Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate. Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type. The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

Motor skill acquisition across short and long time scales: a meta-analysis of neuroimaging data.

In this systematic review and meta-analysis, we explore how the time scale of practice affects patterns of brain activity associated with motor skill acquisition. Fifty-eight studies that involved skill learning with healthy participants (117 contrasts) met inclusion criteria. Two meta-contrasts were coded: decreases: peak coordinates that showed decreases in brain activity over time; increases: peak coordinates that showed increases in activity over time. Studies were grouped by practice time scale: short (≤1 h; 25 studies), medium (>1 and ≤24 h; 18 studies), and long (>24h to 5 weeks; 17 studies). Coordinates were analyzed using Activation Likelihood Estimation to show brain areas that were consistently activated for each contrast. Across time scales, consistent decreases in activity were shown in prefrontal and premotor cortex, the inferior parietal lobules, and the cerebellar cortex. Across the short and medium time scales there were consistent increases in supplementary and primary motor cortex and dentate nucleus. At the long time scale, increases were seen in posterior cingulate gyrus, primary motor cortex, putamen, and globus pallidus. Comparisons between time scales showed that increased activity in M1 at medium time scales was more spatially consistent across studies than increased activity in M1 at long time scales. Further, activity in the striatum (viz. putamen and globus pallidus) was consistently more rostral in the medium time scale and consistently more caudal in the long time scale. These data support neurophysiological models that posit that both a cortico-cerebellar system and a cortico-striatal system are active, but at different time points, during motor learning, and suggest there are associative/premotor and sensorimotor networks active within each system.

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress.

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents ß-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.

Reactive oxygen species and oxidative DNA damage mediate the cytotoxicity of tungsten-nickel-cobalt alloys in vitro.

Tungsten alloys (WA) have been introduced in an attempt to find safer alternatives to depleted uranium and lead munitions. However, it is known that at least one alloy, 91% tungsten-6% nickel-3% cobalt (WNC-91-6-3), causes rhabdomyosarcomas when fragments are implanted in rat muscle. This raises concerns that shrapnel, if not surgically removable, may result in similar tumours in humans. There is therefore a clear need to develop rapid and robust in vitro methods to characterise the toxicity of different WAs in order to identify those that are most likely to be harmful to human health and to guide development of new materials in the future. In the current study we have developed a rapid visual in vitro assay to detect toxicity mediated by individual WA particles in cultured L6-C11 rat muscle cells. Using a variety of techniques (histology, comet assay, caspase-3 activity, oxidation of 2'7'-dichlorofluorescin to measure the production of reactive oxygen species and whole-genome microarrays) we show that, in agreement with the in vivo rat carcinogenicity studies, WNC-91-6-3 was the most toxic of the alloys tested. On dissolution, it produces large amounts of reactive oxygen species, causes significant amounts of DNA damage, inhibits caspase-3, triggers a severe hypoxic response and kills the cells in the immediate vicinity of the alloy particles within 24h. By combining these in vitro data we offer a mechanistic explanation of the effect of this alloy in vivo and show that in vitro tests are a viable alternative for assessing new alloys in the future.

Activity of OGG1 variants in the repair of pro-oxidant-induced 8-oxo-2'-deoxyguanosine.

Cells are continuously exposed to damaging reactive oxygen species (ROS), which are produced from both endogenous and exogenous sources. 8-Oxodeoxyguanosine (8-oxodG) is an abundant base lesion formed during oxidative stress which, if not repaired, can give rise to G:C-->T:A transversions in DNA. The 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated base excision repair (BER) pathway operates to remove 8-oxodG lesions. Ogg1 deletion and polymorphism may result in a hypermutator phenotype and susceptibility to oxidative pathologies including cancer. Limited and conflicting evidence exists regarding the repair capacity of a prevalent human OGG1 (hOGG1) polymorphism, the Cys326-hOGG1 variant. The formamidopyrimidine DNA glycosylase (FPG)-modified comet assay was used to investigate the ability of sodium dichromate, potassium bromate and Ro19-8022 (+light) to induce DNA damage in mogg1(-/-) null (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs) and to assess hOGG1 variant-initiated BER capacities under conditions of oxidative stress. Treatment of WT MEFs with these pro-oxidant agents induced direct DNA strand breaks in a concentration-dependent manner, whereas, identical treatment of KO MEFs produced no effect. In contrast, KO MEFs accumulated significantly more FPG-sensitive sites than WT MEFs. Expression of hOGG1 in KO MEFs restored the WT phenotype in response to all pro-oxidants tested. The results suggest OGG1-initiated BER generates direct DNA strand breaks detected by the conventional comet assay, thus it is important that researchers do not interpret these as direct damage per se but rather a reflection of the repair process. The data also indicate Cys326-hOGG1-initiated BER is transiently impaired with respect to Ser326-hOGG1 (wild-type)- and Gly326-hOGG1 (artificial)-initiated BER following pro-oxidant treatment, possibly via hOGG1 cysteine 326 oxidation. This finding suggests the homozygous cys326/cys326 genotype may be classified as a biomarker of disease susceptibility, which is in support of a growing body of epidemiological evidence.

