Changes In Intracellular Concentration Research Articles

Accumulation of Li+ Ions Triggers Changes in FOS, JUN, EGR1, and MYC Transcription in the LiCl-Treated Human Umbilical Vein Endothelial Cells (HUVEC).

Changes in intracellular concentrations of Na+ and K+ are shown to alter gene expression. Another monovalent cation, Li+, is well known as a medicine for treatment of psychiatric disorders, but mechanism of its action is obscure. Thus, it is important to evaluate the effect of Li+ on gene expression in endothelial cells. Here we studied influence of the increased intracellular Na+ or Li+ concentrations on transcription of Na+i/K+i-sensitive genes. Treatment of the human endothelial cells (HUVEC) with LiCl for 1.5h resulted in accumulation of Li+ in the cells. This was followed by increase in the FOS and EGR1 mRNAs levels and decrease in the JUN and MYC mRNA levels. Treatment of HUVEC with the Na+-ionophore monensin led to accumulation of Na+ and loss of K+ ions. However, monensin had no significant effect on gene expression. Incubation of HUVEC with elevated extracellular NaCl concentration increased intracellular K+ concentration and transcription of the ATF3 gene, while transcription of the JUN gene decreased. These results indicate that Na+ and Li+ ions have different effects on the gene expression profile in the cells that is likely associated with the fact that they affect differently the intracellular monovalent cations ratio.

In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy

BackgroundThe analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. ResultsIn this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. Significance and noveltyBy using the method of small flow perfusion (≤20 μL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

Blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption.

Genetically encoded calcium ion () indicators (GECIs) are powerful tools for monitoring intracellular concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon imaging in hippocampal slices. The -bound state of T-GECO1 has an excitation peak maximum of 468nm, an emission peak maximum of 500nm, an extinction coefficient of , a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The -dependent fluorescence increase is 15-fold, and the apparent for is 82nM. With two-photon excitation conditions at 850nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Function of TRP channels in monocytes/macrophages.

The transient receptor potential channel (TRP channel) family is a kind of non- specific cation channel widely distributed in various tissues and organs of the human body, including the respiratory system, cardiovascular system, immune system, etc. It has been reported that various TRP channels are expressed in mammalian macrophages. TRP channels may be involved in various signaling pathways in the development of various systemic diseases through changes in intracellular concentrations of cations such as calcium and magnesium. These TRP channels may also intermingle with macrophage activation signals to jointly regulate the occurrence and development of diseases. Here, we summarize recent findings on the expression and function of TRP channels in macrophages and discuss their role as modulators of macrophage activation and function. As research on TRP channels in health and disease progresses, it is anticipated that positive or negative modulators of TRP channels for treating specific diseases may be promising therapeutic options for the prevention and/or treatment of disease.

Effects of the gaseous signalling molecule nitroxyl (HNO) on myenteric neurons governing intestinal motility.

The main function of myenteric neurons is the control of gut motility. As we recently showed that nitroxyl (HNO) induces intestinal smooth muscle relaxation, it was of interest to evaluate the effects of this signalling molecule on myenteric neurons in order to distinguish its properties in regard to myocytes. Myenteric neurons isolated from the ileum of 4-10days old rats were used. HNO-induced changes in intracellular concentration of Ca2+ or membrane potential and ion currents were measured using the Ca2+-sensitive fluorescent dye fura-2 AM or by electrophysiological whole-cell recordings, respectively. Changes in intracellular thiol groups pool were evaluated using thiol tracker violet. Angeli's salt was used as HNO donor. The HNO donor Angeli's salt induced a significant increase in the cytosolic Ca2+ concentration at the concentration 50µM and a membrane hyperpolarization from a resting membrane potential of-56.1±8.0mV to-63.1±8.7mV (n=7). Although potassium channels primarily drive membrane potential changes in these cells, outwardly rectifying potassium currents were not significantly affected by 50µM Angeli's salt. Fast inward sodium currents were slightly but not significantly reduced by HNO. In more sensitive cells, HNO tended to reduce the pool of thiol groups. As in the case of smooth muscle cells, HNO causes hyperpolarization of myenteric neurons, an effect also associated with an increase in intracellular Ca2+ concentration. Pathways other than activation of potassium currents appear to drive the hyperpolarization evoked by HNO.

Monitoring Tacrolimus Concentrations in Whole Blood and Peripheral Blood Mononuclear Cells: Inter- and Intra-Patient Variability in a Cohort of Pediatric Patients.

Tacrolimus (TAC) is a first-choice immunosuppressant for solid organ transplantation, characterized by high potential for drug-drug interactions, significant inter- and intra-patient variability, and narrow therapeutic index. Therapeutic drug monitoring (TDM) of TAC concentrations in whole blood (WB) is capable of reducing the incidence of adverse events. Since TAC acts within lymphocytes, its monitoring in peripheral blood mononuclear cells (PBMC) may represent a valid future alternative for TDM. Nevertheless, TAC intracellular concentrations and their variability are poorly described, particularly in the pediatric context. Therefore, our aim was describing TAC concentrations in WB and PBMC and their variability in a cohort of pediatric patients undergoing constant immunosuppressive maintenance therapy, after liver transplantation. TAC intra-PBMCs quantification was performed through a validated UHPLC–MS/MS assay over a period of 2–3 months. There were 27 patients included in this study. No significant TAC changes in intracellular concentrations were observed (p = 0.710), with a median percent change of −0.1% (IQR −22.4%–+46.9%) between timings: this intra-individual variability was similar to the one in WB, −2.9% (IQR −29.4–+42.1; p = 0.902). Among different patients, TAC weight-adjusted dose and age appeared to be significant predictors of TAC concentrations in WB and PBMC. Intra-individual seasonal variation of TAC concentrations in WB, but not in PBMC, have been observed. These data show that the intra-individual variability in TAC intracellular exposure is comparable to the one observed in WB. This opens the way for further studies aiming at the identification of therapeutic ranges for TAC intra-PBMC concentrations.

Physiology of Astroglial Excitability.

Classic physiology divides all neural cells into excitable neurons and nonexcitable neuroglia. Neuroglial cells, chiefly responsible for homeostasis and defense of the nervous tissue, coordinate their complex homeostatic responses with neuronal activity. This coordination reflects a specific form of glial excitability mediated by complex changes in intracellular concentration of ions and second messengers organized in both space and time. Astrocytes are equipped with multiple molecular cascades, which are central for regulating homeostasis of neurotransmitters, ionostasis, synaptic connectivity, and metabolic support of the central nervous system. Astrocytes are further provisioned with multiple receptors for neurotransmitters and neurohormones, which upon activation trigger intracellular signals mediated by Ca2+, Na+, and cyclic AMP. Calcium signals have distinct organization and underlying mechanisms in different astrocytic compartments thus allowing complex spatiotemporal signaling. Signals mediated by fluctuations in cytosolic Na+ are instrumental for coordination of Na+ dependent astrocytic transporters with tissue state and homeostatic demands. Astroglial ionic excitability may also involve K+, H+, and Cl−. The cyclic AMP signalling system is, in comparison to ions, much slower in targeting astroglial effector mechanisms. This evidence review summarizes the concept of astroglial intracellular excitability.

Targeting zinc metalloenzymes in coronavirus disease 2019.

Several lines of evidence support a link between the essential element zinc and the coronavirus disease 2019 (COVID‐19). An important fact is that zinc is present in proteins of humans and of viruses. Some zinc sites in viral enzymes may serve as drug targets and may liberate zinc ions, thus leading to changes in intracellular concentration of zinc ions, while increased intracellular zinc may induce biological effects in both the host and the virus. Drugs such as chloroquine may contribute to increased intracellular zinc. Moreover, clinical trials on the use of zinc alone or in addition to other drugs in the prophylaxis/treatment of COVID‐19 are ongoing. Thereby, we aim to discuss the rationale for targeting zinc metalloenzymes as a new strategy for the treatment of COVID‐19.Linked ArticlesThis article is part of a themed issue on The Pharmacology of COVID‐19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc

Mass transport of lipopolysaccharide induced H2O2 detected by an intracellular carbon nanoelectrode sensor

Hydrogen peroxide is a key component of the innate immune response, regulating how a cell responds to a bacterial threat; however, being transient in nature makes it extremely difficult to detect. We show the development of an improved biosensor capable of the rapid detection of the hydrogen peroxide produced intracellularly in response to both smooth and rough lipopolysaccharides (LPS) structures. The arising signal and mass transport behaviour to the electrodes were characterised. This response was detected utilising a single walled carbon nanotube-based sensor that has been functionalised with an osmium complex for specificity and detecting the change in intracellular concentrations of hydrogen peroxide through chronoamperometry. This was conducted within murine macrophage (RAW264.7) cells and using ultra-pure LPS extracted from two different serotypes of bacteria (0111:B4 and Re495). This allowed the comparison of the immune response when infected with different structures of LPS. We demonstrate that the hydrogen peroxide signal can be electrochemically detected within 3 seconds post injection. Combining the nature of the mass transport of hydrogen peroxide and concentration characteristics, a bacterial ‘fingerprint’ was identified. The impact of this work will be demonstrated in allowing us to develop a rapid diagnostic for bacterial detection.

