Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR

Dual Functions of ASCIZ in the DNA Base Damage Response and Pulmonary Organogenesis

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis

Novel function of the DNA damage response protein ASCIZ as an essential transcription factor

© 2012 Dr. Sabine JuradoDNA damage repair pathways are important to preserve genome integrity and thereby prevent the onset of cancer. In addition, these pathways are necessary to promote physiological processes such as B cell diversification. ASCIZ is a recently discovered protein with reported roles in the DNA damage response and antibody diversification process. The aim of this thesis was to obtain a better understanding of ASCIZ’ function(s). My original hypothesis was that ASCIZ plays a role in the repair of accidental and programmed DNA damage, and that it is specifically involved in the base excision repair (BER) pathway. Supporting this hypothesis, primary cells derived from Asciz KO mice exhibited a modest hypersensitivity to DNA base damaging agents MMS and H2O2 compared to WT and senesced earlier than WT under normoxic conditions, reflecting an impaired oxidative damage response. In addition, germline deletion of Asciz lead to late embryonic lethality and growth retardation, a phenotype not rescued by additional deletion of p53, indicating that ASCIZ might have DNA damage independent functions. This was confirmed in detailed embryology analyses, which revealed a complete absence of lungs in Asciz KO embryos. This defect might be due to a novel function of ASCIZ as a classical Zn2+ finger transcription factor. The main transcriptional target of ASCIZ appears to be the multifunctional protein dynein light chain 1 (Dynll1). Interestingly, DYNLL1 can in turn directly bind to ASCIZ and modulate its transcription factor activity, allowing for a feedback loop to control DYNLL1 levels within the cell. Based on ASCIZ’ function as a BER repair protein, it was expected that ASCIZ-deficiency would lead to defective antibody maturation in the periphery upon antigen-presentation. This hypothesis was tested in mice where ASCIZ was conditionally deleted in order to bypass the embryonic lethality. However, ASCIZ deficiency lead to dramatically reduced peripheral B cell numbers due to impaired B cell development from the pre-B cell stage in the bone marrow. Again, this defect was not rescued by additional p53 deletion, but was suppressed by ectopic restoration of DYNLL1 expression or deletion of its downstream target Bim, indicating that ASCIZ regulates B cell development mainly through its transcriptional activity. Taken together, the results presented in this thesis indicate that ASCIZ has dual functions as a DNA base damage response protein and as an essential transcription factor

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and doublestranded RNA into ~21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic

Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development

Humidity-induced CO 2 capture enhancement in Mg-CUK-1

International audienceKinetic CO2 adsorption measurements in the water-stable and permanently microporous Metal–organic framework material, Mg-CUK-1, reveal a 1.8-fold increase in CO2 capture from 4.6 wt% to 8.5 wt% in the presence of 18% relative humidity. Thermodynamic CO2 uptake experiments corroborate this enhancement effect, while grand canonical Monte Carlo simulations also support the phenomenon of a humidity-induced increase in the CO2 sorption capacity in Mg-CUK-1. Molecular simulations were implemented to gain insight into the microscopic adsorption mechanism responsible for the observed CO2 sorption enhancement. These simulations indicate that the cause of increasing CO2 adsorption enthalpy in the presence of H2O is due to favorable intermolecular interactions between the co-adsorbates confined within the micropores of Mg-CUK-1

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn(2+)-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis

The transcription factor ASCIZ and its target DYNLL1 are essential for the development and expansion of MYC-driven B cell lymphoma

How MYC promotes the development of cancer remains to be fully understood. Here, we report that the Zn2+-finger transcription factor ASCIZ (ATMIN, ZNF822) synergizes with MYC to activate the expression of dynein light chain (DYNLL1, LC8) in the murine Eμ-Myc model of lymphoma. Deletion of Asciz or Dynll1 prevented the abnormal expansion of pre-B cells in pre-cancerous Eμ-Myc mice and potentiated the pro-apoptotic activity of MYC in pre-leukemic immature B cells. Constitutive loss of Asciz or Dynll1 delayed lymphoma development in Eμ-Myc mice, and induced deletion of Asciz in established lymphomas extended the survival of tumor-bearing mice. We propose that ASCIZ-dependent upregulation of DYNLL1 levels is essential for the development and expansion of MYC-driven lymphomas by enabling the survival of pre-neoplastic and malignant cells