Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant gamma-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, gamma-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.     

Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells

Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine-rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG-63) and well-differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real-time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non-metastatic, well-differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG-63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down-regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG-63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.     

Obesity and sleep disturbances: meaningful sub-typing of obesity

Obesity, excessive daytime sleepiness (EDS), and self-reported short sleep duration appear to be on the rise, while there is evidence that obesity and these sleep disorders are strongly connected. In this paper, we review data that challenge the common belief that the sleep apnoea and sleep loss, frequently associated with obesity, are the primary determinants of obesity-related objective daytime sleepiness and subjective fatigue (tiredness without increased sleep propensity). Specifically, obesity is associated with objective and subjective EDS regardless of the presence of sleep apnoea. The association between obesity and EDS was confirmed in recent studies of large random samples of the general population or clinical samples, which showed that the primary determinants of subjective EDS were depression, metabolic disturbances, i.e. obesity/diabetes and insulin resistance, and lack of physical activity, and, secondarily, sleep apnoea or sleep loss. Paradoxically, within the obese, with or without sleep apnoea, those who slept objectively better at night are sleepier (objectively) during the day than those who slept worse. The distinguishing factor between those that slept better vs. those that slept worse appears to be level of emotional stress. Furthermore, many studies reported that obesity is associated with self-reported short sleep duration; however, it appears that short sleep duration is a marker of emotional stress rather than a reflection of true sleep loss. Based on these data, we propose that obesity-related deeper sleep and objective EDS are primarily related to metabolic disturbances, whereas obesity-related poorer sleep and subjective fatigue appear to be the result of psychological distress. Furthermore, based on data from studies in normal controls and patients with sleep disorders, it appears that the interaction of the hypothalamic-pituitary-adrenal (HPA) axis and pro-inflammatory cytokines determines the level of sleep/arousal within the 24-hour cycle, i.e. "eucortisolemia" or "hypocortisolemia" plus hypercytokinemia is associated with high sleep efficiency and objective sleepiness, whereas "hypercortisolemia" plus hypercytokinemia is associated with low sleep efficiency and fatigue. In conclusion, we propose that the above-reviewed data provide the basis for a meaningful phenotypic and pathophysiologic sub-typing of obesity. One subtype is associated with emotional distress, poor sleep, fatigue, HPA axis "hyperactivity," and hypercytokinemia while the other is associated with non-distress, better sleep but more sleepiness, HPA axis "normo or hypoactivity," and hypercytokinemia. This proposed sub-typing may lead to novel, preventive and therapeutic strategies for obesity and its associated sleep disturbances.     

The role of erythropoietin as an inhibitor of tissue ischemia

Erythropoietin is a hypoxia-induced cytokine that stimulates erythropoiesis through the promotion of erythroid precursor cell proliferation and differentiation. Recent evidence supports that erythropoietin has a broad spectrum of tissue protecting actions affecting other systems than hemopoietic. Lately, research has focused on the nonhemopoietic effects of erythropoietin against tissue ischemia due to the unexpected observations of erythropoietin receptor expression by various cells, such as endothelial cells, neuronal cells, cardiac myocytes, and vascular smooth muscle cells. It has been shown that erythropoietin exerts its cardioprotective action during cardiac ischemic injury through reducing the infract size and enhancing new vessel formation over a longer time frame. Erythropoietin plays a crucial role in neuroprotection in many types of ischemic injury in the central and the peripheral nervous system. It is also strongly believed that erythropoietin exhibits a critical role in many other disorders that are pathogenetically related to acute tissue ischemia. This article reviews the proposed implications of erythropoietin in tissue ischemia and discusses the possible mechanisms for this action along with its potential therapeutic applications.     

A novel monoacylglycerol lipase inhibitor with analgesic and anti-inflammatory activity

A variety of long chain 1,2-diamines and related compounds were synthesized and tested for their activity on fatty acid amide hydrolase (FAAH) and monoacyglycerol lipase (MGL). (2S,9Z)-Octadec-9-ene-1,2-diamine selectively inhibits MGL (K(i) 21.8 microM) without significantly affecting FAAH. This compound exhibited interesting in vivo analgesic and anti-inflammatory properties, suggesting that selective inhibitors of MGL may be valuable novel agents for the treatment of inflammatory pain.     