A hybrid model to evaluate the impact of active uptake transport on hepatic distribution of atorvastatin in rats

1. Mathematical modeling remains a useful tool to study the impact of transporters on overall and intracellular drug disposition. The impact of organic anion transporter protein mediated uptake on atorvastatin systemic and intracellular pharmacokinetics, specifically distribution volume, was studied in rats with mathematical modeling and conducting in vivo pharmacokinetic studies for atorvastatin in presence and absence of rifampicin. A previously developed 5-compartment explicit membrane model for the liver was combined with a compartmental model to develop a semi-physiological hybrid model for atorvastatin disposition. 2. Rifampicin treatment resulted in a decrease in systemic clearance and steady-state distribution volume, and an increase in half-life of atorvastatin. The hybrid model predicted higher unbound intracellular liver atorvastatin concentrations than unbound plasma concentrations in both rifampicin treated and untreated groups, indicating involvement of uptake transporters. The intracellular unbound concentrations during the distributive phase were unaffected by rifampicin. The dependence of clearance on blood flow as well as hepatic uptake for atorvastatin (a moderate-to-high extraction ratio drug) can explain this lack of change in intracellular concentrations if there is incomplete inhibition of transport at the tested rifampicin dose. 3. The hybrid model successfully allowed the evaluation of effect of active uptake on intracellular and plasma atorvastatin disposition.

Calcium Mobilization in Endothelial Cell Functions.

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca2+]i, which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca2+ mobilization to ECs in health and disease.

Astroglial Calcium Signaling in Aging and Alzheimer's Disease.

Astrocytes are the homeostatic and protective cells of the central nervous system (CNS). In neurological diseases, astrocytes undergo complex changes, which are subclassified into (1) reactive astrogliosis, an evolutionary conserved defensive rearrangement of cellular phenotype aimed at neuroprotection; (2) pathological remodeling, when astrocytes acquire new features driving pathology; and (3) astrodegeneration, which is manifested by astroglial atrophy and loss of homeostatic functions. In aging brains as well as in the brains affected by Alzheimer's disease (AD), astrocytes acquire both atrophic and reactive phenotypes in a region- and disease-stage-dependent manner. Prevalence of atrophy overreactivity, observed in certain brain regions and in terminal stages of the disease, arguably facilitates the development of neurological deficits. Astrocytes exhibit ionic excitability mediated by changes in intracellular concentration of ions, most importantly of Ca2+ and Na+, with intracellular ion dynamics triggered by the activity of neural networks. AD astrocytes associated with senile plaques demonstrate Ca2+ hyperactivity in the form of aberrant Ca2+ oscillations and pathological long-range Ca2+ waves. Astroglial Ca2+ signaling originating from Ca2+ release from the endoplasmic reticulum is a key factor in initiating astrogliotic response; deficient Ca2+ signaling toolkits observed in entorhinal and prefrontal cortices of AD model animals may account for vulnerability of these regions to the pathology.

The level of intracellular tacrolimus in T cell is affected by CD44+ABCB1+activities triggered by inflammation

Rejection is an important issue in kidney transplant. Although with adequate trough level of tacrolimus, acute rejection occurs, and we are focused on these cases. We hypothesized that the lower concentration of tacrolimus in the peripheral blood mononuclear cell would be a cause of rejection; in this regard, we describe ABCB1, which regulates intracellular concentration of tacrolimus. The effect of inflammation on the intracellular concentration of tacrolimus was evaluated, as was the association between that concentration and ABCB1 and CD44 activities. Seven kidney recipients experiencing acute rejection were prospectively enrolled. Both the whole blood concentration of tacrolimus and intracellular concentration of tacrolimus were measured at the time of enrollment and after stabilization. A human T lymphoblastoid cell line (Jurkat T cell) was treated with various concentrations of tacrolimus for 21 h and then stimulated for 3 h. The levels of mRNA interleukin-2, interleukin-8, and interferon-γ decreased dose dependently by tacrolimus. Furthermore, a fluorescence-activated cell sorter was used to count cells expressing CD44 and ABCB1; changes in intracellular concentration of tacrolimus were explored after tacrolimus treatment and stimulation. Also, B6 splenocytes were tested in the same manner as previous Jurkat T cell experiments. The tacrolimus ratio of three patients was lower at the time of acute rejection than when patients were stabilized. In vitro, the intracellular concentration of tacrolimus decreased after stimulation. Under the same conditions, CD44+ABCB1+cells increased in proportion on tacrolimus treatment and stimulation. This work supports the hypothesis that inflammation reduces the intracellular tacrolimus level, possibly via drug efflux mediated by CD44+ABCB1+and inflammation could lead to acute rejection.

Large-Scale Functional Analysis of CRP-Mediated Feed-Forward Loops.

