Proton export upregulates aerobic glycolysis.

INTRODUCTION: Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the "Warburg Effect." It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. RESULTS: To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. CONCLUSIONS: Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.

Evolutionary Analysis of TCGA Data Using Over- and Under- Mutated Genes Identify Key Molecular Pathways and Cellular Functions in Lung Cancer Subtypes.

We identify critical conserved and mutated genes through a theoretical model linking a gene's fitness contribution to its observed mutational frequency in a clinical cohort. "Passenger" gene mutations do not alter fitness and have mutational frequencies determined by gene size and the mutation rate. Driver mutations, which increase fitness (and proliferation), are observed more frequently than expected. Non-synonymous mutations in essential genes reduce fitness and are eliminated by natural selection resulting in lower prevalence than expected. We apply this "evolutionary triage" principle to TCGA data from EGFR-mutant, KRAS-mutant, and NEK (non-EGFR/KRAS) lung adenocarcinomas. We find frequent overlap of evolutionarily selected non-synonymous gene mutations among the subtypes suggesting enrichment for adaptations to common local tissue selection forces. Overlap of conserved genes in the LUAD subtypes is rare suggesting negative evolutionary selection is strongly dependent on initiating mutational events during carcinogenesis. Highly expressed genes are more likely to be conserved and significant changes in expression (>20% increased/decreased) are common in genes with evolutionarily selected mutations but not in conserved genes. EGFR-mut cancers have fewer average mutations (89) than KRAS-mut (228) and NEK (313). Subtype-specific variation in conserved and mutated genes identify critical molecular components in cell signaling, extracellular matrix remodeling, and membrane transporters. These findings demonstrate subtype-specific patterns of co-adaptations between the defining driver mutation and somatically conserved genes as well as novel insights into epigenetic versus genetic contributions to cancer evolution.

Slow update of internal representations impedes synchronization in autism.

Autism is a neurodevelopmental disorder characterized by impaired social skills, motor and perceptual atypicalities. These difficulties were explained within the Bayesian framework as either reflecting oversensitivity to prediction errors or - just the opposite - slow updating of such errors. To test these opposing theories, we administer paced finger-tapping, a synchronization task that requires use of recent sensory information for fast error-correction. We use computational modelling to disentangle the contributions of error-correction from that of noise in keeping temporal intervals, and in executing motor responses. To assess the specificity of tapping characteristics to autism, we compare performance to both neurotypical individuals and individuals with dyslexia. Only the autism group shows poor sensorimotor synchronization. Trial-by-trial modelling reveals typical noise levels in interval representations and motor responses. However, rate of error correction is reduced in autism, impeding synchronization ability. These results provide evidence for slow updating of internal representations in autism.

Novel evolutionary dynamics of small populations in breast cancer adjuvant and neoadjuvant therapy.

Disseminated cancer cells (DCCs) are detected in the circulation and bone marrow of up to 40% of breast cancer (BC) patients with clinically localized disease. The formation of metastases is governed by eco-evolutionary interactions of DCCs with the tissue during the transition from microscopic populations to macroscopic disease. Here, we view BC adjuvant and neoadjuvant treatments in the context of small population extinction dynamics observed in the Anthropocene era. Specifically, the unique eco-evolutionary dynamics of small asexually reproducing cancer populations render them highly vulnerable to: (1) environmental and demographic fluctuations, (2) Allee effects, (3) genetic drift and (4) population fragmentation. Furthermore, these typically interact, producing self-reinforcing, destructive dynamics-termed the Extinction Vortex-eradicating the population even when none of the perturbations is individually capable of causing extinction. We propose that developing BC adjuvant and neoadjuvant protocols may exploit these dynamics to prevent recovery and proliferation of small cancer populations during and after treatment-termed "Eco-evolutionary rescue" in natural extinctions. We hypothesize more strategic application of currently available agents based on the extinction vulnerabilities of small populations could improve clinical outcomes.

Eradicating Metastatic Cancer and the Eco-Evolutionary Dynamics of Anthropocene Extinctions.

