostasis in response to extracellular stimuli is poorly understood. Previously, we found Aspergillus nidulans FigA, a homolog of Fig1 in S. cerevisiae, similar to the high-affinity calcium uptake system, is required for normal growth under low-Ca2+ minimal medium. In this study, using the calcium-sensitive photoprotein aequorin to monitor cytosolic free calcium concentration ([Ca2+]c) in living cells, we found that the FigA dysfunction decreases the transient [Ca2+]c induced by a high extracellular calcium stress. Furthermore, FigA acts synergistically with CchA (a high-affinity Ca2+ channel) to coordinate cytoplasmic Ca2+ influx in response to an extracellular Ca2+ stimulus. Moreover, FigA mediates ER stress-induced transient [Ca2+]c in the presence or absence of extracellular calcium. Most importantly, these [Ca2+]c responses mediated by FigA are closely related to its conserved claudin superfamily motif, which is also required for hyphal growth and asexual development in A. nidulans. Finally, the function of FigA in Aspergillus fumigatus, the most common airborne human fungal pathogen was studied. The result showed that the two FigA homologous in A. nidulans and A. fumigatus have a large degree of functional homology not only in asexual development but also in regulating transient [Ca2+]c. Our study expands the knowledge of claudin family protein FigA in Ca2+ homeostasis in response to extracellular stimuli.The claudin family protein Fig1 is a unique fungal protein that is involved in pheromone-induced calcium influx and membrane fusion during the mating of  The HACS was activated in response to mating pheromones, alkaline pH, oxidative stress and compounds such as azole-class antifungal agents or ER-stress agents  and the stretch-activated calcium channel/regulatory protein Mid1 (mating-induced death)  in eukaryotes. At resting state, the cytosolic Cao 100 nM . In respartments . In Saccs agents . The HACd death) . Deletionditions . Besidesalbicans .2+ concentration but suppressed under high external Ca2+ concentration, so that the LACS becomes essential for this response  have been characterized in A. nidulans. Consistent with the function of Cch1 and Mid1 as a member of the HACS, CchA and MidA are required for conidial development, hyphal polarity establishment, and cell wall integrations in low-calcium environmental conditions . To further characterize the relationship between figA and midA/cchA in mediating calcium uptake, ΔfigAΔmidA and ΔfigAΔcchA double deletion mutants were grown on MM and the hyphal radial growth were observed. The phenotypic defect in hyphal radial growth was exacerbated in the ΔfigAΔcchA and ΔfigAΔmidA double deletion mutants compared to their respective parental single mutants ΔfigA,ΔcchA, or ΔmidA , which suggesting both CchA/MidA and FigA are required for fungal growth under low calcium conditions. Interestingly, the addition of 20 mM calcium to MM restored the radial growth defects (but not conidiation) in the ΔfigAΔmidA mutant but not in the ΔfigAΔcchA mutant, suggesting non-overlapping roles for MidA and CchA. FigA and CchA appear to act synergistically to coordinate calcium uptake under calcium-limited conditions. To further explore the roles of FigA in the regulation of [Ca2+]c, real-time monitoring of [Ca2+]c in living hyphal cells was performed. The pre-stimulatory resting [Ca2+]c in A. nidulans is typically between 0.05 and 0.1 μM , the highest [Ca2+]c in wild-type cells increased to approximately 0.9 μM. Interestingly, it was the ΔcchA mutant (p = 0.0062) but not the ΔmidA mutant (p = 0.1241) that showed a significant reduction of the transient [Ca2+]c compared to its wild-type strain (100%) . Surprisingly, the transient [Ca2+]c in the ΔfigA mutant showed a similar decrease as the ΔcchA mutant under the same stimulus. Moreover, the ΔfigAΔcchA mutant, but not the ΔfigAΔmidA mutant exhibited a dramatic further reduction in transient [Ca2+]c compared to the ΔfigA mutant under the same stimulus . Collectively, these data suggest that FigA and CchA synergistically coordinate cytoplasmic Ca2+ influx in response to an extracellular calcium stimulus.Our previous data has shown that loss of CchA/MidA or FigA caused a reduced hyphal growth under calcium-limited minimal medium (MM) in pe in MM  . When the parental wild-type strain was treated with 5 μg/mL TM, an immediate transient increase in [Ca2+]c to 0.6 μM was observed. Consistent with our previous result, the transient [Ca2+]c in the ΔcchA mutant but not in ΔmidA mutant showed a significant 27% reduction compared to the parental wild-type strain in response to TM . Notably, the transient [Ca2+]c in the ΔfigA mutant decreased by approximately 24% compared to the parental wild-type strain under the same stimulus . The results suggest that both CchA and FigA are involved in mediating the ER stress-induced calcium influx in A. nidulans.Endoplasmic reticulum (ER) is a multifunctional organelle required for calcium storage, protein folding and processing. Our previous data indicate that CchA, but not MidA influences ER stress-induced calcium influx in nidulans . To expl2+]c resulting from the TM treatment is dependent on external calcium or internal calcium stores. Exposure of the ΔfigA mutant cells to the calcium chelator EGTA (1 mM) prior to the TM treatment showed a significant 22% reduction in the transient [Ca2+]c compared to wild-type. However, there was no significant difference between ΔmidA or ΔcchA and the wild-type strain . Collectively, these data showed that both CchA and FigA mediate ER stress-induced calcium influx in A. nidulans. Moreover, FigA also contributes to ER stress-induced transient [Ca2+]c in the absence of extracellular calcium.Furthermore, we tested whether the transient c in response to external Ca2+, we determined the role of the conserved claudin motif of FigA on transient [Ca2+]c. As is shown in Figure 4C, the [Ca2+]c in the figAmt-4G/CA point mutation strain decreased significantly to 55% compared with that in the wild-type, suggesting a role for the claudin motif on [Ca2+]c regulation. In conclusion, we demonstrated that the conserved claudin motif is required for