Evaluation of methods for DNA extraction from Clostridium tyrobutyricum spores and its detection by qPCR.

Clostridium tyrobutyricum is the major agent that causes the blowing defect in cheese due to the germination of its dormant spores during the ripening stage. As a result, many of the affected cheeses show cavities and cracks, which cause the product loss in most cases. Nowadays, there is not a fast method capable of detecting milk contaminated with C. tyrobutyricum spores. The aim of this study has been to develop a fast and reliable method based on real time PCR (qPCR) to detect C. tyrobutyricum spores in raw milk. One of the main limitations has been to find a good procedure for the spore disruption to extract the DNA due to its high resistance. For this reason, different disruption methods have been tested, including chemical agents, bead beating, enzymatic and microwave treatment. Furthermore, an enzymatic treatment with subtilisin was applied for milk clarification and recovery of spores. The comparison of the assayed methods has been made using sterile milk spiked with C. tyrobutyricum spores, obtained in solid or liquid medium. The results showed that microwave treatment followed by a standard DNA purification step was found to be the best disruption method. The Ct values obtained for spores were higher than those found for vegetative cells by qPCR, for the same quantity of DNA. This difference could be due to the action of the Small Acid Soluble Proteins (SASP) in the DNA packaging of spores. Moreover, spores obtained in agar plate were found more resistant to disruption than those obtained in liquid medium. Subtilisin and microwave treatments were found to be successful for DNA extraction from C. tyrobutyricum spores in milk and subsequent identification by qPCR. However, the differences observed between the amplification of DNA from spores obtained in different media and from vegetative cells have to be taken into account to optimize a method for C. tyrobutyricum detection.

Detection of egg and milk residues on work surfaces in school canteens in the Hortaleza District, Madrid and their relevance to children with allergy to these food groups

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Immunotherapy with liposome-bound TRAIL overcomes partial protection to soluble TRAIL-induced apoptosis offered by down-regulation of Bim in leukemic cells

Purpose. Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. Methods/patients. Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. Results. Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. Conclusion. Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics (AU)

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Immunotherapy with liposome-bound TRAIL overcomes partial protection to soluble TRAIL-induced apoptosis offered by down-regulation of Bim in leukemic cells.

PURPOSE: Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. METHODS/PATIENTS: Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. RESULTS: Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. CONCLUSION: Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics.

Two death pathways induced by sorafenib in myeloma cells: puma-mediated apoptosis and necroptosis

Purpose. Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. Methods. Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. Results. Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. Conclusion. Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells (AU)

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Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis.

PURPOSE: Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. METHODS: Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. RESULTS: Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. CONCLUSION: Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.

AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation.

The alkylating DNA-damage agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX(-/-) cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.

BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation.

Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.