Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

From mitochondrial dynamics to arrhythmias

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Δψ_m, visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Δψ_m, NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Δψ_m under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.     

Spectral analysis of photoplethysmographic signals: The importance of preprocessing

Heart rate variability (HRV) is an important and useful index to assess the responses of the autonomic nervous system (ANS). HRV analysis is performed using electrocardiography (ECG) or photoplethysmography (PPG) signals which are typically subject to noise and trends. Therefore, the elimination of these undesired conditions is very important to achieve reliable ANS activation results. The purpose of this study was to analyze and compare the effects of preprocessing on the spectral analysis of HRV signals obtained from PPG waveform. Preprocessing consists of two stages: filtering and detrending. The performance of linear Butterworth filter is compared with nonlinear weighted Myriad filter. After filtering, two different approaches, one based on least squares fitting and another on smoothness priors, were used to remove trends from the HRV signal. The results of two filtering and detrending methods were compared for spectral analysis accomplished using periodogram, Welch's periodogram and Burg's method. The performance of these methods is presented graphically and the importance of preprocessing clarified by comparing the results. Although both filters have almost the same performance in the results, the smoothness prior detrending approach was found more successful in removing trends that usually appear in the low frequency bands of PPG signals. In conclusion, the results showed that trends in PPG signals are altered during spectral analysis and must be removed prior to HRV analysis.     

Biological parameters and current status of European eel (Anguilla anguilla Linnaeus, 1758) from Asi River,Northeastern Mediterranean region,Turkey

The biological characteristics of eels from the Asi River, Turkey, were assessed between December 2017 and November 2018. Eels were sampled monthly using fyke nets (operated by professional fishermen), yielding a total of 509 specimens. Total length and weight were measured, sex, age and maturity stages (silver or yellow eel) were determined. Catch Per Unit Effort (CPUE) was calculated for both biomass per unit effort and numbers caught on a monthly and annual basis. The length-weight relationships (LWRs) of silver and yellow eel was W = 0.009*TL^3.22 (n = 262) and W = 0.0106*L^3.09 (n = 247), respectively. The age of the sampled fish ranged from year class II to VI. Von Bertalanffy growth parameters were estimated as L_∞=69.25 cm, K = 0.43 1/year, t₀= −0.41 and phi prime index ( ǿ )= 3.31 for all samples. The overall eel fishing mortality rate (F) was 0.31 year^-1, and the exploitation and survival rates of silver stage eels were estimated with 30% and 39%, respectively.     

A new colorimetric DPPH• scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs

2,2-Diphenyl-1-picrylhydrazyl (DPPH^?) radical scavenging, the most commonly used antioxidant method with more than seventeen thousand articles cited, is very practical; however, as with most assays, it has the major disadvantage of dependence on a spectrophotometer. To overcome this drawback, the colorimetric determination of the antioxidant activity using a scanner and freely available Image J software was developed. In this new method, the mixtures of solutions of DPPH^? and standard antioxidants or extracts of common medicinal herbs were dropped onto TLC plates, after an incubation period. The spot images were evaluated with Image J software to determine CSC₅₀ values, the sample concentrations providing 50% colour reduction, which were very similar with the SC₅₀ values obtained with spectrophotometric method. The advantages of the new method are the use of lower amounts of reagents and solvents, no need for costly spectrophotometers, and thus significantly lowered costs, and convenient implementation in any environment and situation.     

Wilson disease mutations associated with uncommon haplotypes in mediterranean patients

This study reports 12 novel mutations of the Wilson disease (WD) gene which have been detected by the molecular analysis of 29 patients of Mediterranean descent carrying uncommon chromosomal haplotypes at the WD locus. These mutations include two nonsense, one splice site and nine missense. The missense mutations lie in regions of the WD gene critical for its function, such as the transmembrane region, the transduction domain and the ATP loop and ATP-binding domain, indicating that they are disease-causing mutations. These new findings improve our knowledge for the role played by functional domains on the ATP7B function. Received: 20 March 1996