Widespread mitochondrial depletion via mitophagy does not compromise necroptosis

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence

Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing

Background As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients’ samples. Method We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals’ sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. Results Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0–60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6–56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13–60 vs. pooled sputum MAR 25.3, SD 14.6, range 1–54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12–3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. Conclusion We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population “bubble”

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia chromosome-positive cells

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease

ERCC1 expression correlated with EGFR and clinicopathological variables in patients with non-small cell lung cancer. An immunocytochemical study on fine-needle aspiration biopsies samples

Purpose: Expression of ERCC1 has not been well described in fine-needle aspiration biopsies (FNABs) in patients with non-small cell lung cancer (NSCLC). We investigated the expression of ERCC1 in correlation with EGFR expression and clinicopathological factors in patients with NSCLC in order to determine if these play a role in the prognosis of the disease. Methods: We studied 45 patients, 34 with adenocarcinoma and 11 with squamous cell carcinoma. Of these 45 patients, 35 were males and 10 females, aged between 45 and 83 years, 30 smokers and 15 non-smokers. Eighteen (18) tumors were of stage I, twelve (12) stage II and fifteen (15) stage III. To investigate the expression of ERCC1 and EGFR (scores 0, 1, 2, 3), immunocytochemistry was performed on air dried specimens (FNABs) using monoclonal antibodies by alkaline-phosphatase (APAAP) method. Results: ERCC1 expression was detected in tumors from 27 patients (60%) and EGFR in 10 patients (22.2%). ERCC1 was expressed more frequently in males (65.7%) in patients >65 years old (64%), in smokers (66.7%) and in stage I (66.7%). Negative ERCC1 expression was significantly associated with the presence of EGFR. EGFR was expressed only in adenocarcinomas and more frequently in women (70%) and non smokers (53.3%). Conclusions: ERCC1 expression was identified as positive (scores 2+ and 3+) in the majority of NSCLCs and seems to be an independent prognostic marker of longer survival. In addition EGFR expression was positive (scores 2+ and 3+) in the minority of NSCLCs and only in adenocarcinomas, more frequently in ERCC1-negative (scores 0 and 1+) tumors, suggesting that it is not an independent prognostic marker for the outcome of the patients suffering from NSCLC. Resumo: Objetivo: A expressão de ERCC1 não foi ainda suficientemente descrita em biópsias aspirativas por agulha fina (FNAB) em doentes com carcinoma pulmonar de não pequenas células (NSCLC). Investigámos a expressão de ERCC1 em correlação com a expressão EGFR e os fatores clinicopatológicos em doentes com NSCLC para determinar se estes desempenham um papel no prognóstico da doença. Métodos: Estudámos 45 doentes, 34 com adenocarcinoma e 11 com carcinoma de células escamosas. Desses 45 doentes, 35 eram homens e 10 mulheres, com idades entre os 45-83 anos, 30 fumadores e 15 não fumadores. Dezoito tumores encontravam-se no estádio I, 12 no estádio II e 15 no estádio III. Para investigar a expressão de ERCC1 e EGFR (resultados 0, 1, 2, 3), foi realizada imunocitoquímica em espécimes a seco (FNAB), usando anticorpos monoclonais pelo método de fosfatase alcalina (APAAP). Resultados: A expressão ERCC1 foi detetada em tumores de 27 doentes (60%) e a do EGFR em 10 doentes (22,2%). O ERCC1 foi expresso com maior frequência em homens (65,7%), em doentes com mais de 65 anos (64%), em fumadores (66,7%) e no estádio I (66,7%). A expressão ERCC1 negativa foi significativamente associada à presença de EGFR. O EGFR foi expresso apenas em adenocarcinomas e com maior frequência em mulheres (70%) e não fumadores (53,3%). Conclusões: A expressão de ERCC1 foi identificada como positiva (resultados 2+ e 3+) na maioria dos NSCLC e parece ser um marcador de prognóstico independente de maior sobrevivência. Além disso, a expressão de EGFR foi positiva (resultados 2+ e 3+) numa minoria dos NSCLCs e apenas em adenocarcinomas, com maior frequência em tumores ERCC1-negativos (resultados 0 e 1+), sugerindo que não é um marcador de prognóstico independente na evolução de doentes que sofram de NSCLC. Keywords: NSCLC, ERCC1, EGFR, FNABs, Palavras-chave: NSCLC, ERCC1, EGFR, FNAB

Evidence for association of the G1733A polymorphism of the androgen receptor gene with recurrent spontaneous abortions

Objective: To determine whether the G1733A polymorphism of the androgen receptor gene is associated with an increased risk for recurrent spontaneous abortion (RSA). Design: Case-control study. Setting: Division of Genetics and Biotechnology, Department of Biology, University of Athens. Patient(s): A total of 131 women with at least three unexplained spontaneous abortions before 20 weeks&apos; gestation, with the same partner, composed the study group. Intervention(s): Subjects were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. Main Outcome Measure(s): G1733A polymorphism genotypes and allele frequencies. Result(s): The observed frequencies of GG, GA, and AA genotypes of the G1733A polymorphism were 0.57, 0.27, and 0.16, respectively, for the patient group and 0.76, 0.15, and 0.09, respectively, for the control group. Allele frequencies were 0.70 and 0.84, respectively, for the patient and control groups for the G allele (wild type) and 0.30 and 0.16, respectively, for the patient and control groups for the A allele (mutant). Statistical analysis of these results indicated significant differences between the two groups. Conclusion(s): These results indicated for the first time that the androgen receptor G1733A polymorphism is strongly associated with increased risk for RSA. © 2008 American Society for Reproductive Medicine

Autophagy in chronic myeloid leukaemia: stem cell survival and implication in therapy

The insensitivity of Chronic Myeloid Leukaemia (CML) stem cells to Tyrosine Kinase Inhibitor (TKI) treatment is now believed to be the main reason for disease persistence experienced in patients. It has been shown that autophagy, an evolutionarily conserved catabolic process that involves degradation of unnecessary or harmful cellular components via lysosomes, is induced following TKI treatment in CML cells. Of clinical importance, autophagy inhibition, using the anti-malarial drug hydroxychloroquine (HCQ), sensitised CML cells, including primitive CML stem cells, to TKI treatment. In this review we discuss the role of autophagy in the maintenance and survival of stem cells in more detail, with a focus on its role in survival of CML stem cells and the possibility to inhibit this pathway as a way to eliminate persistent CML stem cells in vitro and in patients

Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans

Transposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 108 conidiospores, we obtain up to ~0.8×102 transposition events leading to the expected revertant phenotype (niaD+), while ~16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation. © 2015 Elsevier Inc

Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans.

International audienceTransposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 10(8) conidiospores, we obtain up to ∼0.8×10(2) transposition events leading to the expected revertant phenotype (niaD(+)), while ∼16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation