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Abstract The correlation between altered extracellular pH and various pathological conditions, including cancer, inflammation and metabolic disorders, is well known. Bulk pH measurements cannot report the extracellular pH value at the cell surface. However, there is a limited number of suitable tools for measuring the extracellular pH of cells with high spatial resolution, and none of them are commonly used in laboratories around the world. In this study, a versatile ratiometric nanosensor for the measurement of extracellular pH was developed. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with the pH-inert reference dye Nile red and is surface functionalized with a pH-responsive fluorescein dye. Equipped with a targeting moiety, the nanosensor can adhere to cell membranes, allowing direct measurement of extracellular pH at the cell surface. The nanosensor exhibits a sensitive ratiometric pH response within the range of 5.5–9.0, with a calculated pKa of 7.47. This range optimally covers the extracellular pH (pHe) of most healthy cells and cells in which the pHe is abnormal, such as cancer cells. In combination with the nanosensors ability to target cell membranes, its high robustness, reversibility and its biocompatibility, the pHe nanosensor proves to be well suited for in-situ measurement of extracellular pH, even over extended time periods. This pH nanosensor has the potential to advance biomedical research by improving our understanding of cellular microenvironments, where extracellular pH plays an important role.

A unique feature of the tumor microenvironment is extracellular acidosis in relation to intracellular milieu. Metabolic reprogramming in tumors results in overproduction of H+ ions (and lactate), which are extruded from the cells to support tumor survival and progression. As a result, the transmembrane pH gradient (ΔpH), representing the difference between intracellular pH (pHi) and extracellular pH (pHe), is posited to be larger in tumors compared with normal tissue. Controlling the transmembrane pH difference has promise as a potential therapeutic target in cancer as it plays an important role in regulating drug delivery into cells. The current study shows successful development of an MRI/MRSI‐based technique that provides ΔpH imaging at submillimeter resolution. We applied this technique to image ΔpH in rat brains with RG2 and U87 gliomas, as well as in mouse brains with GL261 gliomas. pHi was measured with Amine and Amide Concentration‐Independent Detection (AACID), while pHe was measured with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). The results indicate that pHi was slightly higher in tumors (7.40–7.43 in rats, 7.39–7.47 in mice) compared with normal brain (7.30–7.38 in rats, 7.32–7.36 in mice), while pHe was significantly lower in tumors (6.62–6.76 in rats, 6.74–6.84 in mice) compared with normal tissue (7.17–7.22 in rats, 7.20–7.21 in mice). As a result, ΔpH was higher in tumors (0.64–0.81 in rats, 0.62–0.65 in mice) compared with normal brain (0.13–0.16 in rats, 0.13–0.16 in mice). This work establishes an MRI/MRSI‐based platform for ΔpH imaging at submillimeter resolution in gliomas. [ABSTRACT FROM AUTHOR]

Plant cells must precisely regulate extracellular pH (pH e) by sensing subtle changes caused by the environment or neighboring cells. This allows plants to acclimatize to their environment and modulate growth and development. pH e controls the chemistry and rheology of the cell wall to adjust its elasticity and spatiotemporally regulate cell expansion in plants. Plasma membrane (PM)-localized H+-ATPases, cell-wall components, and cell wall-remodeling enzymes jointly maintain pH e homeostasis. Although the acid growth theory was proposed five decades ago, the mechanism underlying auxin-induced extracellular acidification has only recently been deciphered: TRANSMEMBRANE KINASES (TMKs) are the key signaling components that mediate the rapid effect of auxin on PM H+-ATPase. PM-localized cell-surface peptide–receptor pairs sense pH e during plant development, reproduction, and immunity. In plants, pH determines nutrient acquisition and sensing, and triggers responses to osmotic stress, whereas pH homeostasis protects the cellular machinery. Extracellular pH (pH e) controls the chemistry and rheology of the cell wall to adjust its elasticity and regulate cell expansion in space and time. Plasma membrane (PM)-localized proton pumps, cell-wall components, and cell wall-remodeling enzymes jointly maintain pH e homeostasis. To adapt to their environment and modulate growth and development, plant cells must sense subtle changes in pH e caused by the environment or neighboring cells. Accumulating evidence indicates that PM-localized cell-surface peptide–receptor pairs sense pH e. We highlight recent advances in understanding how plants perceive and maintain pH e , and discuss future perspectives. [ABSTRACT FROM AUTHOR]

Action of numerous adverse environmental factors on higher plants is spatially-heterogenous; it means that induction of a systemic adaptive response requires generation and transmission of the stress signals. Electrical signals (ESs) induced by local action of stressors include action potential, variation potential, and system potential and they participate in formation of fast physiological changes at the level of a whole plant, including photosynthetic responses. Generation of these ESs is accompanied by the changes in activity of H+-ATPase, which is the main system of electrogenic proton transport across the plasma membrane. Literature data show that the changes in H+-ATPase activity and related changes in intra- and extracellular pH play a key role in the ES-induced inactivation of photosynthesis in non-irritated parts of plants. This inactivation is caused by both suppression of CO2 influx into mesophyll cells in leaves, which can be induced by the apoplast alkalization and, probably, cytoplasm acidification, and direct influence of acidification of stroma and lumen of chloroplasts on light and, probably, dark photosynthetic reactions. The ES-induced inactivation of photosynthesis results in the increasing tolerance of photosynthetic machinery to the action of adverse factors and probability of the plant survival. [ABSTRACT FROM AUTHOR]

[Display omitted] Surface topography has become a powerful tool to control cell behaviors, however, it's still difficult to monitor cellular microenvironment changes during topography-induced cell responses. Here, a dual-functional platform integrating cell alignment with extracellular pH (pHe) measurement is proposed. The platform is fabricated by assembling gold nanorods (AuNRs) into micro pattern via wettability difference interface method, which provides topographical cues and surface-enhanced Raman scattering (SERS) effect for cell alignment and biochemical detection respectively. Results demonstrate that contact guidance and cell morphology changes are achieved by the AuNRs micro pattern, and pHe are also obtained by the changes of SERS spectra during cell alignment, where the pHe near cytoplasm is lower than nucleus, revealing the heterogeneity of extracellular microenvironment. Moreover, a correlation between lower extracellular pH and higher cell migration ability is revealed, and AuNRs micro pattern can differentiate cells with different migration ability, which may be an inheritable character during cell division. Furthermore, mesenchymal stem cells response dramatically to AuNRs micro pattern, showing different morphology and increased pHe level, offering the potential of impacting stem cell differentiation. This approach provides a new idea for the research of cell regulation and response mechanism. [ABSTRACT FROM AUTHOR]

An acidic extracellular pH value (pHe) is characteristic of many cancers, in contrast to the physiologic pHe found in most benign cells. This difference in pH offers a unique opportunity to design and engineer chemicals that can be employed for pH-selective reactions in the extracellular fluid of cancer cells. The viability of human skin melanoma and corresponding fibroblasts exposed to CaS dispersions is reported. The viability of melanoma cells decreases with CaS dispersion concentration and reaches 57% at 3%, a value easily distinguishable from melanoma control experiments. In contrast, the viability of benign fibroblasts remains nearly constant within experimental error over the range of dispersion concentrations studied. The CaS dispersions facilitate vinculin delocalization in the cytoplasmic fluid, a result consistent with improved focal adhesion kinase (FAK) regulation in melanoma cells. Thermodynamic considerations are consistent with the formation of H2S from CaS in the presence of protons. The thermodynamic prediction is verified in independent experiments with solid CaS and acidic aqueous solutions. The amount of H2S formed decreases with pH. An activation energy for the process of (30 ± 10) kJ/mol in the temperature range of 280 to 330 K is estimated from initial rate measurements as a function of temperature. The total Gibbs energy minimization approach was employed to establish the distribution of sulfides—including H2S in the gas and aqueous phases—from the dissociation of CaS as a function of pH to mimic physiologically relevant pH values. Theoretical calculations suggest that partially protonated CaS in solution can be stable until the sulfur atom bonds to two hydrogen atoms, resulting in the formation of Ca2+ and H2S, which can be solvated and/or released to the gas phase. Our results are consistent with a model in which CaS is dissociated in the extracellular fluid of melanoma cells selectively. The results are discussed in the context of the potential biomedical applications of CaS dispersions in cancer therapies.

