DNA damage associated with ultrastructural alterations in rat myocardium after loud noise exposure.

Noise exposure causes changes at different levels in human organs, particularly the cardiovascular system, where it is responsible for increasing heart rate, peripheral vascular resistance, and blood pressure. In this study, we evaluated the effect of noise exposure on DNA integrity and ultrastructure of rat cardiomyocytes. The exposure to loud noise (100 dBA) for 12 hr caused a significant increase of DNA damage, accompanied by swelling of mitochondrial membranes, dilution of the matrix, and cristolysis. These alterations were concomitant with increased in situ noradrenaline levels and utilization. Genetic and ultrastructural alterations did not decrease 24 hr after the cessation of the stimulus. An elevated oxyradical generation, possibly related to altered sympathetic innervation, is hypothesized as responsible for the induction and persistence of noise-induced cellular damage

DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions

Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues

Proteolysis-inducing factor, a cachexia-inducing tumour product, is an N-glycosylated peptide with homology to the unglycosylated neuronal survival peptide Y-P30 and a predicted product of the dermcidin gene, a pro-survival oncogene in breast cancer. We aimed to investigate whether dermcidin is pro-survival in liver cells, in which proteolysis-inducing factor induces catabolism, and to determine the role of potentially glycosylated asparagine residues in this function. Reverse cloning of proteolysis-inducing factor demonstrated ∼100% homology with the dermcidin cDNA. This cDNA was cloned into pcDNA3.1+ and both asparagine residues removed using site-directed mutagenesis. In vitro translation demonstrated signal peptide production, but no difference in molecular weight between the products of native and mutant vectors. Immunocytochemistry of HuH7 cells transiently transfected with V5-His-tagged dermcidin confirmed targeting to the secretory pathway. Stable transfection conferred protection against oxidative stress. This was abrogated by mutation of both asparagines in combination, but not by mutation of either asparagine alone. These findings suggest that dermcidin may function as an oncogene in hepatic as well as breast cells. Glycosylation does not appear to be required, but the importance of asparagine residues suggests a role for the proteolysis-inducing factor core peptide domain

HUWE1 E3 ligase promotes PINK1/PARKINindependent mitophagy by regulating AMBRA1 activation via IKKa

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al

Starvation Induced Cell Death in Autophagy-Defective Yeast Mutants Is Caused by Mitochondria Dysfunction

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants

Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo

Pancreatic cancer is highly resistant to the currently available chemotherapeutic agents. Less than 5% of patients diagnosed with this disease could survive beyond 5 years. Thus, there is an urgent need for the development of novel, efficacious drugs that can treat pancreatic cancer. Herein we report the identification of artesunate (ART), a derivative of artemisinin, as a potent and selective antitumor agent against human pancreatic cancer cells in vitro and in vivo. ART exhibits selective cytotoxic activity against Panc-1, BxPC-3 and CFPAC-1 pancreatic cancer cells with IC50 values that are 2.3- to 24-fold less than that of the normal human hepatic cells (HL-7702). The pan caspase inhibitor zVAD-fmk did not inhibit the cytotoxic activity of ART. Electron microscopy of ART-treated cells revealed severe cytoplasmic swelling and vacuolization, swollen and internally disorganized mitochondria, dilation (but not fragmentation) of the nuclei without chromatin condensation, and cell lysis, yielding a morphotype that is typical of oncosis. The ART-treated cells exhibited a loss of mitochondrial membrane potential (ΔΨm) and ART-induced cell death was inhibited in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC). Importantly, ART produced a dose-dependent tumor regression in an in vivo pancreatic cancer xenografts model. The in vivo antitumor activity of ART was similar to that of gemcitabine. Taken together, our study suggests that ART exhibits antitumor activity against human pancreatic cancer via a novel form of oncosis-like cell death, and that ART should be considered a potential therapeutic candidate for treating pancreatic cancer

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

LRRK2 deficiency induced mitochondrial Ca2+ efflux inhibition can be rescued by Na+/Ca2+/Li+ exchanger upregulation

Variants of leucine-rich repeat kinase 2 (lrrk2) are associated with an increased risk in developing Parkinson’s disease (PD). Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012