the proper function of FigA, including normal hyphal growth, conidiation, and the regulation of transient [Ca2+]c.The conserved motif in FigA, GΦΦGxC(n)C, is a feature of the claudin superfamily. The glycine and cysteine residues in the conserved motif are located near the end of the first predicted extracellular loop region, between TM1 and TM2   has 82% identity with its homolog in A. nidulans. Both proteins have conserved topology and contain the claudin motif. To explore the function of the FigA in A. fumigatus, an AffigA deletion strain was constructed by homologous gene replacement employing the N. crassa pyr4 gene as a selectable marker . The resulting strain, ΔAffigA, was confirmed by diagnostic PCR and Southern blot . As is shown in Figure 5A, the loss of figA in A. fumigatus caused reduced conidia production on MM. Consistent with our observations in A. nidulans, the conidiation defects in the ΔAffigA mutant could not be rescued by the addition of extracellular calcium . However, it seems that FigA plays a more important role in conidiation in A. nidulans than that in A. fumigatus. Conidia production in the ΔAffigA mutant was 20% of the reference strain (100%). In comparison, conidiation was almost completely abolished in the A. nidulans figA deletion strain. In addition, loss of figA in A. fumigatus did not affect the hyphal radial and polarized growth .or human . FigA inA. nidulans and A. fumigatus, two wild-type figA genes were used to cross complement each figA deletion strain. Functional assays showed that the A. fumigatus FigA could completely restore the asexual conidiation and hyphal growth defects seen in the A. nidulans figA deletion strain and vice versa under all test conditions including MM, MM plus EGTA or MM plus calcium . The results indicated that the two FigA homologs in A. fumigatus and A. nidulans have a large degree of functional homology.To verify the functional homology between the two FigA homologs in figA affects calcium influx in A. nidulans. In order to explore whether A. fumigatus FigA regulates calcium influx, we monitored the transient [Ca2+]c in response to extracellular calcium and TM by expressing codon-optimized aequorin in living cells of A. fumigatus wild-type A1160 and the ΔAffigA mutant. As expected, the [Ca2+]c in the ΔAffigA mutant exposed to the 0.1 M CaCl2 stimulus significantly decreased by 26% compared to that of the parental wild-type strain . When treated with the ER stressor TM, the [Ca2+]c in the ΔAffigA mutant significantly decreased by 16% compared to that of the parental wild-type strain . In summary, the above data showed that FigA possess a conserved role in [Ca2+]c regulation in A. fumigatus.The above results suggested that loss of 2+-mediated signaling pathway plays a crucial role in fungal growth and survival under various stresses. In yeast, the HACS components Cch1 and Mid1 are the primary channels involved in Ca2+ homeostasis under low-Ca2+ conditions. The function of LACS component or regulator Fig1 seems to be confined to the pheromone-induced calcium influx in the rich medium which the function of the HACS is inhibited by calcineurin  FigA is required for the calcium uptake under low-Ca2+ conditions. Loss of figA causes a similar phenotype for retardation of hyphal growth to that of cchA and midA deletion mutants under low calcium conditions. And that growth defects are able to be restored by the addition of exogenous calcium. (II) FigA and CchA act synergistically to coordinate calcium uptake under calcium-limited conditions since the exogenous calcium-based restoration is abolished in a figA and cchA double deletion mutant. (III) FigA is required for the transient [Ca2+]c in response to an extracellular calcium stimulus. The transient [Ca2+]c in the ΔfigA mutant showed a similar amplitude decrease as the ΔcchA mutant following treatment with a high extracellular calcium stress stimulus. Moreover, double deletion of figA and cchA further decreases transient [Ca2+]c than that in ΔcchA or ΔfigA mutants under the same stimulating condition. (IV) FigA also regulates the transient [Ca2+]c in response to the ER stressor TM. It has been reported that ER stress can stimulate transient [Ca2+]c through the HACS to promote fungal cell survival  that shares sequence similarity with members of the claudin superfamily. In mammals, the functions of claudin superfamily members are involved in epithelial tight-junction formation, which allow ions and other solutes to pass between cells . All fungal strains were routinely cultured on MM with supplements to support the growth of relevant auxotrophic strains, as described previously  of indicated strains were spotted onto MM and MM plus 0.75 μg/mL Congo Red. All strains were cultured at 37°C for 2.5 days and then the colonies were observed and imaged.To assess the influence of extracellular calcium on fungal growth, MM was supplemented with 20 mM CaClA. nidulans wild-type as the template, a 2.7 kb figA DNA fragment including a 1 kb upstream promoter region, a 0.9 kb coding sequence, and a 3′ flanking sequence was amplified with the primer pair FigAF/FigAR. The resulting fragment was digested with SmaI and PstI and was cloned into the SmaI and PstI digested plasmid pQa-pyroA to generate plasmid pFigA-pyroA. The Mut Express II Fast Mutagenesis kit (VazymeTM) was used to construct plasmids carrying site-directed mutations. In brief, using the resulting plasmid pAnFigA-pyroA as a template, a DNA fragment with the complete ORF including the site-directed mutation , promoter sequence and 3′ UTR was amplified with the primers G97AF/G97AR. A similar strategy was used to construct plasmids carrying other mutations, including glycine100-alanine, cysteine102-alanine, cysteine112-alanine and the combined four mutated sites. All recombinant plasmids were individually transformed into the ΔfigA mutant strain to generate relevant FigA point mutants.Using genomic DNA (gDNA) of AffigA deletion cassette in A. fumigatus, a fusion PCR based method was used as described previously . The fragment amplified with primers AffigA – southern – F and AffigA – southern – R was used as a probe to detect