Curative therapy for metastatic cancers is equivalent to causing extinction of a large, heterogeneous, and geographically dispersed population. Although eradication of dinosaurs is a dramatic example of extinction dynamics, similar application of massive eco-evolutionary force in cancer treatment is typically limited by host toxicity. Here, we investigate the evolutionary dynamics of Anthropocene species extinctions as an alternative model for curative cancer therapy. Human activities can produce extinctions of large, diverse, and geographically distributed populations. The extinction of a species typically follows a pattern in which initial demographic and ecological insults reduce the size and heterogeneity of the population. The surviving individuals, with decreased genetic diversity and often fragmented ecology, are then vulnerable to small stochastic perturbations that further reduce the population until extinction is inevitable. We hypothesize large, diverse, and disseminated cancer populations can be eradicated using similar evolutionary dynamics. Initial therapy is applied to reduce population size and diversity and followed by new treatments to exploit the eco-evolutionary vulnerability of small and/or declining populations. Mathematical models and computer simulations demonstrate initial reductive treatment followed immediately by demographic and ecological perturbations, similar to the empirically derived treatment of pediatric acute lymphocytic leukemia, can consistently achieve curative outcomes in nonpediatric cancers. SIGNIFICANCE: Anthropocene extinctions suggest a strategy for eradicating metastatic cancers in which initial therapy, by reducing the size and diversity of the population, renders it vulnerable to extinction by rapidly applied additional perturbations.

The Warburg effect as an adaptation of cancer cells to rapid fluctuations in energy demand.

To maintain optimal fitness, a cell must balance the risk of inadequate energy reserve for response to a potentially fatal perturbation against the long-term cost of maintaining high concentrations of ATP to meet occasional spikes in demand. Here we apply a game theoretic approach to address the dynamics of energy production and expenditure in eukaryotic cells. Conventionally, glucose metabolism is viewed as a function of oxygen concentrations in which the more efficient oxidation of glucose to CO2 and H2O produces all or nearly all ATP except under hypoxic conditions when less efficient (2 ATP/ glucose vs. about 36ATP/glucose) anaerobic metabolism of glucose to lactic acid provides an emergency backup. We propose an alternative in which energy production is governed by the complex temporal and spatial dynamics of intracellular ATP demand. In the short term, a cell must provide energy for constant baseline needs but also maintain capacity to rapidly respond to fluxes in demand particularly due to external perturbations on the cell membrane. Similarly, longer-term dynamics require a trade-off between the cost of maintaining high metabolic capacity to meet uncommon spikes in demand versus the risk of unsuccessfully responding to threats or opportunities. Here we develop a model and computationally explore the cell's optimal mix of glycolytic and oxidative capacity. We find the Warburg effect, high glycolytic metabolism even under normoxic conditions, is represents a metabolic strategy that allow cancer cells to optimally meet energy demands posed by stochastic or fluctuating tumor environments.

1,3-Dinitrobenzene neurotoxicity - Passage effect in immortalized astrocytes.

Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2-8) and high passage DI-TNC1 astrocytes (HP; #17-28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K(+)-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K(+)-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K(+) in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9-16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells.

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

Immediate-release methylphenidate for attention deficit hyperactivity disorder (ADHD) in adults.