Visual search and coordination changes in response to video and point-light demonstrations without KR.

The authors examined the observational learning of 24 participants whom they constrained to use the model by removing intrinsic visual knowledge of results (KR). Matched participants assigned to video (VID), point-light (PL), and no-model (CON) groups performed a soccer-chipping task in which vision was occluded at ball contact. Pre- and posttests were interspersed with alternating periods of demonstration and acquisition. The authors assessed delayed retention 2-3 days later. In support of the visual perception perspective, the participants who observed the models showed immediate and enduring changes to more closely imitate the model's relative motion. While observing the demonstration, the PL group participants were more selective in their visual search than were the VID group participants but did not perform more accurately or learn more.

Activation of c-Jun N-terminal kinase in A549 lung carcinoma cells by sodium dichromate: role of dissociation of apoptosis signal regulating kinase-1 from its physiological inhibitor thioredoxin.

Changes in the components of the Jun N-terminal kinase (JNK) signalling pathway were investigated in human A549 lung carcinoma cells treated with sodium dichromate. Sodium dichromate (100 microM, 0-6h) failed to activate nuclear factor kappa B (NF-kappaB) as determined by a lack of nuclear translocation of p65 but resulted in Jun N-terminal kinase activation as assessed by phospho-Jun N-terminal kinase Western blotting in a dose-dependent (>25 microM) and time-dependent (>1h) manner. In addition, c-Jun, a downstream target of Jun N-terminal kinase signalling was also activated with a similar dose- and time-dependency at the level of both protein expression and degree of phosphorylation. In contrast, sodium dichromate treatment had no effect on levels of phospho-p38. Immunoprecipitation demonstrated that apoptosis signal regulating kinase-1 (ASK-1), an upstream activator of Jun N-terminal kinase was dissociated from its inhibitory partner thioredoxin (Trx) in response to sodium dichromate (100 microM, 4h) treatment. This treatment was also associated with a transient (2h) increase in cytosolic levels of thioredoxin but no nuclear translocation of thioredoxin was observed. In conclusion, sodium dichromate had a stimulatory effect on the Jun N-terminal kinase signalling pathway in A549 cells, resulting in activation of downstream effector molecules. We hypothesise that dissociation of apoptosis signal regulating kinase-1 from thioredoxin may be at least partially responsible for Jun N-terminal kinase activation.

Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells.

Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including Cr(V), Cr(IV) and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine (hOGG1) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0-100 microM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RT-PCR and RNase protection assay. Sodium dichromate at 25 microM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 microM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H(2)O(2) (0-200 microM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H(2)O(2). Western blotting demonstrated that sodium dichromate (100 microM; 16 h and >25 microM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 microM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.

Induction of DNA-strand breaks in human peripheral blood lymphocytes and A549 lung cells by sodium dichromate: association with 8-oxo-2-deoxyguanosine formation and inter-individual variability.

Hexavalent chromium [Cr(VI)] is a genotoxic carcinogen for which inhalation is a major potential route of exposure in occupational settings. In the present study, the ability of sodium dichromate to cause DNA strand breaks in three populations of cells, human whole blood cells, isolated human peripheral blood lymphocytes and cultured A549 lung epithelial cells, was investigated. Treatment with non-cytotoxic concentrations of sodium dichromate (for 1 h) resulted in a concentration-dependent increase in the number of DNA strand breaks as measured by the Comet assay. The lowest concentrations of sodium dichromate that resulted in a statistically significant (P < 0.01) increase in the number of DNA strand breaks were 500, 50 and 10 microM, respectively, in these cells. The use of formamidopyrimidine glycosylase increased the sensitivity of detection of strand breaks in A549 cells 10-fold, suggesting a role for DNA base oxidation in the mechanism of dichromate-induced DNA strand breaks. In support of this hypothesis, immunocytochemistry indicated an elevation of 8-oxodeoxyguanosine in A549 cells treated with 10 and 500 microM sodium dichromate for 1 h. We also demonstrated 2.11- and 2.5-fold ranges in the level of control and dichromate (500 microM)-induced DNA strand breaks, respectively, in cells of whole blood within a group of healthy volunteers (n = 26). A statistically significant (P < 0.001) positive Pearson's correlation (r = 0.606) was found between control and treated levels of DNA strand breaks, suggesting that factors responsible for relatively low levels of DNA strand breaks in untreated PBL may also offer protection against the formation of dichromate-induced DNA strand breaks.

Learning a coordination skill: interactive effects of instruction and feedback.

Information prior to and during the acquisition of a continuous bimanual task was manipulated. Participants practiced a difficult coordination pattern, which produced circular shapes on the computer, when they moved their arms correctly. Four groups were examined, which differed in the type and amount of information provided. Either limb or circle feedback was provided in the presence or absence of instructions detailing how to move the limbs. Circle feedback facilitated learning relative to the limb feedback in which the explicit displacements of the limbs was displayed. Under circle feedback conditions, instructions hindered acquisition. Little instructional effects were observed under limb feedback conditions, despite the prediction that instructions would benefit learning when the feedback was more compatible. Findings are discussed in relation to the complexity of the feedback and processing demands.