the ΔAffigA and wild-type strains, respectively. Labeling and visualization were performed using a digoxigenin (DIG) DNA labeling and detection kit according to the manufacturer’s instructions .To perform Southern blotting, the genomic DNA from the wild-type and the ΔfigA genes from A. fumigatus and A. nidulans were cloned into pAN7-1 vector, which contained a hygromycin selection marker. Using gDNA of A. fumigatus wild-type A1160 as a template, a 2796 bp DNA fragment was amplified with the primer pair AfFigA-recon-P1/AfFigA-recon-P3, and then the fragment was cloned into plasmid pAN7-1 using the ClonExpress II OneStep Cloning Kit (VazymeTM) to generate the plasmid pAfFigA-hygB. A similar strategy was used to clone A. nidulans figA into plasmid pAN7-1 to generate pFigA-hygB plasmid using the primer pair FigA-recon-P1/FigA-recon-P3 with gDNA of A. nidulans TN02A7 as a template.The A. fumigatus figA into the pQa-pyroA vector, a 2.7 kb DNA fragment was amplified with the primer pair AfFigAF/AfFigAR using A. fumigatus A1160 gDNA as a template. The fragment was digested with SmaI and PstI and ligated into SmaI and PstI digested pQa-pyroA to generate the recombinant plasmid pAfFigA-pyroA. The plasmids pAfFigA-hygB and pFigA-hygB were transformed into the ΔAffigA mutant, and the plasmids pAfFigA-pyroA and pFigA-pyroA were transformed into ΔfigA mutant to generate the relevant complemented strains.To clone 6 spores in liquid media were dispensed in each well of a 96-well microtiter plate . Each experiment was performed as six replicates in the same multi well plate. Aequorin was reconstituted by incubating mycelia in 100 μl PGM containing 2.5 μM coelenterazine native  for 4 h at 4°C in the dark. After reconstitution, mycelia were washed with two 1 ml washes in PGM and allowed to recover to room temperature for 1 h  ethanol in the presence of excess Ca2+ (3 M CaCl2) to determine the total aequorin luminescence of each culture. Luminescence was measured with an LB 96P Microlumat Luminometer , which was controlled by a dedicated computer running the Microsoft Windows-based Berthold WinGlow software. Relative light unit (RLU) values were converted into [Ca2+]c concentrations using the following empirically derived calibration formula: pCa = 0.332588 (-logk) + 5.5593, where k is luminescence (in RLU) s-1/total luminescence (in RLU) (2+]c compared to that of wild-type (100%).Strains expressing the codon-optimized aequorin gene were grown on MM for 2.5 days to achieve maximal conidiation. 1 × 10 for 1 h . To chel(in RLU) . Error bp-Values were calculated with one-way ANOVA for multiple comparisons and adjusted with Tukey correction and non-paired Student’s t-test where two groups were compared.Statistical differences were analyzed using GraphPad Prism 6 software (GraphPad Software). HQ, SZ, and LL: conception and design of the investigation and work. HQ and QC: completion of the experiments. HQ and SZ: evaluation and analysis of the results and manuscript writing. HQ, QC, SZ, and LL: final approval of manuscript.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.


Acute myeloid leukaemia (AML) in early childhood is characterised by a high frequency of recurrent genomic aberrations associated with distinct myeloid subtypes, clinical outcomes and pathogenesis. Genomic instability is the first step of pathogenic mechanism in early childhood AML. A sum of adverse events is necessary to the development of infant AML (i-AML), which includes latency of biochemical-molecular and cellular effects. Inherited genetic susceptibility associated with exposures to biotransformation substances can modulate the risk of DNA damage and it is a very important piece in the pathogenic puzzle. In this review, we have aimed to explore the chain of events in the time-points of the natural history of i-AML, which includes maternal exposures during pregnancy, the speculations about the formation of somatic mutations during foetal life and the secondary genomic aberrations associated with i-AML. The modulation of risk conferred by xenobiotic metabolism´s genes variants is the bottom line of the pathogenic process. Since we have conducted observational and molecular investigations in early childhood leukaemia, the data focused here is based on Brazilian findings with summarised results of our experience with epidemiological and molecular studies in early-age leukaemia. Acute myeloid leukaemia (AML) accounts for about 20% of childhood leukaemia. There are increased incidence rates occurring in infants . The incidence declines dramatically in children between five and nine years of age at diagnosis, and then it increases again in adolescents and young adults [et al [AML is a heterogeneous disease that develops through the transformation of a haematopoietic progenitor cell as a result of a block in differentiation and uncontrolled proliferation, leading to an accumulation of immature cells and the decrease of mature blood cells. It was postulated that AML might be originated by mutations in genes involved in proliferative and signal transduction pathways, as well as transcription factors of progenitor myeloid cells , 3. The et al .RUNX1-RUNX1T1 and inv(16)/CBFβ-MYH11. Acute promyelocytic leukaemia (APL) with t/PML-RARA is also frequent in children [RUNX1-RUNX1T1, CBFβ-MYH1 and KMT2A (or MLL) rearrangements (KMT2A-r) and PML-RARA occurred in frequencies of 12.2%, 17.4%, 67.6% and 3.2%, respectively [KMT2A-r is highly prevalent in our series of i-AML cases. Other chromosomal translocations, such as the t/MNX1-ETV6, t/MYST3-CREBBP, t/RBM15-MKL1 and inv(16)(p13.3q24.3)/CBFA2T3-GLIS2, are all specifically associated with i-AML in variable prevalent frequencies that encompass the remaining 30% of aberrations [The most frequent chromosomal abnormalities in children with AML (c-AML) older than two years are those associated with the core-binding factor (CBF) aberrations, such as t/) with t1;17, a clonal pre-leukaemia condition that occurs in about 10% of neonates with DS, presenting at a median age of 3–7 days with the accumulation of megakaryoblasts, increased leukocytes and thrombocytopenia. The majority of TAM regresses spontaneously without treatment; however, about 20% of these patients will develop megakaryocytic leukaemia in the first four years of life [The great majority of i-AML clinically presents with high white blood cell counts, hepatosplenomegaly, often central nervous system involvement and chloroma. A subset of i-AML has pre-leukaemia or constitutional syndromes that suggest heterogeneous etiologies for these cases. Indeed, approximately 10% of i-AML has myelodysplastic syndrome with monosomy 7 or del(7q), has Down Syndrome (DS), has Noonan syndrome or carry neurofibromatosis type 1 mutations, which all predispose to early AML , 13. Chiutations –16. GATAferation , 15. Evital life . GATA-1  of life , 19. ThiSince we have performed clinical, epidemiological and molecular studies in early-age childhood leukaemia, this review focuses on the development and data from maternal exposures during pregnancy and molecular events associated with i-AML .KTM2A-AFF1 and ETV6-RUNX1, in dried neonatal blood spots of children who subsequently developed leukaemia [RAS mutation was associated with i-AML and chemical exposures [Given the young age of most children with i-AML, it was proposed and it is consensually accepted that early-age leukaemia is initiated before the birth of the child. The main evidence for a prenatal origin of childhood leukaemia was described in identical twins with concordant leukaemia. The same genomic fusion gene sequences were identified in twin pairs with acute leukaemia, which led to the assumption of a common clonal origin. The plausible basis for this finding is that in one twin foetus, clonal progeny spread to the co-twin via the vascular net. This assumption was endorsed by the identification of clonotypic gene fusion sequences, such as eukaemia , 21. It xposures .KMT2A-r caused by topoisomerase II (Topo-II) inhibitors [KMT2A-r. In t-MN investigations, the dose-effect associations can be well documented with qualitative and quantitative measurements, while in i-AML, the associations’ estimates are a presumption of biological plausibility.Based on biologic similarities between the therapy-related myeloid neoplasm (t-MN) and infant leukaemia with similar hibitors , the t-MKTM2A genomic breakpoint junction sequences has shown duplicated regions up to several hundred bases long from KTM2A and/or its partner gene on either derivative chromosomes, or deletions of several hundred bases. Topo-II creates 4-base staggered double-stranded breaks (DSB) in DNA, but also introduces single-stranded nicks as kinetic intermediates of DSB. The precision of the breakpoint junction sequences and the results of DNA Topo-II cleavage assays in treatment-related leukaemia suggest a mechanism in which two DNA Topo-II introduce separate single-stranded nicks in duplex DNA that are staggered by up to several hundred bases. Subsequent template-directed polymerisation of the single-stranded overhangs between the staggered nicks would then generate the sequence duplications. This leads to a DNA damage-repair model in which various naturally occurring DNA Topo-II poisons induce DNA Topo II-mediated damage in leukaemia pathogenesis [Patients with a t-MN have clonal genomic abnormalities in their bone marrow cells, which frequently are correlated with the type of preceding cytotoxic compound, the amount of drug exposed and the latency period. The classical t-MNs occurred after exposure to alkylating agents, Topo-II inhibitors and/or radiation therapy, and were described after a latency period of 5–10 years, often preceded by a myelodysplastic phase . Nowadayogenesis , 26.KTM2A-r than the latency period of t-MN with unbalanced aberrations, for example, monosomy-5 or monosomy-7 [IDH1, ASXL1, SRSF2, SF3B1, SETBP1, TP53 and KTM2A-r in childhood t-MN [One interesting observation concerns the latency of t-MN and early-age leukaemia. The latency period to develop t-MN is much shorter for nosomy-7 . Importanosomy-7 . Recent ood t-MN . These mAccepting the minimal two-step model for AML, in which the disruptive effect of abnormal gene rearrangements within cell signalling pathways originates a clone, failure in DNA repair system and/or modulation of susceptibility genetic factor, particularly with continued exposure to genotoxic substances, have been the pathogenic ripple, which needs to be uncovered.KTM2A drives essential biological processes through its DNA bind domains either directly (through sequences enriched for AT rich or nonmethylated CpG) or indirectly (through sequence specific transcription factors such as E2Fs), providing interfaces for the assembly of multiprotein complexes and methylate histone H3 at lysine 4 [MLL, nowadays called KMT2A gene, were associated with a dismal outcome in ALL, while in AML, some reports have demonstrated that some KMTA2 partner gene displays either a good or intermediate prognosis [KMT2A occur mainly in 8-kb area between exon 8 and 13 (breakpoint cluster region) and accounts for 5%–10% of all acute leukaemia. In AML, KMT2A-r is recurrently associated with 35%–50% of infants and 5%–10% of therapy-related cases. The identification of the partner gene is necessary to provide optimal treatment stratification at diagnosis and prognosis.lysine 4 . Chromosrognosis , 29. TheMLL recombinome study that has been allowing us to identify new reciprocal translocations, complex rearrangements, internal duplications, deletions or inversions on the 11q23 region where the KMT2A is located and inserted into other chromosomes [MLLrecombinome, the most frequent rearrangements occur either with MLLT3/AF9, MLLT1/ENL, ELL, MLLT10/AF10, MLLT4/AF6 or AFF1/AF4 genes, or are derived from gene internal duplications (MLL-PTDs) representing ~90% of AML cases. Amongst infants, breakpoints occur more frequently in KMT2A intron 11 [MLL-AF6 fusions displayed a clear preference for KMT2A intron 9 recombinations. Their occurrence differed significantly in the cohorts of infant, paediatric and adult leukaemia patients [KMT2A-r in AML differs from ALL, either in infants, paediatric or adult patients, suggesting different biological mechanisms that may also reflect diverging factors. The extensive MLL