Feed-forward loops (FFLs) represent an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs, such as araBAD and araFGH operons and adapts to fluctuating environmental nutrients, thereby enhancing the survivability of E. coli. Although computational simulations have been conducted to explore the potential functionality of FFLs, a comprehensive study on the functions of all structural types on the basis of in vivo data is lacking. Moreover, the regulatory role of CRP-mediated FFLs (CRP-FFLs) remains obscure. We identified 393 CRP-FFLs in E. coli using EcoCyc and RegulonDB. Dose–response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, which were further divided into five functional groups. The microarray and reported regulatory relationships identified 202 CRP-FFLs that were directly regulated by CRP in these eight types of FFLs. Interestingly, 34% (147/432) of genes were directly regulated by CRP and CRP-regulated TFs, which indicates that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3’,5’-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3’-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The ‘downregulated’ group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes in local availability of thyroid hormones might favor a shift from a differentiated to a more proliferation-prone state of cancer tissues and cell lines.

Exercise increases lactoferrin, but decreases lysozyme in salivary granulocytes.

Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise. To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45+CD15+ cells. 11 males (20.3 ± 0.8years, 57.2 ± 7.6 mL/kg/min V̇O2pk, 11.1 ± 3.9% body fat) ran for 45min at 75% of VO2pk. 12mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45+) and granulocytes (CD45+CD15+) using flow cytometry. Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p <0.05). Lys MFI decreased with exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p <0.05). Acute running resulted in an increased Lac concentration which could lead to a decrease in inflammation, adding further evidence of the anti-inflammatory effects of exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

Myoinositol as a Biomarker in Recurrent Glioblastoma Treated with Bevacizumab: A 1H-Magnetic Resonance Spectroscopy Study.

BackgroundAntiangiogenic treatment of glioblastomas with Bevacizumab lacks predictive markers. Myoinositol (MI) is an organic osmolyte, with intracellular concentration changes depending on the extracellular osmolality. Since Bevacizumab markedly reduces tumor edema and influences the tumor microenvironment, we investigated whether the MI concentration in the tumor changes during therapy.MethodsWe used 1H-magnetic resonance spectroscopy to measure the MI concentrations in the tumor and contralateral control tissue of 39 prospectively recruited patients with recurrent glioblastomas before and 8–12 weeks after starting therapy. 30 patients received Bevacizumab and 9 patients were treated with CCNU/VM26 as control. We performed a survival analysis to evaluate MI as a predictive biomarker for Bevacizumab therapy.ResultsMI concentrations increased significantly during Bevacizumab therapy in tumor (p < .001) and control tissue (p = .001), but not during CCNU/VM26 treatment. For the Bevacizumab cohort, higher MI concentrations in the control tissue at baseline (p = .021) and higher differences between control and tumor tissue (delta MI, p = .011) were associated with longer survival. A Kaplan-Meier analysis showed a median OS of 164 days for patients with a deltaMI < 1,817 mmol/l and 275 days for patients with a deltaMI > 1,817 mmol/l. No differences were observed for the relative changes or the post treatment concentrations. Additionally calculated creatine concentrations showed no differences in between subgroups or between pre and post treatment measurements.ConclusionOur data suggest that recurrent glioblastoma shows a strong metabolic reaction to Bevacizumab. Further, our results support the hypothesis that MI might be a marker for early tumor cell invasion. Pre-therapeutic MI concentrations are predictive of overall survival in patients with recurrent glioblastoma treated with Bevacizumab.

Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate.

We have developed a method for rapid quenching of samples taken from chemostat cultures of Escherichia coli that gives reproducible and reliable measurements of extracellular and intracellular metabolites by 1H NMR and have applied it to study the major central metabolites during the transition from anaerobic to aerobic growth. Almost all metabolites showed a gradual change after perturbation with air, consistent with immediate inhibition of pyruvate formate-lyase, dilution of overflow metabolites and induction of aerobic enzymes. Surprisingly, although pyruvate showed almost no change in intracellular concentration, the extracellular concentration transiently increased. The absence of intracellular accumulation of pyruvate suggested that one or more glycolytic enzymes might relocate to the cell membrane. To test this hypothesis, chromosomal pyruvate kinase (pykF) was modified to express either PykF-green fluorescent protein or PykF-FLAG fusion proteins. Measurements showed that PykF-FLAG relocates to the cell membrane within 5 min of aeration and then slowly returns to the cytoplasm, suggesting that on aeration, PykF associates with the membrane to facilitate secretion of pyruvate to maintain constant intracellular levels.

Synchronous Drosophila circadian pacemakers display nonsynchronous Ca²⁺ rhythms in vivo.

In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brainwide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca(2+) that were entrained by environmental cues and timed by molecular clocks. However, these rhythms were not synchronous, as each group exhibited its own phase of activation. Ca(2+) rhythms displayed by pacemaker groups that were associated with the morning or evening locomotor activities occurred ~4 hours before their respective behaviors. Loss of the receptor for the neuropeptide PDF promoted synchrony of Ca(2+) waves. Thus, neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers.