Lipidomics reveals insights on the biological effects of copper oxide nanoparticles in a human colon carcinoma cell line.

Engineered nanomaterials have unique properties compared to their bulk counterparts. Copper oxide nanoparticles (CuO NPs) are one example of nanomaterials used in a wide range of consumer products due to their conductivity and biocidal properties. While CuO NPs can induce toxicity in various organisms, their interactions with different organisms and how they affect cellular homeostasis is yet to be fully understood. In this work, the toxicity of CuO NPs was evaluated in different human cell lines (colorectal carcinoma, cervical cancer, embryonic kidney, and lung fibroblast), showing a dose-dependent toxicity. An untargeted lipidomics approach using liquid chromatography-quadrupole time of flight mass spectrometry was employed in a human colon carcinoma cell line to investigate the impact of CuO NP exposure at the cellular level. A 24 h CuO NP exposure at 2.5 and 5 µg mL-1 resulted in upregulation of different metabolites: triacylglycerols, phosphatidylcholines, and ceramides accumulated. The most profound increase in a dose-dependent manner was observed in ceramides, specifically in C18:0, C18:1, and C22:0 species, with up to ∼10 fold accumulations. Further experiments suggested that activation of autophagy and oxidative stress could be responsible for the toxicity observed in these cell lines. Increases in the level of glutathione oxide (∼7 fold) also supported the activation of oxidative stress upon CuO NP treatment. Based on the changes in different metabolites induced by CuO NP exposure and previous studies from our laboratory, we propose that autophagy and oxidative stress could play a role in CuO NP-induced toxicity.

Orthotopic Liver Transplantation in an Adult With Biliary Atresia, Situs Inversus, and Inferior Cava Vein Absence: A Case Report.

BACKGROUND: We report the case of a 34-year-old man who underwent Kasai portoenterostomy for biliary atresia at 6 weeks of age. In 2011, pulmonary hypertension was diagnosed and he began treatment with sildenafil. In 2012, he presented with an episode of upper gastrointestinal bleeding secondary to esophageal varices resistant to treatment. Later, he exhibited liver dysfunction. He was included on the waiting list for transplantation on May 29, 2013, with a Model for End-stage Liver Disease score of 24. METHODS: He underwent liver transplantation with an isogroup graft from a brain dead donor on June 9, 2013. Native hepatectomy was laborious owing to important collateral circulation and adhesions after previous operations, which had injured loops of the small bowel (SB). Orthotopic implantation was accomplished with direct anastomosis of the upper liver cava vein to the right atrium of the receiver. Portal and arterial anastomoses were performed as usual. Biliary reconstruction surgery by hepatojejunostomy was delayed 24 hours owing to SB loops injuries. RESULTS: Graft viability was confirmed by normal hepatic function. Postoperative complications included abdominal compartment syndrome treated by decompressing laparotomy, severe pulmonary alveolar hemorrhage resolved with artery embolization and endotracheal intubation, intraabdominal abscess requiring percutaneous drain, and stroke requiring long-term rehabilitation. He is currently asymptomatic, presents normal graft function, and receives sildenafil because of pulmonary hypertension. CONCLUSIONS: The association of situs inversus and biliary atresia is low. There is no consensus on the optimal operative approach to liver transplantation. An individualized assessment and multidisciplinary patient management are required.

Pontin is a critical regulator for AML1-ETO-induced leukemia.

The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Our previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Here, using an in vivo RNA interference screen for suppressors of AML1-ETO activity, we identified pontin/RUVBL1 as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. We further show that PONTIN inhibition strongly impaired the growth of human t(8;21)(+) or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promoted the transcription of PONTIN. Moreover, transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together our data suggest that the upregulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells.

Leukemic fusion genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the telomerase subunit TERT.

MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respectively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogenicity, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal.

Previous antimalarial therapy in patients diagnosed with lupus nephritis: influence on outcomes and survival.

The aim of this study was to analyze the effect of exposure to antimalarial drugs at diagnosis of lupus nephritis on the outcome of the disease, especially renal failure, comorbid processes, and survival. We analyzed a cohort of 206 consecutive patients with biopsy-proven lupus nephritis. Renal biopsies were categorized according to the classification proposed by the ISN/RPS in 2003. Exposure to antimalarial drugs (chloroquine and hydroxychloroquine) was defined as the use of these drugs before the diagnosis of lupus nephritis independent of dose and duration. Fifty-six (27%) patients had received antimalarials before the diagnosis of lupus nephritis. During the follow-up, these patients had a lower frequency of creatinine values >4 mg/dL (2% vs 11%, P = 0.029) and end-stage renal failure (2% vs 11%, P = 0.044) in comparison with those never treated with antimalarials. Patients exposed to antimalarials also had a lower frequency of hypertension (32% vs 50%, P = 0.027), infections (11% vs 29%, P = 0.006), and thrombotic events (5% vs 17%, P = 0.039). Twenty patients (10%) died during the study period. Patients exposed to antimalarials had a lower mortality rate at the end of the follow-up (2% vs 13% for those not exposed to antimalarials, P = 0.029). Multivariate analysis identified thrombosis and infections as statistically significant independent variables. Kaplan-Meier plots showed a lower rate of end-stage renal failure (log rank = 0.04) in patients exposed to antimalarials. In conclusion, exposure to antimalarials before the diagnosis of lupus nephritis was negatively associated with the development of renal failure, hypertension, thrombosis and infection, and with a better survival rate at the end of the follow-up. This, together with other published data, suggests that antimalarials should be considered a mandatory therapeutic option in all patients diagnosed with systemic lupus erythematosus.

siRNA-mediated AML1/MTG8 depletion affects differentiation and proliferation-associated gene expression in t(8;21)-positive cell lines and primary AML blasts.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

Optimised helper virus-free production of high-quality adeno-associated virus vectors.

BACKGROUND: Clinical development of adeno-associated virus (AAV) requires standardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several strategies have been reported for the approach to the manufacturing problem. We report a helper virus-free process that produces high quality rAAV stocks. METHODS: rAAV were produced by triple transfection, a helper virus-free process. After lysis of the cells in the presence of nuclease, the rAAV produced were purified by HPLC through two ion-exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological activity and for the presence of residual contaminants. The titer of infectious particles and of rep + particles was determined by dRA assay. Contaminating DNA and RNA were determined by fluorescent dye binding and real-time PCR. The protein content of the rAAV stocks was characterised by SDS-PAGE, ELISA test, Western blot and specific enzymatic assays for putative residual contaminating protein. The in vivo biological activity of the stocks was evaluated in mouse muscle. RESULTS: rAAV stocks obtained following this procedure elicit: 2-5 x 10(12) pp/ml; 3-6 x 10(10) ip/ml; < 10(3) rep + particles/ml; <0.3 mUeq/ml of residual benzonase activity; non-detectable Ad or beta-galactosidase proteins; <35 pg/ml of cellular genomic DNA; in vivo expression in mouse muscle without any immune reaction detected. CONCLUSIONS: This work demonstrates the possibility of producing purified high-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors.

[Sodium saccharin effect on the mice large intestine]. / Efecto de la sacarina de sodio en el intestino grueso del ratón.

Sodium saccharin has found to be a tumoral promoter in the rat's bladder epithelium, property not demonstrated in humans. Nevertheless, at present there's no references on the possible alterations produced by sodium saccharin in the epithelium of the mice colon. In this work we describe the alterations produced by low doses of sodium saccharin in the epithelium of the mice colon. The changer produced by sodium saccharin consist in pleomorphic microvill with variations of form, length diameter and curvature and demonstrate by transmission electron microscopy.