recombinome analyses at the molecular level have allowed the identification of more than 71 different KMT2A-fusion partners [KMT2A-r, a multiplex RT-PCR (mRT-PCR) technique was standardised to identify the most frequent partners genes, such as MLLT1, MLLT3, MLLT4, MLLT10, AFF1 and the partial tandem duplication (MLL-PTD) [KMT2A fusion partners in our series of i-AML are: MLLT3/AF9 (35.7%), MLLT1/ENL (15.3%), MLLT4/AF6 (14.1%), MLLT10/AF10 (10%), AFF1/AF4 and MLL-PTD (6.1%). KMT2A with other partner genes or nonidentified are still underestimated because of the difficulty in their detection using only conventional cytogenetic technique, FISH and mRT-PCR. The KMT2A-r was morphologically more associated with AML-M4/M5, although was also identified in other AML subtypes, e.g., AML-M7. These groups altogether were associated with adverse prognosis [German investigators with a huge worldwide collaboration have created the omosomes . Accordintron 11 . Howeverpatients . It is wpartners . To systMLL-PTD) . In thisrognosis .The genome-wide technologies that demonstrated the genomic landscape of acute leukaemia may unravel the prenatal origin, throughout mutational signatures associated with environmental exposures.Consensual evidence about risk factors and etiology of c-AML is still a challenge. It is well accepted that ionizing radiation exposures during gestation and genetic syndromes, e.g., DS, and familial monosomy 7 increase the risk of AML . HoweverIt is well known that children are more susceptible to environmental carcinogens than adults due to physiological vulnerability . The immTwo meta-analyses showed that prenatal maternal occupational exposure to pesticides was strongly associated with c-AML. The risk estimates were  and  in each study , 38. Indp for trend = 0.008). Seven out of 204 c-AML had mothers exposed to pesticides for more than 1,000 days; for either parent exposed for more than 1,000 days, the association with c-AML was significant  [p-dioxin used during the Vietnam War. A total of 605 c-AML and individually matched controls were included in the analysis, and results showed that c-AML risk was slightly increased among the offspring of veterans who had served in Vietnam  [A hospital-based case-control study from Children’s Cancer Study Group (CCG), which gathered 204 AML cases younger than 18 years and single-matched controls, showed a suggestive dose-dependent association of maternal occupational exposure to pesticides with c-AML risk in the offspring (1.5–9.7) . Another1.0–2.9) . More re1.0–2.9) . This gr1.0–2.9) .Residential exposure to pesticides during pregnancy was also associated with increased risk to childhood leukaemia, considering unspecified pesticides , insecticides  and herbicides . Specifically, for c-AML, an increased risk association was observed with insecticides  . The samet al that gathered patients with different costumes and genetic backgrounds  and accrued 74 i-AML cases (29 with KMT2A-r identified); they have shown that maternal pesticide exposure during pregnancy was associated with i-AML  [Regarding i-AML, four observational studies demonstrated associations of infant leukaemia with maternal exposure to pesticide during pregnancy, leading to the speculation of transplacental foetal exposure with DNA damage and chromosomal translocation as early pre-leukemic events –48. The 4–14.04) . Then th4–14.04) , 48.RUNX1-RUNXT1 in cord blood samples was about 2-fold higher among those with detectable meconium levels of the methylcarbamate insecticide propoxur [RAS mutations and antecedents of exposure to solvents was observed  [RAS mutation genes have been observed with variable frequencies (~15%–20%) in AML [RAS mutations in early-age leukaemia were modulated by NQO1 rs1800566 (C609T), emphasizing the critical role of genetic susceptibility with somatic mutations in the mechanistic pathway leading to leukaemia in childhood [Children exposure to pesticides occurs in different ways, from foetal period to late childhood, either through contamination of their parents’ work clothes or direct household residues in water, air, soil and food , 49. Newpropoxur . Another.2–18.8) , 53. Thi) in AML  and RAS hildhood .et al have measured the internal dose levels of organochlorines in paired settings of dyads, mother blood and cord blood samples from a rural village  located in the vicinity of an organochlorine-compound factory plant. All newborns presented detectable levels of organochlorine compounds, with higher values in samples from Flix than nearby villages [The applications of various pesticides in agricultural and public health programs have caused severe environmental pollution and health hazards. It is a challenge to evaluate the exposure of individual substances , although, Sala villages . This stet al [Brazil has been the world’s top pesticide market consumer as very well described by Albuquerque et al . Therefoin vitro studies (human cell lines) have demonstrated the genotoxic potential of OP inducing DSB and hypermethylation in CDKN1A [KMT2A-r through caspase 3-pathway with genomic instability and Topo-II inhibition in human foetal liver haematopoietic CD34+ cells [Pesticides are chemical compounds that include insecticides , herbicides , fungicides (dithiocarbamates and captan), fumigants (ethylene dibromide and methyl bromide), rodenticides (anticoagulants to control rodents), and algicides, among others. Since the class of organochlorine came in restricted use, the second line of pesticides, i.e., OP and pyrethroids, became the most common group available . For insn CDKN1A . The genn CDKN1A . It has 4+ cells .in vitro experiments , and are found also in pesticide’s manufacturing workers and sprayers [It has been shown that pesticides may induce oxidative stress by producing free radicals, enhancing lipid peroxidation, causing a drastic activity of antioxidant enzymes in mammalian systems . Moreovesprayers , 64. Thusprayers , 66. Lonsprayers .Pesticide structures may be very stable, which means that they do not break down easily and can remain in the environment long after application and in organisms long after exposure. Some of these compounds