BACKGROUND: Symptoms of attention deficit hyperactivity disorder (ADHD), diagnosed mainly in children, often persist into adulthood. Adults in this group have a high rate of other psychiatric problems and functional difficulties in a number of key areas such as academic achievement, interpersonal relationships, and employment. Although the usefulness of immediate-release methylphenidate in children has been extensively studied, studies in adults, which are few, demonstrate varying results. OBJECTIVES: To evaluate the efficacy and tolerability of immediate-release methylphenidate versus placebo in the treatment of adults with ADHD. SEARCH METHODS: We searched the following databases in November 2013: CENTRAL, Ovid MEDLINE, EMBASE, PsycINFO, Database of Abstracts of Reviews of Effects (DARE), and two trials registers. Biosis was searched in December 2013. We inspected references of all relevant papers to identify more studies and contacted authors of recently published trials. SELECTION CRITERIA: We included all randomized trials comparing immediate-release methylphenidate versus placebo in participants aged 18 years or older with ADHD. We excluded trials conducted on subpopulations of adults with ADHD such as adults with both ADHD and substance dependence. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, extracted data, and assessed trial risk of bias. We contacted authors of trials to ask for additional and missing data. For dichotomous outcomes, we calculated risk ratios (RRs) and 95% confidence intervals (CIs). For continuous outcomes, we calculated mean differences (MDs) or standardized mean differences (SMDs) with 95% CIs. MAIN RESULTS: Results from the 11 randomized controlled trials (474 participants, counting participants from cross-over studies as a single arm, and counting both arms from parallel studies) included in the review demonstrated improvement in core clinical ADHD symptoms of hyperactivity, impulsivity, and inattentiveness, and overall improvement. We were able to pool results from 10 studies, which included 466 participants.Most included studies were judged to have unclear risk of bias for most categories. However, as all studies were randomized, double-blind, and placebo-controlled and, in general, did not contain factors that significantly decreased the quality of the body of evidence, the quality of evidence was assessed as "high" for most outcomes according to the GRADE (Grades of Recommendation, Assessment, Development, and Evaluation) approach. For one outcome-inattentiveness-most information came from studies at unclear risk of bias, and so the quality of evidence for this outcome was judged as "moderate."Results are given as SMD for each of the core clinical symptoms of ADHD. In all cases, participant numbers were calculated by counting participants in a single arm from cross-over studies and in both arms from parallel studies. The SMD for the outcome of hyperactivity was -0.60 (95% CI -1.11 to -0.09, 6 studies, number of participants (n) = 245, high-quality evidence) in favor of immediate-release methylphenidate; the SMD for impulsivity was -0.62 (95% CI -1.08 to -0.17, 5 studies, n = 207, high-quality evidence) in favor of immediate-release methylphenidate; and the SMD for inattentiveness was -0.66 (95% CI -1.02 to -0.30, 7 studies, n = 391, moderate-quality evidence) in favor of immediate-release methylphenidate. Moderate to extreme statistical heterogeneity was detected for all outcomes. Subgroup analysis comparing high versus low doses did not indicate that higher doses of immediate-release methylphenidate were associated with greater efficacy.For overall change, the SMD was -0.72 (95% CI -1.12 to -0.32, 9 studies, n = 455, high-quality evidence) in favor of immediate-release methylphenidate.The effects of immediate-release methylphenidate on anxiety and depression as parameters of general changes in mental state were equivocal. Some trials reported reduction in depression and anxiety, others detailed no change, and still others described an increase in depressive and anxious symptoms.The most common adverse effect was loss of appetite, in some cases with weight loss. Although no study reported either of these effects as problematic or severe, the included studies were of short duration; thus clinical significance could not be properly assessed. Five studies reported changes in systolic or diastolic blood pressure, and three reported increases in heart rate. None of these results were judged to present cause for concern. No study reported clinically significant adverse effects-cardiovascular or other. Three studies did not mention adverse effects. We were unable to determine whether adverse effects were not discussed by study authors because none occurred, or because no data on adverse effects were collected. AUTHORS' CONCLUSIONS: Data from randomized controlled trials suggest that immediate-release methylphenidate is efficacious for treating adults with ADHD with symptoms of hyperactivity, impulsivity, and inattentiveness, and for improving their overall clinical condition. Trial data suggest that adverse effects from immediate-release methylphenidate for adults with ADHD are not of serious clinical significance, although this conclusion may be limited, certainly in the case of weight loss, by the short duration of published studies.

Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane.

BACKGROUND: Cancer cells, and a variety of normal cells, exhibit aerobic glycolysis, high rates of glucose fermentation in the presence of normal oxygen concentrations, also known as the Warburg effect. This metabolism is considered abnormal because it violates the standard model of cellular energy production that assumes glucose metabolism is predominantly governed by oxygen concentrations and, therefore, fermentative glycolysis is an emergency back-up for periods of hypoxia. Though several hypotheses have been proposed for the origin of aerobic glycolysis, its biological basis in cancer and normal cells is still not well understood. RESULTS: We examined changes in glucose metabolism following perturbations in membrane activity in different normal and tumor cell lines and found that inhibition or activation of pumps on the cell membrane led to reduction or increase in glycolysis, respectively, while oxidative phosphorylation remained unchanged. Computational simulations demonstrated that these findings are consistent with a new model of normal physiological cellular metabolism in which efficient mitochondrial oxidative phosphorylation supplies chronic energy demand primarily for macromolecule synthesis and glycolysis is necessary to supply rapid energy demands primarily to support membrane pumps. A specific model prediction was that the spatial distribution of ATP-producing enzymes in the glycolytic pathway must be primarily localized adjacent to the cell membrane, while mitochondria should be predominantly peri-nuclear. The predictions were confirmed experimentally. CONCLUSIONS: Our results show that glycolytic metabolism serves a critical physiological function under normoxic conditions by responding to rapid energetic demand, mainly from membrane transport activities, even in the presence of oxygen. This supports a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Cells use efficient but slow-responding aerobic metabolism to meet baseline, steady energy demand and glycolytic metabolism, which is inefficient but can rapidly increase adenosine triphosphate (ATP) production, to meet short-timescale energy demands, mainly from membrane transport activities. In this model, the origin of the Warburg effect in cancer cells and aerobic glycolysis in general represents a normal physiological function due to enhanced energy demand for membrane transporters activity required for cell division, growth, and migration.

ADHD subjects fail to suppress eye blinks and microsaccades while anticipating visual stimuli but recover with medication.

Oculomotor behavior and parameters are known to be affected by the allocation of attention and could potentially be used to investigate attention disorders. We explored the oculomotor markers of Attention-deficit/hyperactivity disorder (ADHD) that are involuntary and quantitative and that could be used to reveal the core-affected mechanisms, as well as be used for differential diagnosis. We recorded eye movements in a group of 22 ADHD-diagnosed patients with and without medication (methylphenidate) and in 22 control observers while performing the test of variables of attention (t.o.v.a.). We found that the average microsaccade and blink rates were higher in the ADHD group, especially in the time interval around stimulus onset. These rates increased monotonically over session time for both groups, but with significantly faster increments in the unmedicated ADHD group. With medication, the level and time course of the microsaccade rate were fully normalized to the control level, regardless of the time interval within trials. In contrast, the pupil diameter decreased over time within sessions and significantly increased above the control level with medication. We interpreted the suppression of microsaccades and eye blinks around the stimulus onset as reflecting a temporal anticipation mechanism for the transient allocation of attention, and their overall rates as inversely reflecting the level of arousal. We suggest that ADHD subjects fail to maintain sufficient levels of arousal during a simple and prolonged task, which limits their ability to dynamically allocate attention while anticipating visual stimuli. This impairment normalizes with medication and its oculomotor quantification could potentially be used for differential diagnosis.

Photothermal therapy of cancer cells mediated by blue hydrogel nanoparticles.

AIM: The aim of this study was to investigate in vitro the utility of biologically compatible, nontoxic and cell-specific targetable hydrogel nanoparticles (NPs), which have Coomassie® Brilliant Blue G dye (Sigma-Aldrich, MO, USA) covalently linked into their polyacrylamide matrix, as candidates for photothermal therapy (PTT) of cancer cells. MATERIALS & METHODS: Hydrogel NPs with Coomassie Brilliant Blue G dye covalently linked into their polyacrylamide matrix were fabricated using a reverse micelle microemulsion polymerization method and were found to be 80-95 nm in diameter, with an absorbance value of 0.52. PTT-induced hyperthermia/thermolysis was achieved at 37°C using an inexpensive, portable, light-emitting diode array light source (590 nm, 25 mW/cm(2)). RESULTS & CONCLUSION: Hydrogel NPs with Coomassie Brilliant Blue G dye linked into their polyacrylamide matrix are effective in causing PTT-induced thermolysis in immortalized human cervical cancer cell line (HeLa) cells for varying NP concentrations and treatment times. These multifunctional particles have previously been used in cancer studies to enable delineation, for glioma surgery and in photoacoustic imaging studies. The addition of the PTT function would enable a three-pronged theranostic approach to cancer medicine, such as guided tumor surgery with intra-operative photoacoustic imaging and intra-operative PTT.

Cell volume changes during apoptosis monitored in real time using digital holographic microscopy.

Cellular volume changes play important roles in many processes associated with the normal cell activity, as well as various diseases. Consequently, there is a considerable need to accurately measure volumes of both individual cells and cell populations as a function of time. In this study, we have monitored cell volume changes in real time during apoptosis using digital holographic microscopy. Cell volume changes were deduced from the measured phase change of light transmitted through cells. Our digital holographic experiments showed that after exposure to 1 µM staurosporine for 4 h, the volumes of KB cells were reduced by ~50-60%, which is consistent with previous results obtained using electronic cell sizing and atomic force microscopy. In comparison with other techniques, digital holographic microscopy is advantageous because it employs noninvasive detection, has high time resolution, real time measurement capability, and the ability to simultaneously investigate time-dependent volume changes of both individual cells and cell populations.

Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free calcium ions.

Ca(2+) is a universal second messenger and plays a major role in intracellular signaling, metabolism, and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.

Two-photon nano-PEBBLE sensors: subcellular pH measurements.

Intracellular pH mapping is of great importance as it plays a critical role in many cellular events. Also, in tissue, pH mapping can be an indicator for the onset of cancer. Here we describe a biocompatible, targeted, ratiometric, fluorescent, pH sensing nano-PEBBLE (Photonic Explorer for Biomedical use with Biologically Localized Embedding) that is based on two-photon excitation. Two-photon excitation minimizes the photobleaching and cell autofluorescence drastically, leading to an increase in the signal-to-noise ratio. PEBBLE nanosensors provide a novel approach for introducing membrane impermeant dyes, like HPTS, into cells. We use both non-targeted and F3 peptide targeted PEBBLE nanosensors for intracellular pH measurement of 9L cells. The intracellular measurements suggest that the non-targeted nanosensors are mostly trapped in endosomes, whereas the F3 peptide targeting enables them to escape/avoid these acidic compartments. Combining the advantages of pH sensitive PEBBLE nanoparticles, including their specific targeting, with the advantages of two-photon microscopy provides an attractive and promising prospect for non-invasive real-time monitoring of pH inside cancer cells and tissues.