Efficient computation of the phylogenetic likelihood function on multi-gene alignments and multi-core architectures

The continuous accumulation of sequence data, for example, due to novel wet-laboratory techniques such as pyrosequencing, coupled with the increasing popularity of multi-gene phylogenies and emerging multi-core processor architectures that face problems of cache congestion, poses new challenges with respect to the efficient computation of the phylogenetic maximum-likelihood (ML) function. Here, we propose two approaches that can significantly speed up likelihood computations that typically represent over 95 per cent of the computational effort conducted by current ML or Bayesian inference programs. Initially, we present a method and an appropriate data structure to efficiently compute the likelihood score on 'gappy' multi-gene alignments. By 'gappy' we denote sampling-induced gaps owing to missing sequences in individual genes (partitions), i.e. not real alignment gaps. A first proof-of-concept implementation in RAXML indicates that this approach can accelerate inferences on large and gappy alignments by approximately one order of magnitude. Moreover, we present insights and initial performance results on multi-core architectures obtained during the transition from an OpenMP-based to a Pthreads-based fine-grained parallelization of the ML function.     

Development and implementation of microbial sensors for efficient process control in wastewater treatment plants

This paper demonstrates the functionality, laboratory testing and field application of a microbial sensor, which can be modified to monitor organic pollution extent, toxicity and over-(under)load of wastewaters both under anaerobic and aerobic conditions. Since nitrification is related to protons formation and the addition of alkaline is necessary for pH control, an aerobic biosensor monitoring Na₂CO₃ consumption was developed and practically implemented to control the nitrification process. As CO₂ is the respiration product from aerobic degradation which can be correlated to the organic pollution extent, the previous biosensor was modified to monitor and measure the online toxicity and BOD/COD. Under anaerobic conditions, the online measurement of NaOH consumption and biogas production allowed the detection of toxicity incidents and over-(under)load in the influent. Such toximeters get in contact with the wastewater the earliest possible, providing sufficient time for protection of sensitive biological wastewater treatment processes and for the implementation of control and management strategies.     

TOM software toolbox: acquisition and analysis for electron tomography

Automated data acquisition procedures have changed the perspectives of electron tomography (ET) in a profound manner. Elaborate data acquisition schemes with autotuning functions minimize exposure of the specimen to the electron beam and sophisticated image analysis routines retrieve a maximum of information from noisy data sets. "TOM software toolbox" integrates established algorithms and new concepts tailored to the special needs of low dose ET. It provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis. Designed as a collection of computational procedures it is a complete software solution within a highly flexible framework. TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications.     

How lipophilic cannabinergic ligands reach their receptor sites

It is postulated that lipophilic ligands reach their sites of action on membrane-bound functional proteins through fast lateral diffusion across the membrane bilayer. We have shown using NMR experiments that such ligands when incorporated in a membrane system assume a preferred orientation and conformation. While occupying a specific location within the bilayer, these molecules undergo fast lateral diffusion which allows them to engage in productive interactions with their respective protein sites of action. The proposed model is discussed using a group of classical and non-classical cannabinoids as well as the endogenous cannabinoid ligand anandamide.     

Neuromodulation via conditional release of endocannabinoids in the spinal locomotor network

Endocannabinoids act as retrograde signals to modulate synaptic transmission. Little is known, however, about their significance in integrated network activity underlying motor behavior. We have examined the physiological effects of endocannabinoids in a neuronal network underlying locomotor behavior using the isolated lamprey spinal cord. Our results show that endocannabinoids are released during locomotor activity and participate in setting the baseline burst rate. They are released in response to mGluR1 activation and act as retrograde messengers. This conditional release of endocannabinoids can transform motoneurons and crossing interneurons into modulatory neurons by enabling them to regulate their inhibitory synaptic inputs and thus contribute to the modulation of the locomotor burst frequency. These results provide evidence that endocannabinoid retrograde signaling occurs within the locomotor network and contributes to motor pattern generation and regulation in the spinal cord.