present with slow degradation and subsequent bioaccumulation. In summary, the biological evidence linking pesticide compounds suggest that drugs interfering with DNA remodelling by Topo-II can mediate the formation of specific chromosomal translocation breakpoints in a similar pathway as chemotherapy agents.Genetic susceptibility has emerged as an important risk factor for childhood leukaemia, mainly because gene variants have modulated the attributable risk between environmental exposures and diseases. GWS have been providing lists of gene mutations, single nucleotide variations (SNP), expression frequencies and copy number variation (CNV) of candidate genes as potential biomarkers for genomic instability and novel therapeutic targets for children and adult cancer , 69. HowGenetic syndromes are well established factors associated with high predisposition for childhood leukaemia and it accounts for about 10% of cases , 70. A sGSTM1, GSTT1 and NQO1 [CYP1A1 are associated with an increased risk for paediatric leukaemia (ALL and AML) whose parents are smokers (OR: 2.1) [CYP2E1* 5 allele in heterozygosity was associated with the risk of AML in children in Turkey (OR: 4.9) [GSTM1 and GSTT1 genes encode for glutathione S-transferases μ1 and θ1, respectively, which function in detoxifying processes of electrophilic xenobiotics, such as chemical carcinogens, and environmental pollutants, and inactivate secondary metabolites from endogenous oxidative stress. GSTM1 and GSTT1 alleles can both be deleted (null genotypes), which result in the absence of the protein [GSTM1/T1 null genotype conferred increased risks to AML (aOR: 2.14).The equilibrium between the activities of Phase I and II enzymes of the xenobiotic system is essential for the organism’s response to environmental insults to haemostasis, efficiency and maintenance of human genetic material. A systematic review that included the great majority of the studies carried out in this topic has identified about 50 studies that found consistent results with the estimated rates of genetic susceptibility and risk of pediatric leukaemia. The polymorphisms most frequently investigated were those located in the genes of the cytochrome P450 family (CYPs), and NQO1 . Some stOR: 2.1) , while COR: 4.9) . The GST protein –77. In oNQO1 609C > T (rs1800566) known as NQO1*2 allele encodes a nonsynonymous mutation (Pro187Ser) that confers the lack of enzyme activity, and has been associated with the increased risk for benzene poisoning among benzene-exposed workers (7.6-fold rate) [NQO1gene encodes for a cytosolic flavoenzyme, NAD(P)H dehydrogenase quinone 1, that catalyses the two-electron reduction of quinones to hydroquinones, preventing the formation of ROS generated by redox cycling of semiquinones, thus functioning as a detoxifying enzyme [The genetic variant ld rate) . The NQOg enzyme .NQO1 C609T (P187S) polymorphisms have been strongly associated with the risk of childhood ALL particularly for IL with KMT2A-r [NQO1 C609T modified risk in pediatrics leukaemia depending on age range and the variant genotype in combinations with other gene polymorphisms, such as Paraoxonase 1 (PON1) gene variant [PON1 polymorphisms A21439G (PON1 Q192R) and T12801A (PON1 L55M) cause variability in enzyme activity, affecting its sensitivity to xenobiotic metabolism in young individuals [NQO1 and PON1 polymorphisms associated with early-age leukaemia considering acquiring genomic aberrations and age at the onset of the disease. We have found that infant leukaemia with KTM2A-r was strongly associated with NQO1 C609T variant genotypes (OR: 2.93), while PON1L55M polymorphism increased the risk of ALL in children aged ≥13 months (OR: 3.2) [The detoxification enzyme NAD(P)H: quinoneoxidoreductase (NQO1) is a flavoenzyme that detoxifies benzene metabolites, quinines and azo-dyes. NQO1 has an important function to protect cells against mutagenecity from free radicals and oxygen metabolites . The NQO KMT2A-r . We have variant . PON1 fu variant . The mosividuals . We haveOR: 3.2) .KTM2A positive. BER is crucial in the elimination process of single-strand damages with reinsertion of band religation. In homeostasis, the XRCC1 is responsible for recruitment and anchoring ligase-enzymes during the double-strand religation. So far, XRCC1 399A > G confers an increased risk for i-AML as whole. In addition, in our investigation, the DIP3-XRCC4 was found associated with infants KTM2A-r (OR: 3.2). In a model system to investigate the function of XRCC genes, the DIP3  of XRCC4 had substantially decreased the XRCC4 protein levels leading to reduced cellular ligase IV activity in vitro and in vivo studies.Genetic polymorphisms conferred by SNPs in the DNA repair genes pathways have also been associated with genetic susceptibility to paediatric leukaemia. For instance, a Taiwanian study, which included 266 cases of paediatric leukaemia, found that XRCC4 (x-ray repair cross complementing 4) haplotypes, composed of SNPs rs6869366 and rs28360071, were associated with the increased risk for the disease, without distinguishing subtypes [KTM2A-r and is associated with transplacental exposures. We have comprehensively summarised the epidemiologic and biological evidence that sustains the hypothesis of leukaemogenesis initiating upon transplacental maternal exposures. Few observational studies have estimated the risk association of i-AML with maternal exposures during pregnancy to pesticide compounds. We have put together the results of the Brazilian studies and the review of mechanistic investigations that support the biological plausibility of observational findings. The genomewide technologies that demonstrated the genomic landscape of acute leukaemia might unravel the prenatal origin throughout mutational signatures, and the challenge is to combine cell biology associated with environmental exposures. Thus, future work should explore the associations of genes involved in cell detoxification concerning the myeloid compartment and target for AML.In this review, we have revisited early-age AML, a group that comprises distinct AML with The authors declare that they have no conflicts of interest.


