Francisco Pinho

Sulforaphane induces DNA damage and mitotic abnormalities in human osteosarcoma MG-63 cells: correlation with cell cycle arrest and apoptosis.

Osteosarcoma is a recalcitrant bone malignancy with poor responsiveness to treatments; therefore, new chemotherapeutic compounds are needed. Sulforaphane (SFN) has been considered a promising chemotherapeutic compound for several types of tumors by inducing apoptosis and cytostasis, but its effects (e.g., genotoxicity) in osteosarcoma cells remains exploratory. In this work, the MG-63 osteosarcoma cell line was exposed to SFN up to 20 μM for 24 and 48 h. SFN induced G2/M phase arrest and decreased nuclear division index, associated with disruption of cytoskeletal organization. Noteworthy, SFN induced a transcriptome response supportive of G2/M phase arrest, namely a decrease in Chk1- and Cdc25C-encoding transcripts, and an increase in Cdk1-encoding transcripts. After 48-h exposure, SFN at a dietary concentration (5 μM) contributed to genomic instability in the MG-63 cells as confirmed by increased number of DNA breaks, clastogenicity, and nuclear and mitotic abnormalities. The increased formation of nucleoplasmic bridges, micronuclei, and apoptotic cells positively correlated with loss of viability. These results suggest that genotoxic damage is an important step for SFN-induced cytotoxicity in MG-63 cells. In conclusion, SFN shows potential to induce genotoxic damage at low concentrations and such potential deserves further investigation in other tumor cell types.

Cadmium-induced genotoxicity in human osteoblast-like cells.

Cadmium (Cd) is a widespread heavy metal used in numerous industrial processes. Cd exerts toxicological effects mostly in kidney and liver. Bone is also an important target of Cd, however, the cellular mechanisms of Cd toxicological effects in the bone cells are still poorly understood. Therefore, the present work aimed to investigate the putative cytotoxic and genotoxic effects of Cd to human bone cells. For that, the osteoblast-like MG-63 cells were exposed to 20 and 50μM Cd for 24 and 48h. Results showed a dose-dependent increase in Cd accumulation in cells and a decrease in cell viability, especially after 48h. Cell cycle analysis showed a delay at S phase concomitant with a decrease in cells at G0/G1 phase. After 24h, Cd treatment downregulated the expression of CHEK1, CHEK2 and CDK2 genes and upregulated the expression of CCNE1 gene. After 48h, the expression of ATM and CCNB1 genes were downregulated. Also, a 3.3 fold increase on the expression of gene CCNE1 was detected. Both Cd doses induced DNA fragmentation at 48h, while an increase in micronuclei (MN) and nucleoplasmic bridges (NPBs) together with an increase in the percentage of apoptotic/necrotic cells was detected for both time periods. Overall, our results demonstrate the cytotoxicity and genotoxicity of Cd in human bone cells. Also, the cytokinesis-block micronucleus (CBMN) assay parameters (MN, NPBs and the percentage of cells under apoptosis or necrosis) together with the cell cycle appear as the most sensitive to Cd cyto- and genotoxicity, being early affected even with the lowest Cd dose. Therefore, these cyto-/genotoxic techniques may be selected for early detection of Cd-induced toxicity.

The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.

Age effects on EEG correlates of the Wisconsin Card Sorting Test

Body and brain undergo several changes with aging. One of the domains in which these changes are more remarkable relates with cognitive performance. In the present work, electroencephalogram (EEG) markers (power spectral density and spectral coherence) of age‐related cognitive decline were sought whilst the subjects performed the Wisconsin Card Sorting Test (WCST). Considering the expected age‐related cognitive deficits, WCST was applied to young, mid‐age and elderly participants, and the theta and alpha frequency bands were analyzed. From the results herein presented, higher theta and alpha power were found to be associated with a good performance in the WCST of younger subjects. Additionally, higher theta and alpha coherence were also associated with good performance and were shown to decline with age and a decrease in alpha peak frequency seems to be associated with aging. Additionally, inter‐hemispheric long‐range coherences and parietal theta power were identified as age‐independent EEG correlates of cognitive performance. In summary, these data reveals age‐dependent as well as age‐independent EEG correlates of cognitive performance that contribute to the understanding of brain aging and related cognitive deficits.