Potentiation of epidermal growth factor-induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation.

A transient induction of S phase DNA synthesis is a common feature of non-genotoxic rodent hepatocarcinogens when administered in vivo. In the present study the ability of phenobarbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated. In the absence of serum or growth factors PB was not a mitogen per se. However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-culture with PB. This effect was both time and concentration dependent. The lowest concentration of PB that significantly enhanced the effect of EGF was 10 microM and the effect was maximal at 1.0 mM. At a concentration of 2.0 mM PB no longer enhanced EGF-induced S phase DNA synthesis. Hepatocyte cultures pretreated with PB (0.1 mM) for 2 days were more responsive to the induction of S phase DNA synthesis by EGF for the subsequent 2 days. Despite the inhibition of PB enhancement of S phase DNA synthesis by the antioxidant dimethylthiourea, reduced glutathione was not depleted by PB treatment nor were oxidized glutathione or lipid peroxides elevated. Western blotting analysis showed that PB had no effect on epidermal growth factor receptor (EGFR) autophosphorylation per se after 1 and 48 h culture, enhanced sensitization of EGFR therefore does not appear to contribute to the enhancement of S phase DNA synthesis by PB. In contrast, treatment of hepatocytes with PB for 12 h resulted in a small but statistically significant activation of p42/44 MAP kinase activity and activation of protein kinase C, as measured by redistribution of enzyme activity from a soluble to a particulate compartment of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.

The roles of talent, physical precocity and practice in the development of soccer expertise.

Here we consider the potential contributions of talent, physical precocity and deliberate practice in the development of soccer expertise. After presenting a working definition of 'talent', we examine how coaches perceive and select potential talent. Our findings suggest that much of what coaches see as early talent may be explained by physical precocity associated with a relative age advantage. Finally, as a test of the model of Deliberate Practice, we review the results of studies that assessed the progress of international, national and provincial players based on accumulated practice, amount of practice per week and relative importance and demands of various practice and everyday activities. A positive linear relationship was found between accumulated individual plus team practice and skill. Various practical suggestions can be made to improve talent detection and selection and to optimize career practice patterns in soccer.

Effects of aging on automatic and effortful processes in bimanual coordination.

Two experiments are reported that compared younger and older adults on their performance of two bimanual temporal coordination tasks at varying movement speeds. In many cases, older adults performed as well as younger adults at all speeds of an in-phase coordination pattern and at slow speeds of an anti-phase pattern for both coordination accuracy and stability. Age differences tended to emerge most prominently at high speeds for the anti-phase pattern. These findings are consistent with the aging literature regarding automatic and effortful processing distinctions, suggesting that relative age differences become magnified when effortful resources are required for motor performance.

Developing a standardized test for the assessment of suturing skill in novice microsurgeons.

Suturing performance was assessed for 13 novice microsurgeons throughout a 4-5 day microsurgical training course. Time to complete a suture (from needle insertion to completion of tie-off) was assessed on a standardized suture task, for two sutures at the beginning and end of each training day. For days 2-4, suturing performance with actual tissue was also assessed at both the beginning and end of each day. An average learning curve for suturing performance on the standardized test was developed and demonstrated huge performance improvement. A consistent and significant relationship existed between trainees' performance on the standardized suturing test and suturing of actual tissue. Thus the standardized test appears both to reflect actual suturing performance and to be sensitive to improvements in suturing skill that result from practice.

Contextual interference in learning new patterns of bimanual coordination.

Two experiments are reported in which the question of whether or not contextual interference effects are found in motor tasks that require the acquisition of new coordination patterns was examined. Participants (N = 18, Experiment 1; N = 12, Experiment 2) practiced 3 novel bimanual patterns (45 degrees , 90 degrees , and 135 degrees relative phase) in either a random or a blocked order. No statistically significant acquisition or retention differences between groups were found when all 3 patterns were practiced on each of 2 days (Experiment 1). When the blocked group practiced 1 pattern on each of 3 acquisition days (Experiment 2), however, typical contextual interference effects were found: The blocked group performed better than the random group in practice, but the random group performed better than the blocked group in a delayed (by I week) retention test. The experiments revealed that contextual interference effects can arise in motor tasks that require the acquisition of new coordination patterns and are not limited to tasks involving novel scaling of a previously existing pattern.

Hand, space and attentional asymmetries in goal-directed manual aiming.

Two experiments were conducted to explore the interaction of the two cerebral hemispheres in motor control, by examining hand, space and attentional asymmetries in goal-directed aiming. In Experiment 1, right-handed subjects moved to targets more quickly with their right hand than their left hand. In addition, each hand was faster when moving in its own hemispace. Although in a control condition, movements were initiated more quickly with the left hand, visual distractors disrupted left hand performance more than right hand performance. For contralateral aiming, ipsilateral distractors caused the greatest interference. In Experiment 2, when targets and distractors were all presented at the midline, a right hand advantage was found for movement time along with a left hand advantage for reaction time, independent of target and distractor location. Our findings are discussed in terms of a right hemisphere role in movement preparation and the allocation of attention in space, and greater left hemisphere involvement in movement execution.