However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)–biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.Ca


It is suggested that intracellular calcium mainly contributes to the cold-induced [Ca2+]cyt response by entering into the cytosol. Two calcium-permeable mechanosensitive channels, MCA1 and MCA2 (mid1-complementing activity), have been identified in Arabidopsis. Here, we demonstrate that MCA1 and MCA2 are involved in a cold-induced increase in [Ca2+]cyt. The cold-induced [Ca2+]cyt increase in mca1 and mca2 mutants was markedly lower than that in wild types. The mca1 mca2 double mutant exhibited chilling and freezing sensitivity, compared to wild-type plants. Expression of At5g61820, At3g51660, and At4g15490, which are not regulated by the CBF/DREB1s transcription factor, was down-regulated in mca1 mca2. These results suggest that MCA1 and MCA2 are involved in the cold-induced elevation of [Ca2+]cyt, cold tolerance, and CBF/DREB1-independent cold signaling.Cold shock triggers an immediate rise in the cytosolic free calcium concentration ([Ca The stimulus-specific spatiotemporal patterning of [Ca2+]cyt dynamics is called the Ca2+ signature4, and to create these signatures, Ca2+ influx channels and Ca2+ efflux transporters that permit transient increases in [Ca2+]cyt are required5.Calcium ions are used as secondary messengers in eukaryotic cells. The cytosolic Ca2+ signals remains unknown. To identify the spatiotemporal patterning of [Ca2+]cyt dynamics, recombinant aequorin has been introduced as a reporter of [Ca2+]cyt changes in plant systems6. In Arabidopsis plants expressing aequorin in the cytoplasm, low temperature triggers an immediate and transient rise in [Ca2+]cyt8. The final temperature and cooling rate are important for sensing low temperature in Arabidopsis9. In mammals, many TRP  channels, which are a specific class of ion channels, function as intracellular Ca2+ release channels10. Some of these channels also function as thermosensors10, and TRPA1 seems to act as a sensor for cold13. Although no proteins with high similarity to TRP channels have been identified in land-plant genomes, the genes for Cr-TRP proteins are encoded in the genomic sequence of the alga Chlamydomonas reinhardtii and show functional properties that are similar to those of mammalian TRP channels14.How plant cells generate stimulus-specific Ca2+-permeable mechanosensitive channels, named MCA1 and MCA2 (mid1-complementing activity 1 and 2), have been identified in Arabidopsis19. Both MCA1 and MCA2 complement deficiency of Ca2+ uptake in yeast cells lacking a Ca2+ channel composed of the Mid1 and Cch1 subunits16. It should be noted that this complementation activity is detected under conditions that allow the Mid1/Cch1 channel to function as the sole Ca2+ influx system in yeast cells, suggesting that MCA1 and MCA2 can directly mediate Ca2+ influx in the cells lacking both Mid1 and Cch1. Electrophysiological studies have shown that both MCA1 and MCA2 produce stretch-activated currents when expressed in Xenopus laevis oocytes17. These results with yeast cells and Xenopus oocytes suggest that MCA1 and MCA2 mediate Ca2+ influx as mechanosensitive channels, and are not accessory factors that facilitate Ca2+ influx. Overexpression of MCA1 enhances an increase in [Ca2+]cyt upon hypoosmotic shock15. The mca2 mutant exhibits a defect in Ca2+ uptake from the roots16. Structurally, MCA1 and MCA2 have 74% identity and 89% similarity in amino acid sequences15. Both have a single transmembrane segment and an EF-hand-like motif and coiled-coil motif in the N-terminal region, as well as a plac8 motif in the C-terminal region18. MCA1-GFP and MCA2-GFP are localized to the plasma membrane15. MCA1 and MCA2 form a homotetramer20. Topological analysis has indicated that the EF-hand-like motif, the coiled-coil motif, and the plac8 motif are present in the cytoplasm18, suggesting that both channels recognize intracellular Ca2+. The MCA genes are conserved in the plant kingdom21, and an increase in [Ca2+]cyt as a result of hypo-osmotic shock is mediated by MCA proteins in rice and tobacco23.Two Ca2+-permeable channels are transiently activated by cold shock in Arabidopsis mesophyll cells7. In plants, extracellular freezing causes dehydration and mechanical stresses on the plasma membrane, and cold-acclimated plant plasma membranes become resistant to mechanical stress24. Expression of CBF2 is induced not only by cold, but also by mechanical stress25. Therefore, it is assumed that mechanical stress may be one of the factors involved in cold acclimation.Application of the patch-clamp technique has demonstrated that Ca26. CBF/DREB1 genes are rapidly and transiently induced after cold treatment27, and overexpression of CBF/DREB1 constitutively enhances freezing tolerance29. Under cold stress, CBF/DREB1 proteins bind to CRT/DRE cis-elements in the promoter of cold-regulated (COR) genes and induce transcription28. However, gene expression analyses reveals that only 6.5% of the total COR genes are regulated by CBF/DREB130. In addition to CBF/DREB1 genes, 27 transcription factors that were up-regulated at an early stage after cold treatment were considered as first-wave transcription factors30. Use of the cbf1/2/3 triple mutant showed that six first-wave transcription factors are partially regulated by CBF/DREB1, whereas the transcription factors HSFC1, ZAT12, and CZF1, which regulate cold-regulated genes31, are not regulated by CBF/DREB132. As acclimated cbf1/2/3 triple mutants are more tolerant of freezing stress than non-acclimated ones33, and the expression of a large number of cold-regulated genes is not affected by the cbf1/2/3 triple mutation32, a CBF/DREB1-independent pathway may control cold tolerance. Overexpression of HSFC1 enhances cold tolerance without an increase in expression of CBF1, CBF2, or CBF330, suggesting that HSFC1 is one of the important transcription factors controlling non-CBF/DREB1 regulons and cold tolerance.Three CBF/DREB1 (C-repeat binding factor/DRE binding factor 1) transcription factors have been extensively studied. They belong to the AP2/ERF  superfamily and are important factors for cold acclimation in plants2+]cyt upon cold shock. The cold-induced increase in [Ca2+]cyt was smaller in the mca1 and mca2 mutants than in the Col-0 wild type. The mca1 mca2 double mutant exhibited increased sensitivity to chilling and freezing stresses. These results suggest that MCA1 and MCA2 are involved in cold-induced Ca2+ influx and that the reduced [Ca2+]cyt increase caused by the mca1 and mca2 mutations affects cold acclimation. As the CBF/DREB1 genes and their regulon genes were not down-regulated in the mca1 mca2 mutant, MCA may not be involved in the regulation of CBF/DREB1-dependent cold signaling.Here, we demonstrate that MCA1 and MCA2 are involved in a transient rise in [Ca2+]cyt (the cytosolic concentration of Ca2+), Arabidopsis seedlings expressing aequorin, a Ca2+ indicator15, that had been immersed in MS medium (400 µl) at 22 °C were exposed to low temperatures by the addition of MS medium (500 µl) kept at 3, 10, or 22 °C. [Ca2+]cyt in the wild type was significantly increased by a 3 °C shock  that is insensitive to the blockers we used in the plasma membrane, or that is in the intracellular compartment.Since MCA1 and MCA2 are present in the plasma membraneGTA Fig. , La3+  than that of the mca single mutants and the wild type, even before it was subjected to freezing temperatures, and it increased as the freezing temperature was lowered  must exist in the plasma membrane and/or organellar membranes. Indeed, it is reported that the vacuole, the major intracellular Ca2+ store, is involved in a cold-induced Ca2+ release8. In the present study, we did not calibrate the bioluminescent intensity of aequorin for [Ca2+]cyt because of difficulties in the precise calibration, although we noted a report describing a successful calibration specific for the isoform of aequorin and temperature that the authors used8. It should be mentioned that although the present study has clearly suggested the involvement of MCA1 and MCA2 in cold-induced [Ca2+]cyt increases, it remains to be examined whether complementation lines of mca1, mca2 and mca1 mca2 mutants expressing aequorin show a wild-type level of cold-induced [Ca2+]cyt increases.Different stimuli produce different patterns of Ca2+-regulated gene expression36. However, it is still unclear how cold-induced [Ca2+]cyt increases are recognized. One possible mechanism involves calmodulin-binding transcription factors (CAMTAs). CAMTAs possess calmodulin (CaM)-binding domains37 and CAMTAs play a role in the regulation of gene expression in response to Ca2+ signals38. CAMTA3 is a positive regulator of CBF2/DREB1C expression and binds to the consensus sequence of a CGCG core motif, a cis-element for CAMTAs, in the promoter of CBF2/DREB1C39. The camta2 camta3 double mutant is sensitive to freezing temperatures39. Microarray analyses demonstrated that the expression level of HSFC1 (At3g24520) in the camta1/2/3 mutant is lower than that in the wild type40. CAMTA is one of possibilities how increased [Ca2+]cyt is recognized.Plants employ several kinds of mechanisms to control Camca1 mca2 double mutation reduced about 40% of the [Ca2+]cyt increase. The transient increase in [Ca2+]cyt may be conducted by other Ca2+-permeable channels that are responsible for 60% of the transient increase. One such channel could be AtGLR3.4, a member of the Arabidopsis homologs of ionotropic glutamate receptors, whose expression is up-regulated under cold stress41. Another such channel could be the cyclic nucleotide-gated ion channel (CNGC) family. In rice, the expressions of 10 out of 16 CNGC genes are induced under cold stress42.As shown in Fig. mca1 mca2 double mutant exhibited increased sensitivity to chilling and freezing stresses, even though the single mutants did not exhibit a severe phenotype  of Arabidopsis and its isogenic, transgenic lines −2 s−1 light intensity were used to monitor [Ca2+]cyt changes upon cold shock. A seedling was harvested 14 days after sowing and incubated overnight at 22 °C in 2 ml of MS medium containing 2.5 µM coelenterazine in the dark to reconstitute aequorin. The seedling was transferred to fresh MS medium (400 µl) kept at 22 °C in a tube  and received an additional 500 µl of the same medium kept at 3, 10, or 22 °C. Luminescence (L) from aequorin in the whole seedlings was measured using a luminometer . At the end of each monitoring, 1 ml of 20% ethanol/2 M CaCl2 solution was added to the medium  to measure the maximum luminescence (Lmax). The luminescence ratios (L/Lmax) are presented in Figs Apoaequorin-expressing seedlings grown at 22 °C on MS medium supplemented with 0.8% agar and 1% sucrose under 16-h light conditions at 40–60 µM mmca1, mca2, and mca1 mca2 plants were grown at 24 °C for 3 weeks in soil with fluorescent lighting (16 h/8 h light/dark photoperiod). These plants were then incubated at 4 °C for 1 week for acclimation to low temperatures. For non-acclimation, 3-week-old plants were treated with the freezing temperature without incubation at 4 °C. Whole-plant freezing assays were performed as previously described44. Briefly, plants were incubated at 0 °C for 1 h, and the temperature was lowered by 2 °C h−1 until it reached to the indicated temperature, and then held at the desired temperature for 1 h or 4 h for non-acclimated plants or cold-acclimated plants, respectively, in the incubator . After cold acclimation, the plants were incubated at 4 °C overnight and transferred to 24 °C. The survival ratio was determined 1 week after the freezing test.Wild-type (ecotype Col-0), A646 + 7.18 A66345.For the chilling assay, 5-day-old plants were incubated at 4 °C. After incubation for 1 month under constant illumination, the chlorophyll content of the plants was determined. Eighty percent acetone was added to leaves ground with liquid nitrogen. The mixture was shaken at 4 °C. After centrifugation, the absorbances of the supernatant at 663 nm and 646 nm were measured. Total chlorophyll content was calculated as 17.3 47. The sample was incubated in a refrigerated circular bath . The conductivity was measured with a conductivity meter .Electrolyte leakage from fully developed rosettes of leaves of three-week-old plants was measured as previously describedmca1, mca2, and mca1 mca2 plants were subjected to cold treatment at 4 °C for the indicated time. Isolation of total RNA, cDNA synthesis, and quantitative RT-PCR were performed as previously described47. The primers used to detect CBF/DREB1 and its regulon genes were also previously described47. Other genes were detected with gene-specific primers for At5g61820 (5′-GAGGCACCTGCGAGAAGCTTGAG-3′ and 5′ GTAACCATCTTCCCGTTTCTGTC-3′), At3g51660 (5′-GACCTCAAAACTTAGTGATGGTG-3′ and 5′-TTAACTTGTTTGGTGATGCCTCC-3′), At4g15490 (5′-CCTCCCATGGAAGGGACATTTGTAGA-3′ and 5′-ACAAGCAATCGCAGGATGAGCCA-3′), MCA1 (5′-AAGATTGCCACTGCAGCATCC-3′ and 5′-ACGCCATTAGCTCATTACATGCTTC-3′), and MCA2 (5′-AAGATCATTGCAACACCGTGGA-3′ and 5′-GTGTCTTCAAGCAAAGACAAGGTTC-3′).Three-week-old wild-type (ecotype Col-0), Supplementary Information