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

This corrects the article DOI: 10.1038/cdd.2015.26.

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.

Small molecules, big effects: the role of microRNAs in regulation of cardiomyocyte death.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranscriptional regulation of gene expression, and exerting regulatory roles in plethora of biological processes. In recent years, miRNAs have received increased attention for their crucial role in health and disease, including in cardiovascular disease. This review summarizes the role of miRNAs in regulation of cardiac cell death/cell survival pathways, including apoptosis, autophagy and necrosis. It is envisaged that these miRNAs may explain the mechanisms behind the pathogenesis of many cardiac diseases, and, most importantly, may provide new avenues for therapeutic intervention that will limit cardiomyocyte cell death before it irreversibly affects cardiac function. Through an in-depth literature analysis coupled with integrative bioinformatics (pathway and synergy analysis), we dissect here the landscape of complex relationships between the apoptosis-regulating miRNAs in the context of cardiomyocyte cell death (including regulation of autophagy-apoptosis cross talk), and examine the gaps in our current understanding that will guide future investigations.

Cytometry of apoptosis. Historical perspective and new advances.

Characteristic changes in cell morphology paralleled by the appearance of a multitude of molecular and biochemical markers occur during apoptosis. These changes vary depending on the cell type, mechanism of induction of apoptosis, and the time-window at which the process of apoptosis is analyzed. By virtue of the capability of rapid measurement of individual cells the flow- and imaging-cytometry become preferred technologies to detect, identify and record incidence of apoptosis in large cell populations. It also provided a valuable tool to investigate molecular mechanisms in field of necrobiology. This review outlines the progress in development of the most commonly used cytometric methods probing cells death based on analysis of fragmentation of DNA, activation of caspases, analysis of mitochondrial potential, alterations in plasma membrane structure and other features that characterize programmed cell death. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later"

Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin.

We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.

Self-adhesive microculture system for extended live cell imaging.

Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.

Gherlin expression in women with polycystic ovary syndrome--a preliminary study.

The etiology and pathogenesis of polycystic ovary syndrome (PCOS) is still unknown. Using real-time PCR, we detected that polycystic ovaries showed almost ten times lower expression of ghrelin mRNA than normal ovaries, whereas the mRNA levels in blood cells were similar in both study groups. This suggests that the presence of ghrelin in PCOS and normal ovaries may have an autocrine/paracrine modulatory effect on ovary functions and local significance in the etiology of PCOS.

Impact of health education on knowledge and prevention behavior for congenital toxoplasmosis: the experience in Poznan, Poland.

In 1991-1997 educational activities were undertaken in the Poznan region of Poland to promote health education for the prevention of toxoplasmosis. The effect of education was measured in 2710 pregnant women by a questionnaire survey. Knowledge of toxoplasmosis and its prevention was almost doubled within 4 years. Similarly, the proportion of women having antenatal serological tests for toxoplasmosis significantly increased. In the examined population the knowledge of how Toxoplasma gondii is transmitted/acquired was better than the knowledge of individual risk factors for congenital toxoplasmosis. Correct hygienic behaviors in pregnancy were often practised by women who lacked good knowledge of toxoplasmosis. The experience from this study suggests the possible effectiveness of including prevention of toxoplasmosis into the whole package of preventing infectious diseases in pregnancy and into healthy lifestyle promotion. Health educational activities need to be realized by modern promotional technologies in addition to making available traditional written educational texts. There is a considerable role of medical services in promotion of a hygienic behavior in pregnant women preventing congenital toxoplasmosis in their offspring. Health education should be especially tailored to the population of pregnant women below the age of 21.