Effect of single bout versus repeated bouts of stretching on muscle recovery following eccentric exercise

OBJECTIVES To analyze the effects of a single bout and repeated bouts of stretching on indirect markers of exercise-induced muscle damage. DESIGN A randomized controlled clinical trial at a university human research laboratory was conducted. METHODS Fifty-six untrained males were randomly divided into four groups. (I) a single stretching group underwent a single bout of stretching on the quadriceps muscle; (II) an eccentric exercised group underwent eccentric quadriceps muscle contractions until exhaustion; (III) an eccentric exercise group followed by a single bout of stretching; (IV) an eccentric exercised group submitted to repeated bouts of stretching performed immediately and 24, 48, and 72 h post-exercise. Muscle stiffness, muscle soreness, maximal concentric peak torque, and plasma creatine kinase activity were assessed before exercise and 1, 24, 48, 72, and 96 h post-exercise. RESULTS All exercised groups showed significant reduction in maximal concentric peak torque and significant increases in muscle soreness, muscle stiffness, and plasma creatine kinase. There were no differences between these groups in all assessed variables, with the exception of markers of muscle stiffness, which were significantly lower in the eccentric exercise group followed by single or repeated bouts. The single stretching group showed no change in any assessed variables during the measurement period. CONCLUSIONS Muscle stretching performed after exercise, either as single bout or as repeated bouts, does not influence the levels of the main markers of exercise-induced muscle damage; however, repeated bouts of stretching performed during the days following exercise may have favorable effects on muscle stiffness.

myBrain: a novel EEG embedded system for epilepsy monitoring

Abstract The World Health Organisation has pointed that a successful health care delivery, requires effective medical devices as tools for prevention, diagnosis, treatment and rehabilitation. Several studies have concluded that longer monitoring periods and outpatient settings might increase diagnosis accuracy and success rate of treatment selection. The long-term monitoring of epileptic patients through electroencephalography (EEG) has been considered a powerful tool to improve the diagnosis, disease classification, and treatment of patients with such condition. This work presents the development of a wireless and wearable EEG acquisition platform suitable for both long-term and short-term monitoring in inpatient and outpatient settings. The developed platform features 32 passive dry electrodes, analogue-to-digital signal conversion with 24-bit resolution and a variable sampling frequency from 250 Hz to 1000 Hz per channel, embedded in a stand-alone module. A computer-on-module embedded system runs a Linux® operating system that rules the interface between two software frameworks, which interact to satisfy the real-time constraints of signal acquisition as well as parallel recording, processing and wireless data transmission. A textile structure was developed to accommodate all components. Platform performance was evaluated in terms of hardware, software and signal quality. The electrodes were characterised through electrochemical impedance spectroscopy and the operating system performance running an epileptic discrimination algorithm was evaluated. Signal quality was thoroughly assessed in two different approaches: playback of EEG reference signals and benchmarking with a clinical-grade EEG system in alpha-wave replacement and steady-state visual evoked potential paradigms. The proposed platform seems to efficiently monitor epileptic patients in both inpatient and outpatient settings and paves the way to new ambulatory clinical regimens as well as non-clinical EEG applications.

Wireless and wearable eeg acquisition platform for ambulatory monitoring

Electroencephalogram (EEG) Ambulatory monitoring has been regarded as a promising tool to improve diagnosis, classification and medication prescription in patients with epilepsy and other paroxysmal diseases. This study presents the development of a wireless and wearable EEG acquisition system for ambulatory monitoring. The platform comprises 32 active dry electrodes, an analog-to-digital conversion unit with 24 bit resolution, 1 ksps sampling frequency per channel and a module for acquisition, processing and wireless transmission based on IGEP COM embedded system development platform under a Linux™ operative system. The base operating system consists of two software frameworks which interact to ensure the real-time requirements of the acquired signals and parallel recording, processing and data transmission. In order to control the analog-to-digital converters and the synchronous reception of converted data, a Linux™ kernel driver was developed. It was also developed an userspace application for data saving, digital processing and wireless transmission via socket TCP-IP on a 802.11 b/g network topology. An application based on C# from .NET development environment was also developed for PC data reception and visualization. This application consists of a TCP socket server for data reception and a graphic environment for signals visualization. For signal plotting, it was used the open source ZedGraph library. The proposed system may operate on data streaming or event detection modes and presents feasible performance on EEG monitoring of both epileptic inpatients and outpatients.

Epileptic event detection algorithm for ambulatory monitoring platforms

Detecting epileptic electroencephalography (EEG) signals, both automatically and accurately, is significant in ambulatory long-term monitoring patients with epilepsy. In this study, it is presented a novel epileptic-like event detection algorithm based on a mixture of amplitude, frequency and spatial analysis with rule-based decision. In this work, EEG signals from 6 different subjects were searched for epileptic-like and normal data segments. The herein proposed algorithm detects putative epileptic EEG channels by comparing the RMS values of EEG activity with a hysteresis threshold, on a channel basis. The raw EEG signals are filtered with an artefact attenuation technique. The threshold is calculated on a reviewer-visually-selected baseline epoch, free of artefacts. Generalized epileptic activity detection is based on a spatial decision rule. Experimental results have shown detection rates as high as 95% with a false-negative rate as low as 1%. The algorithm seems to show a promising detection performance, even on artifact contaminated datasets. The proposed algorithm is intended to be used in real-time ambulatory monitoring of epileptic patients, with subject personalization, small size window analysis, good artefact immunity and no need for classifier training.

Effects of age-related cognitive deficits on EEG phase coherence

Body and brain undergo several changes with aging. One of these changes is the loss of neuroplasticity, which leads to the decrease of cognitive abilities. Hence the necessity of stopping or reversing these changes is of utmost importance to contemporary society. In the present work, electroencephalogram (EEG) markers of cognitive decline are sought whilst the subjects perform the Wisconsin Card Sorting Test (WCST). Considering the expected age-related cognitive deficits, WCST was applied to young and elder participants. The results suggest that coherence on theta and alpha EEG rhythms decrease with aging and increase with performance. Additionally, theta phase coherence seems more sensitive to performance, while alpha synchronization appears as a potential ageing marker.

Biochemical and transcriptional analyses of cadmium-induced mitochondrial dysfunction and oxidative stress in human osteoblasts

ABSTRACT Cadmium (Cd) accumulation is known to occur predominantly in kidney and liver; however, low-level long-term exposure to Cd may also result in bone damage. Few studies have addressed Cd-induced toxicity in osteoblasts, particularly upon cell mitochondrial energy processing and putative associations with oxidative stress in bone. To assess the influence of Cd treatment on mitochondrial function and oxidative status in osteoblast cells, human MG-63 cells were treated with Cd (up to 65 μM) for 24 or 48 h. Intracellular reactive oxygen species (ROS), lipid and protein oxidation and antioxidant defense mechanisms such as total antioxidant activity (TAA) and gene expression of antioxidant enzymes were analyzed. In addition, Cd-induced effects on mitochondrial function were assessed by analyzing the activity of enzymes involved in mitochondrial respiration, membrane potential (ΔΨm), mitochondrial morphology and adenylate energy charge. Treatment with Cd increased oxidative stress, concomitantly with lipid and protein oxidation. Real-time polymerase chain reaction (qRT-PCR) analyses of antioxidant genes catalase (CAT), glutathione peroxidase 1 (GPX1), glutathione S-reductase (GSR), and superoxide dismutase (SOD1 and SOD2) exhibited a trend toward decrease in transcripts in Cd-stressed cells, particularly a downregulation of GSR. Longer treatment with Cd (48 h) resulted in energy charge states significantly below those commonly observed in living cells. Mitochondrial function was affected by ΔΨm reduction. Inhibition of mitochondrial respiratory chain enzymes and citrate synthase also occurred following Cd treatment. In conclusion, Cd induced mitochondrial dysfunction which appeared to be associated with oxidative stress in human osteoblasts.