Enlightening brain energy metabolism.

Brain tissue metabolism is distributed across several cell types and subcellular compartments, which activate at different times and with different temporal patterns. The introduction of genetically-encoded fluorescent indicators that are imaged using time-lapse microscopy has opened the possibility of studying brain metabolism at cellular and sub-cellular levels. There are indicators for sugars, monocarboxylates, Krebs cycle intermediates, amino acids, cofactors, and energy nucleotides, which inform about relative levels, concentrations and fluxes. This review offers a brief survey of the metabolic indicators that have been validated in brain cells, with some illustrative examples from the literature. Whereas only a small fraction of the metabolome is currently accessible to fluorescent probes, there are grounds to be optimistic about coming developments and the application of these tools to the study of brain disease.

Metabolic Recruitment in Brain Tissue.

Information processing imposes urgent metabolic demands on neurons, which have negligible energy stores and restricted access to fuel. Here, we discuss metabolic recruitment, the tissue-level phenomenon whereby active neurons harvest resources from their surroundings. The primary event is the neuronal release of K+ that mirrors workload. Astrocytes sense K+ in exquisite fashion thanks to their unique coexpression of NBCe1 and α2ß2 Na+/K+ ATPase, and within seconds switch to Crabtree metabolism, involving GLUT1, aerobic glycolysis, transient suppression of mitochondrial respiration, and lactate export. The lactate surge serves as a secondary recruiter by inhibiting glucose consumption in distant cells. Additional recruiters are glutamate, nitric oxide, and ammonium, which signal over different spatiotemporal domains. The net outcome of these events is that more glucose, lactate, and oxygen are made available. Metabolic recruitment works alongside neurovascular coupling and various averaging strategies to support the inordinate dynamic range of individual neurons.

Experimental determination of k0 and Q0 values for 121Sb, 123Sb and 130Ba targets applying covariance analysis.

This work consists of an experimental determination of k0 and Q0 for 121Sb, 123Sb and 130Ba targets. Covariance analysis has been introduced to supply not only the overall uncertainties in these parameters but also their correlations. The irradiations were performed near the core of the IEA-R1 4.5 MW swimming-pool nuclear research reactor of the Nuclear and Energy Research Institute (IPEN-CNEN/SP), in São Paulo, Brazil. The epithermal neutron flux shape parameter, alpha, at the irradiation position is very close to zero, which favors to obtain Q0 values more accurately. Two irradiations were carried out in sequence, using two sets of samples: the first with bare samples and the second inside a Cd cover. The activity measurements were carried out in a previously calibrated HPGe gamma-ray spectrometer. The measurements were corrected for: saturation, decay time, cascade summing, geometry, self-attenuation, measuring time and mass. Standard sources of 152Eu, 133Ba, 60Co and 137Cs traceable to a 4πß-γ primary system were used to obtain the HPGe gamma-ray peak efficiency as a function of the energy. The experimental efficiency curve was performed by a fourth-degree polynomial fit, in the energy range of the standard sources, 121-1408 keV, it contains all correlations between points. For energies above 1408 keV, the efficiencies were obtained by the Monte Carlo Method. The covariance matrix methodology was applied to all uncertainties involved. The final values for k0 and Q0 were compared with the literature.

Visualizing physiological parameters in cells and tissues using genetically encoded indicators for metabolites.

The study of metabolism is undergoing a renaissance. Since the year 2002, over 50 genetically-encoded fluorescent indicators (GEFIs) have been introduced, capable of monitoring metabolites with high spatial/temporal resolution using fluorescence microscopy. Indicators are fusion proteins that change their fluorescence upon binding a specific metabolite. There are indicators for sugars, monocarboxylates, Krebs cycle intermediates, amino acids, cofactors, and energy nucleotides. They permit monitoring relative levels, concentrations, and fluxes in living systems. At a minimum they report relative levels and, in some cases, absolute concentrations may be obtained by performing ad hoc calibration protocols. Proper data collection, processing, and interpretation are critical to take full advantage of these new tools. This review offers a survey of the metabolic indicators that have been validated in mammalian systems. Minimally invasive, these indicators have been instrumental for the purposes of confirmation, rebuttal and discovery. We envision that this powerful technology will foster metabolic physiology.

How expensive is the astrocyte?

The energy cost of information processing is thought to be chiefly neuronal, with a minor fraction attributed to glial cells. However, there is compelling evidence that astrocytes capture synaptic K+ using their Na+/K+ ATPase, and not solely through Kir4.1 channels as was once thought. When this active buffering is taken into account, the cost of astrocytes rises by >200%. Gram-per-gram, astrocytes turn out to be as expensive as neurons. This conclusion is supported by 3D reconstruction of the neuropil showing similar mitochondrial densities in neurons and astrocytes, by cell-specific transcriptomics and proteomics, and by the rates of the tricarboxylic acid cycle. Possible consequences for reactive astrogliosis and brain disease are discussed.

Determination of k0 and Q0 for 74Se, 113In, 186W and 191Ir targets applying covariance analysis.

In the present work, the determinations of k0 and Q0 for 74Se, 113In, 186W and 191Ir targets were performed. The irradiations were conducted near the core of the IEA-R1 4.5 MW swimming-pool nuclear research reactor of the Instituto de Pesquisas Energéticas e Nucleares (IPEN-CNEN/SP - Nuclear and Energy Research Institute), in São Paulo, Brazil. The irradiation position was chosen where the neutron spectrum shape parameter α is very close to zero. For this reason, the correction to be applied for the determination of Q0 is very close to one, thus improving the accuracy of the results. For each experiment, two irradiations were carried out in sequence: the first one with bare samples and the second with a cadmium cover around the samples. All partial uncertainties were considered, applying the covariance matrix methodology. The final results were compared with the literature.

SUMCOR: Cascade summing correction for volumetric sources applying MCNP6.

The main features of code SUMCOR developed for cascade summing correction for volumetric sources are described. MCNP6 is used to track histories starting from individual points inside the volumetric source, for each set of cascade transitions from the radionuclide. Total and FEP efficiencies are calculated for all gamma-rays and X-rays involved in the cascade. Cascade summing correction is based on the matrix formalism developed by Semkow et al. (1990). Results are presented applying the experimental data sent to the participants of two intercomparisons organized by the ICRM-GSWG and coordinated by Dr. Marie-Cristine Lépy from the Laboratoire National Henri Becquerel (LNE-LNHB), CEA, in 2008 and 2010, respectively and compared to the other participants in the intercomparisons.

Transactivation activity and nucleocytoplasmic transport of ß-catenin are independently regulated by its C-terminal end.

The key protein in the canonical Wnt pathway is ß-catenin, which is phosphorylated both in absence and presence of Wnt signals by different kinases. Upon activation in the cytoplasm, ß-catenin can enter into the nucleus to transactivate target gene expression, many of which are cancer-related genes. The mechanism governing ß-catenin's nucleocytoplasmic transport has been recently unvealed, although phosphorylation at its C-terminal end and its functional consequences are not completely understood. Serine 646 of ß-catenin is a putative CK2 phosphorylation site and lies in a region which has been proposed to be important for its nucleocytoplasmic transport and transactivation activity. This residue was mutated to aspartic acid mimicking CK2-phosphorylation and its effects on ß-catenin activity as well as localization were explored. ß-Catenin S6464D did not show significant differences in both transcriptional activity and nuclear localization compared to the wild-type form, but displayed a characteristic granular nuclear pattern. Three-dimensional models of nuclei were constructed which showed differences in number and volume of granules, being those from ß-catenin S646D more and smaller than the wild-type form. FRAP microscopy was used to compare nuclear export of both proteins which showed a slightly higher but not significant retention of ß-catenin S646D. Altogether, these results show that C-terminal phosphorylation of ß-catenin seems to be related with its nucleocytoplasmic transport but not transactivation activity.

Treatment Steps, Surgery, and Hospitalization Rates During the First Year of Follow-up in Patients with Inflammatory Bowel Diseases from the 2011 ECCO-Epicom Inception Cohort.

BACKGROUND AND AIMS: The ECCO-EpiCom study investigates the differences in the incidence and therapeutic management of inflammatory bowel diseases [IBD] between Eastern and Western Europe. The aim of this study was to analyse the differences in the disease phenotype, medical therapy, surgery, and hospitalization rates in the ECCO-EpiCom 2011 inception cohort during the first year after diagnosis. METHODS: Nine Western, five Eastern European centres and one Australian centre with 258 Crohn's disease [CD], 380 ulcerative colitis [UC] and 71 IBD unclassified [IBDU] patients [female/male: 326/383; mean age at diagnosis: 40.9 years, SD: 17.3 years] participated. Patients' data were registered and entered in the web-based ECCO-EpiCom database [www.epicom-ecco.eu]. RESULTS: In CD, 36 [19%] Western Europe/Australian and 6 [9%] Eastern European patients received biological therapy [p = 0.04], but the immunosuppressive [IS] use was equal and high in these regions [Eastern Europe vs Western Europe/Australia: 53% vs 45%; p = 0.27]. Surgery was performed in 17 [24%] CD patients in Eastern Europe and 13 [7%] in Western Europe/Australia [p < 0.001, pLogRank = 0.001]. Of CD patients from Eastern Europe, 24 [34%] were hospitalized, and 39 [21%] from Western Europe/Australia, [p = 0.02, pLogRank = 0.01]. In UC, exposure to biologicals and colectomy rates were low and hospitalization rates did not differ between these regions during the 1-year follow-up period [16% vs 16%; p = 0.93]. CONCLUSIONS: During the first year after diagnosis, surgery and hospitalization rates were significantly higher in CD patients in Eastern Europe compared with Western Europe/Australia, whereas significantly more CD patients were treated with biologicals in the Western Europe/Australian centres.

Incidence and initial disease course of inflammatory bowel diseases in 2011 in Europe and Australia: results of the 2011 ECCO-EpiCom inception cohort.

BACKGROUND AND AIMS: The aim of the present study was to validate the IBD (inflammatory bowel diseases) incidence reported in the 2010 ECCO-EpiCom (European Crohn's and Colitis Organization-Epidemiological Committee) inception cohort by including a second independent inception cohort from participating centers in 2011 and an Australian center to investigate whether there is a difference in the incidence of IBD between Eastern and Western European countries and Australia. METHODS: Fourteen centers from 5 Eastern and 9 Western European countries and one center from Australia participated in the ECCO-EpiCom 2011 inception cohort. Patients' data regarding disease type, socio-demographic factors, extraintestinal manifestations and therapy were entered into the Web-based EpiCom database, www.ecco-epicom.eu. RESULTS: A total of 711 adult patients were diagnosed during the inclusion year 2011, 178 (25%) from Eastern, 461 (65%) from Western Europe and 72 (10%) from Australia; 259 (37%) patients were diagnosed with Crohn's disease, 380 (53%) with ulcerative colitis and 72 (10%) with IBD unclassified. The mean annual incidence rate for IBD was 11.3/100,000 in Eastern Europe, 14.0/100,000 in Western Europe and 30.3/100,000 in Australia. Significantly more patients were diagnosed with complicated disease at diagnosis in Eastern Europe compared to Western Europe (43% vs. 27%, p=0.02). CONCLUSION: Incidence rates, disease phenotype and initial treatment characteristics in the 2011 ECCO-EpiCom cohort were not significantly different from that reported in the 2010 cohort.

East-West gradient in the incidence of inflammatory bowel disease in Europe: the ECCO-EpiCom inception cohort.

OBJECTIVE: The incidence of inflammatory bowel disease (IBD) is increasing in Eastern Europe. The reasons for these changes remain unknown. The aim of this study was to investigate whether an East-West gradient in the incidence of IBD in Europe exists. DESIGN: A prospective, uniformly diagnosed, population based inception cohort of IBD patients in 31 centres from 14 Western and eight Eastern European countries covering a total background population of approximately 10.1 million people was created. One-third of the centres had previous experience with inception cohorts. Patients were entered into a low cost, web based epidemiological database, making participation possible regardless of socioeconomic status and prior experience. RESULTS: 1515 patients aged 15 years or older were included, of whom 535 (35%) were diagnosed with Crohn's disease (CD), 813 (54%) with ulcerative colitis (UC) and 167 (11%) with IBD unclassified (IBDU). The overall incidence rate ratios in all Western European centres were 1.9 (95% CI 1.5 to 2.4) for CD and 2.1 (95% CI 1.8 to 2.6) for UC compared with Eastern European centres. The median crude annual incidence rates per 100,000 in 2010 for CD were 6.5 (range 0-10.7) in Western European centres and 3.1 (range 0.4-11.5) in Eastern European centres, for UC 10.8 (range 2.9-31.5) and 4.1 (range 2.4-10.3), respectively, and for IBDU 1.9 (range 0-39.4) and 0 (range 0-1.2), respectively. In Western Europe, 92% of CD, 78% of UC and 74% of IBDU patients had a colonoscopy performed as the diagnostic procedure compared with 90%, 100% and 96%, respectively, in Eastern Europe. 8% of CD and 1% of UC patients in both regions underwent surgery within the first 3 months of the onset of disease. 7% of CD patients and 3% of UC patients from Western Europe received biological treatment as rescue therapy. Of all European CD patients, 20% received only 5-aminosalicylates as induction therapy. CONCLUSIONS: An East-West gradient in IBD incidence exists in Europe. Among this inception cohort--including indolent and aggressive cases--international guidelines for diagnosis and initial treatment are not being followed uniformly by physicians.

Small is fast: astrocytic glucose and lactate metabolism at cellular resolution.

Brain tissue is highly dynamic in terms of electrical activity and energy demand. Relevant energy metabolites have turnover times ranging from milliseconds to seconds and are rapidly exchanged between cells and within cells. Until recently these fast metabolic events were inaccessible, because standard isotopic techniques require use of populations of cells and/or involve integration times of tens of minutes. Thanks to fluorescent probes and recently available genetically-encoded optical nanosensors, this Technology Report shows how it is now possible to monitor the concentration of metabolites in real-time and in single cells. In combination with ad hoc inhibitor-stop protocols, these probes have revealed a key role for K(+) in the acute stimulation of astrocytic glycolysis by synaptic activity. They have also permitted detection of the Warburg effect in single cancer cells. Genetically-encoded nanosensors currently exist for glucose, lactate, NADH and ATP, and it is envisaged that other metabolite nanosensors will soon be available. These optical tools together with improved expression systems and in vivo imaging, herald an exciting era of single-cell metabolic analysis.

Acute feedback control of astrocytic glycolysis by lactate.

Neuronal activity is accompanied by a rapid increase in interstitial lactate, which is hypothesized to serve as a fuel for neurons and a signal for local vasodilation. Using FRET microscopy, we report here that the rate of glycolysis in cultured mice astrocytes can be acutely modulated by physiological changes in extracellular lactate. Glycolytic inhibition by lactate was not accompanied by detectable variations in intracellular pH or intracellular ATP and was not dependent of mitochondrial function. Pyruvate was also inhibitory, suggesting that the effect of lactate is not mediated by the NADH/NAD(+) ratio. We propose that lactate serves as a fast negative feedback signal limiting its own production by astrocytes and therefore the amplitude of the lactate surge. The inhibition of glucose usage by lactate was much stronger in resting astrocytes than in K(+)-stimulated astrocytes, which suggests that lactate may also help diverting glucose from resting to active zones.

Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: a multiphoton study of cerebellar slices.

Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real-time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several-fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG-P can diffuse horizontally across the molecular layer, presumably through gap junctions between Bergmann glial cells. Our main conclusion is that in acute cerebellar slices, the glucose transport capacity and glycolytic rate of Bergmann glia are several-fold higher than those of Purkinje cells. Given that the cerebellum is largely fueled by glucose and Purkinje neurons are estimated to spend more energy than Bergmann glial cells, these results suggest substantial shuttling of an energy-rich metabolite like lactate between glial cells and neurons.

Current use and future perspectives of diagnostic and therapeutic lasers in Oral Medicine.

Several diagnostic and therapeutic methods are based on the optical properties of lasers. In therapeutic applications, laser light is absorbed in a specific manner, whereas light is scattered, reflected, or transmitted from different structures. Improvements in laser technology allow new procedures and broaden the scope of applications for both diagnosis and therapy. The focus of laser application in Oral Medicine diagnosis should be early detection of oral squamous cell carcinoma. Novel modalities for the detection of oral malignancy are urgently needed, while others must be continuously improved. Optical coherence tomography and laser-induced fluorescence spectroscopy are currently being studied. In addition to diagnosis of non-malignant lesions, laser therapy has been used based upon the biological reactions and molecular wound healing mechanisms as an alternative for the treatment of a variety of oral soft tissue lesions. The aim of the present article is to review current knowledge and future perspectives of lasers in Oral Medicine.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury.

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 æg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09 percent). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50 percent after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50 percent. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.