An acidic extracellular pH value (pHe) is characteristic of many cancers, in contrast to the physiologic pHe found in most benign cells. This difference in pH offers a unique opportunity to design and engineer chemicals that can be employed for pH-selective reactions in the extracellular fluid of cancer cells. The viability of human skin melanoma and corresponding fibroblasts exposed to CaS dispersions is reported. The viability of melanoma cells decreases with CaS dispersion concentration and reaches 57% at 3%, a value easily distinguishable from melanoma control experiments. In contrast, the viability of benign fibroblasts remains nearly constant within experimental error over the range of dispersion concentrations studied. The CaS dispersions facilitate vinculin delocalization in the cytoplasmic fluid, a result consistent with improved focal adhesion kinase (FAK) regulation in melanoma cells. Thermodynamic considerations are consistent with the formation of H2S from CaS in the presence of protons. The thermodynamic prediction is verified in independent experiments with solid CaS and acidic aqueous solutions. The amount of H2S formed decreases with pH. An activation energy for the process of (30 ± 10) kJ/mol in the temperature range of 280 to 330 K is estimated from initial rate measurements as a function of temperature. The total Gibbs energy minimization approach was employed to establish the distribution of sulfides—including H2S in the gas and aqueous phases—from the dissociation of CaS as a function of pH to mimic physiologically relevant pH values. Theoretical calculations suggest that partially protonated CaS in solution can be stable until the sulfur atom bonds to two hydrogen atoms, resulting in the formation of Ca2+ and H2S, which can be solvated and/or released to the gas phase. Our results are consistent with a model in which CaS is dissociated in the extracellular fluid of melanoma cells selectively. The results are discussed in the context of the potential biomedical applications of CaS dispersions in cancer therapies. [ABSTRACT FROM AUTHOR]

Zn2+ is known to be important for the normal brain functions. Disruption of zinc homeostasis and zinc-induced neurotoxicity has been shown to play a role in the development of neurodegenerative diseases. In this work, we investigated the effect of extracellular alkalosis on the zinc ions neurotoxicity in the cultured rat cerebellar granule neurons. Zinc chloride (0.03–0.06 mM, 24 h) added to the culture medium of rat cerebellar granule neurons caused the dose-dependent death of these cells. According to ultrastructural morphological features, the process of cell death could be attributed to necrosis, since it was accompanied by swelling of intracellular organelles and disruption of cell membranes against the background of relatively intact nuclear membranes. Neuronal death was associated with an increase in the level of intracellular free zinc. The toxic effect of zinc ions was significantly decreased when ionotropic glutamate NMDA-receptors were blocked by MK-801 or when the extracellular pH was increased from 7.3 to 7.8, due to a decrease in the zinc overload of the cytoplasm of these cells. The presented results demonstrate that NMDA channels are one of the Zn ion entry pathways in the cultured cerebellar granule neurons. Extracellular alkalosis reduces the zinc overload of the cytoplasm and, consequently, promotes the survival of neurons. Probably, zinc's neurotoxicity is inextricably linked with changes in the intracellular concentration of protons. [ABSTRACT FROM AUTHOR]

There is an unmet clinical need for new and robust imaging biomarkers to distinguish indolent from aggressive prostate cancer. Hallmarks of aggressive tumors such as a decrease in extracellular pH (pHe) can potentially be used to identify aggressive phenotypes. In this study, we employ an optimized, high signal-to-noise ratio hyperpolarized (HP) 13C pHe imaging method to discriminate between indolent and aggressive disease in a murine model of prostate cancer. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice underwent a multiparametric MR imaging exam, including HP [13C] bicarbonate MRI for pHe, with 1H apparent diffusion coefficient (ADC) mapping and HP [1-13C] pyruvate MRI to study lactate metabolism. Tumor tissue was excised for histological staining and qRT-PCR to quantify mRNA expression for relevant glycolytic enzymes and transporters. We observed good separation in pHe between low- and high-grade tumor regions, with high-grade tumors demonstrating a lower pHe. The pHe also correlated strongly with monocarboxylate transporter Mct4 gene expression across all tumors, suggesting that lactate export via MCT4 is associated with acidification in this model. Our results implicate extracellular acidification as an indicator of indolent-to-aggressive transition in prostate cancer and suggest feasibility of HP pHe imaging to detect high-grade, clinically significant disease in men as part of a multiparametric MRI examination.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplasm with high metastatic potential and poor clinical outcome. Like other solid tumors, PDAC in the early stages is often asymptomatic, and grows very slowly under a distinct acidic pHe (extracellular pH) microenvironment. However, most previous studies have only reported the fate of cancerous cells upon cursory exposure to acidic pHe conditions. Little is known about how solid tumors—such as the lethal PDAC originating within the pancreatic duct-acinar system that secretes alkaline fluids—evolve to withstand and adapt to the prolonged acidotic microenvironmental stress. Methods Representative PDAC cells were exposed to various biologically relevant periods of extracellular acidity. The time effects of acidic pHe stress were determined with respect to tumor cell proliferation, phenotypic regulation, autophagic control, metabolic plasticity, mitochondrial network dynamics, and metastatic potentials. Results Unlike previous short-term analyses, we found that the acidosis-mediated autophagy occurred mainly as an early stress response but not for later adaptation to microenvironmental acidification. Rather, PDAC cells use a distinct and lengthy process of reversible adaptive plasticity centered on the early fast and later slow mitochondrial network dynamics and metabolic adjustment. This regulates their acute responses and chronic adaptations to the acidic pHe microenvironment. A more malignant state with increased migratory and invasive potentials in long-term acidosis-adapted PDAC cells was obtained with key regulatory molecules being closely related to overall patient survival. Finally, the identification of 34 acidic pHe-related genes could be potential targets for the development of diagnosis and treatment against PDAC. Conclusions Our study offers a novel mechanism of early rapid response and late reversible adaptation of PDAC cells to the stress of extracellular acidosis. The presence of this distinctive yet slow mode of machinery fills an important knowledge gap in how solid tumor cells sense, respond, reprogram, and ultimately adapt to the persistent microenvironmental acidification.

Obtaining analytical information about chemical species at interfaces is fundamentally important to improving our understanding of chemical reactions and biological processes. pH at solid-liquid interfaces is found to deviate from the bulk solution value, for example, in electrocatalytic reactions at surfaces or during the corrosion of metals. Also, in the vicinity of living cells, metabolic reactions or cellular responses cause changes in pH at the extracellular interface. In this review, we collect recent progress in the development of sensors with the capability to detect pH at or close to solid-liquid and bio interfaces, with spatial and time resolution. After the two main principles of pH detection are presented, the different classes of molecules and materials that are used as active components in these sensors are described. The review then focuses on the reported electroanalytical techniques for local pH sensing. As application examples, we discuss model studies that exploit local pH sensing in the area of electrocatalysis, corrosion, and cellular interfaces. We conclude with a discussion of key challenges for wider use of this analytical approach, which shows promise to improve the mechanistic understanding of reactions and processes at realistic interfaces.

Aim: TASK-1 channels are established regulators of pulmonary artery tone but their contribution to the regulation of vascular tone in systemic arteries is poorly understood. We tested the hypothesis that TASK-1 channel functional impact differs among systemic vascular beds, that this is associated with differences in their expression and may increase with alkalization of the extracellular environment. Therefore, we evaluated the expression level of TASK-1 channels and their vasomotor role in mesenteric and renal arteries. Methods: Pulmonary, mesenteric and renal arteries from male Wistar rats were used for TASK-1 channel mRNA (qPCR) and protein content (Western blotting) measurements. The functional role of TASK-1 channels was studied by wire myography using the TASK-1 channel blocker AVE1231. In some experiments, the endothelium was removed with a rat whisker. Results: Expression levels of both mRNA and protein of the TASK-1 channel pore-forming subunit were highest in pulmonary arteries, lowest in mesenteric arteries and had an intermediate value in renal arteries. Blockade of TASK-1 channels by 1 µM AVE1231 increased U46619-induced contractile responses of pulmonary arteries but did not affect basal tone and contractile responses to methoxamine of mesenteric and renal arteries at physiological extracellular pH (pHo = 7.41). At alkaline extracellular pH = 7.75 (increase of NaHCO3 to 52 mM) AVE1231 evoked the development of basal tone and increased contractile responses to low concentrations of methoxamine in renal but not mesenteric arteries. This effect was independent of the endothelium. Conclusion: In the rat systemic circulation, TASK-1 channels are abundant in renal arteries and have an anticontractile function under conditions of extracellular alkalosis. [ABSTRACT FROM AUTHOR]

Aim: TASK-1 channels are established regulators of pulmonary artery tone but their contribution to the regulation of vascular tone in systemic arteries is poorly understood. We tested the hypothesis that TASK-1 channel functional impact differs among systemic vascular beds, that this is associated with differences in their expression and may increase with alkalization of the extracellular environment. Therefore, we evaluated the expression level of TASK-1 channels and their vasomotor role in mesenteric and renal arteries.Methods: Pulmonary, mesenteric and renal arteries from male Wistar rats were used for TASK-1 channel mRNA (qPCR) and protein content (Western blotting) measurements. The functional role of TASK-1 channels was studied by wire myography using the TASK-1 channel blocker AVE1231. In some experiments, the endothelium was removed with a rat whisker.Results: Expression levels of both mRNA and protein of the TASK-1 channel pore-forming subunit were highest in pulmonary arteries, lowest in mesenteric arteries and had an intermediate value in renal arteries. Blockade of TASK-1 channels by 1 µM AVE1231 increased U46619-induced contractile responses of pulmonary arteries but did not affect basal tone and contractile responses to methoxamine of mesenteric and renal arteries at physiological extracellular pH (pHo = 7.41). At alkaline extracellular pH = 7.75 (increase of NaHCO3 to 52 mM) AVE1231 evoked the development of basal tone and increased contractile responses to low concentrations of methoxamine in renal but not mesenteric arteries. This effect was independent of the endothelium.Conclusion: In the rat systemic circulation, TASK-1 channels are abundant in renal arteries and have an anticontractile function under conditions of extracellular alkalosis.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplasm with high metastatic potential and poor clinical outcome. Like other solid tumors, PDAC in the early stages is often asymptomatic, and grows very slowly under a distinct acidic pHe (extracellular pH) microenvironment. However, most previous studies have only reported the fate of cancerous cells upon cursory exposure to acidic pHe conditions. Little is known about how solid tumors—such as the lethal PDAC originating within the pancreatic duct-acinar system that secretes alkaline fluids—evolve to withstand and adapt to the prolonged acidotic microenvironmental stress. Methods: Representative PDAC cells were exposed to various biologically relevant periods of extracellular acidity. The time effects of acidic pHe stress were determined with respect to tumor cell proliferation, phenotypic regulation, autophagic control, metabolic plasticity, mitochondrial network dynamics, and metastatic potentials. Results: Unlike previous short-term analyses, we found that the acidosis-mediated autophagy occurred mainly as an early stress response but not for later adaptation to microenvironmental acidification. Rather, PDAC cells use a distinct and lengthy process of reversible adaptive plasticity centered on the early fast and later slow mitochondrial network dynamics and metabolic adjustment. This regulates their acute responses and chronic adaptations to the acidic pHe microenvironment. A more malignant state with increased migratory and invasive potentials in long-term acidosis-adapted PDAC cells was obtained with key regulatory molecules being closely related to overall patient survival. Finally, the identification of 34 acidic pHe-related genes could be potential targets for the development of diagnosis and treatment against PDAC. Conclusions: Our study offers a novel mechanism of early rapid response and late reversible adaptation of PDAC cells to the stress of extracellular acidosis. The presence of this distinctive yet slow mode of machinery fills an important knowledge gap in how solid tumor cells sense, respond, reprogram, and ultimately adapt to the persistent microenvironmental acidification. [ABSTRACT FROM AUTHOR]

Acidity and salinity of the extracellular fluid, reflecting degrees of acid and sodium contents respectively, regulate essential cellular functions in health and disease, especially cancer. Tumor invasiveness is enhanced by the acidic extracellular milieu as a consequence of upregulated aerobic glycolysis. But recent discoveries also suggest that enhanced proliferative mitosis, which also hallmarks cancer, is impacted by interstitial salinity. Abnormal transmembrane proton/sodium gradients lead to pathophysiological alterations at the cellular level. These novel perspectives mandate pioneering imaging of extracellular acidity and salinity, preferably monitored simultaneously. By dissecting the interplay between dysregulated pH and electrolyte imbalance within the tumor habitat, these biomarkers hold promise for early cancer diagnosis and tracking therapies, from chemotherapy to immunotherapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important mediator of tumor immune surveillance. In addition, its potential to kill cancer cells without harming healthy cells led to the development of TRAIL receptor agonists, which however did not show the desired effects in clinical trials. This is caused mainly by apoptosis resistance mechanisms operating in primary cancer cells. Meanwhile, it has been realized that in addition to cell death, TRAIL also induces non-apoptotic pro-inflammatory pathways that may enhance tumor malignancy. Due to its late detection and resistance to current therapeutic options, pancreatic ductal adenocarcinoma (PDAC) is still one of the deadliest types of cancer worldwide. A dysregulated pH microenvironment contributes to PDAC development, in which the cancer cells become highly dependent on to maintain their metabolism. The impact of extracellular pH (pHe) on TRAIL-induced signaling in PDAC cells is poorly understood so far. To close this gap, we analyzed the effects of acidic and alkaline pHe, both in short-term and long-term settings, on apoptotic and non-apoptotic TRAIL-induced signaling. We found that acidic and alkaline pHe differentially impact TRAIL-induced responses, and in addition, the duration of the pHe exposition also represents an important parameter. Thus, adaptation to acidic pHe increases TRAIL sensitivity in two different PDAC cell lines, Colo357 and Panc1, one already TRAIL-sensitive and the other TRAIL-resistant, respectively. However, the latter became highly TRAIL-sensitive only by concomitant inhibition of Bcl-xL. None of these effects was observed under other pHe conditions studied. Both TRAIL-induced non-apoptotic signaling pathways, as well as constitutively expressed anti-apoptotic proteins, were regulated by acidic pHe. Whereas the non-apoptotic pathways were differently affected in Colo357 than in Panc1 cells, the impact on the anti-apoptotic protein levels was similar in both cell lines. In Panc1 cells, adaptation to either acidic or alkaline pHe blocked the activation of the most of TRAIL-induced non-apoptotic pathways. Interestingly, under these conditions, significant downregulation of the plasma membrane levels of TRAIL-R1 and TRAIL-R2 was observed. Summing up, extracellular pH influences PDAC cells’ response to TRAIL with acidic pHe adaptation, showing the ability to strongly increase TRAIL sensitivity and in addition to inhibit TRAIL-induced pro-inflammatory signaling.

Extracellular pH and concentration of K+ as well as their gradient across the plasma membrane have a significant impact on the physiology of the yeast cell, but their role in cell death has not been thoroughly investigated. Here we observed that increasing extracellular pH, as well as supplementing with K+ ions had a mitigating effect on cell death in yeast occurring under several conditions. The first is sugar induced cell death (SICD), and the second is death caused by several specific gene deletions, which have been recently identified in a systematic screen. It was shown that in both cases, primary necrosis is suppressed at neutral pH. SICD was also inhibited by the protonophore dinitrophenol (DNP) and 150 mM extracellular K+, with the latter condition also benefiting survival of cell dying due to gene mutations. In the case of SICD, these effects could not be mitigated by perturbing known pH-dependent signaling pathways, and thus are likely to be realized via direct effects on the plasma membrane potential. Thus, (a)—we show that stabilization of external pH at a neutral level can suppress different types of primary necrosis, and (b)—we suggest that changes to the cellular membrane potential can play a central role in yeast cell death caused by different factors. [ABSTRACT FROM AUTHOR]

Purpose: Multiple myeloma (MM) is an incurable disease of malignant plasma cells in the bone marrow (BM). Adaptive responses to hypoxia may be an essential element in MM progression and drug resistance. This metabolic adaptation involves a decrease in extracellular pH (pHe), and it depends on the upregulation of glucose transporters (GLUTs) that is common in hypoxia and in cancer cells. CEST MRI is an imaging technique that assesses pHe indirectly by the exchange rate of magnetic saturation transfer between labile protons on a solute and water. Thus, this study aimed to determine the feasibility of acidoCEST MRI for pHe measurement using an orthotopic mouse model of MM compared with GLUT1 immunofluorescence staining as a reference. Procedures: Orthotopic BM engrafted MM xenografts were established in NSG/NOD mice using the human RPMI8226 myeloma cell line. AcidoCEST MRI was performed approximately 6 weeks after intravenous challenge, before and after intravenous administration of iopamidol. BM pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations. Samples were decalcified, sectioned, and immunostained for GLUT1 expression. Pearson's correlation was used to assess the relationship between pHe and [H3O+] versus GLUT1 expression. Results: Ten mice underwent acidoCEST MRI followed by immunofluorescent histologic analysis. A strong negative correlation was seen between pHe versus GLUT1 expression (r = − 0.75, p < 0.001). After transformation of pH to [H3O+], a strong positive correlation between [H3O+] and GLUT1 expression was observed (r = 0.8, p < 0.001). Conclusions: AcidoCEST MRI can measure the extracellular pH of bone marrow affected by multiple myeloma. In this MM orthotopic mouse model, pHe measured by acidoCEST MRI showed strong correlations with the metabolic phenotype of BM tumor assessed by immunofluorescent histological assessment of GLUT1 overexpression. [ABSTRACT FROM AUTHOR]

Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the "up" and "down" conformations identified by crystallography do not correspond to open and closed states of these channels. [ABSTRACT FROM AUTHOR]

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment. [ABSTRACT FROM AUTHOR]

ATPase, a key enzyme involved in energy metabolism, has not yet been well studied in Clostridium acetobutylicum. Here, we knocked down the atpG gene encoding the ATPase gamma subunit in C. acetobutylicum ATCC 824 using a mobile group II intron system and analyzed the physiological characteristics of the atpG gene knockdown mutant, 824-2866KD. Properties investigated included cell growth, glucose consumption, production of major metabolites, and extracellular pH. Interestingly, in 2-L batch fermentations, 824-2866KD showed no significant difference in metabolite biosynthesis or cell growth compared with the parent ATCC 824. However, the pH value in 824-2866KD cultures at the late stage of the solventogenic phase was abnormally high (pH 6.12), compared with that obtained routinely in the culture of ATCC 824 (pH 5.74). This phenomenon was also observed in batch cultures of another C. acetobutylicum, BEKW-2866KD, an atpG-knockdown and pta-buk double-knockout mutant. The findings reported in this study suggested that ATPase is relatively minor than acid-forming pathway in ATP metabolism in C. acetobutylicum.

Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the “up” and “down” conformations identified by crystallography do not correspond to open and closed states of these channels.

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment.

Abstract Background Rapamycin is a potent inhibitor of the highly conserved TOR kinase, the nutrient-sensitive controller of growth and aging. It has been utilised as a chemotherapeutic agent due to its anti-proliferative properties and as an immunosuppressive drug, and is also known to extend lifespan in a range of eukaryotes from yeast to mammals. However, the mechanisms through which eukaryotic cells adapt to sustained exposure to rapamycin have not yet been thoroughly investigated. Methods Here, S. cerevisiae response to long-term rapamycin exposure was investigated by identifying the physiological, transcriptomic and metabolic differences observed for yeast populations inoculated into low-dose rapamycin-containing environment. The effect of oxygen availability and acidity of extracellular environment on this response was further deliberated by controlling or monitoring the dissolved oxygen level and pH of the culture. Results Yeast populations grown in the presence of rapamycin reached higher cell densities complemented by an increase in their chronological lifespan, and these physiological adaptations were associated with a rewiring of the amino acid metabolism, particularly that of arginine. The ability to synthesise amino acids emerges as the key factor leading to the major mechanistic differences between mammalian and microbial TOR signalling pathways in relation to nutrient recognition. Conclusion Oxygen levels and extracellular acidity of the culture were observed to conjointly affect yeast populations, virtually acting as coupled physiological effectors; cells were best adapted when maximal oxygenation of the culture was maintained in slightly acidic pH, any deviation necessitated more extensive readjustment to additional stress factors.

BACKGROUND High carbon dioxide (CO2) level from indoor environments, such as classrooms and offices, might cause sick building syndrome. Excessive indoor CO2 level increases CO2 level in the blood, and over-accumulation of CO2 induces an adaptive response that requires modulation of gene expression. This study aimed to investigate the adaptive transcriptional response toward hypoxia and oxidative stress in human peripheral blood mononuclear cells (PBMCs) exposed to elevated CO2 level in vitro and its association with cell viability. METHODS PBMCs were treated in 5% CO2 and 15% CO2, representatives a high CO2 level condition for 24 and 48 hours. Extracellular pH (pHe) was measured with a pH meter. The levels of reactive oxygen species were determined by measuring superoxide and hydrogen peroxide with dihydroethidium and dichlorofluorescin-diacetate assay. The mRNA expression levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, nuclear factor (NF)-κB, and manganese superoxide dismutase (MnSOD) were analyzed using a realtime reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell survival was determined by measuring cell viability. RESULTS pHe increased in 24 hours after 15% CO2 treatment, and then decreased in 48 hours. Superoxide and hydrogen peroxide levels increased after the 24- and 48-hour of high CO2 level condition. The expression levels of NF-κB, MnSOD, HIF-1α, and HIF-2α decreased in 24 hours and increased in 48 hours. The increased antioxidant mRNA expression in 48 hours showed that the PBMCs were responsive under high CO2 conditions. Elevated CO2 suppressed cell viability significantly in 48 hours. CONCLUSIONS After 48 hours of high CO2 level condition, PBMCs showed an upregulation in genes related to hypoxia and oxidative stress to overcome the effects of CO2 elevation. [ABSTRACT FROM AUTHOR]

Purpose: Chemical exchange saturation transfer MRI can provide accurate pH images, but the slow scan time (due to long saturation periods and multiple offsets sampling) reduce both the volume coverage and spatial resolution capability, hence the possibility to interrogate the heterogeneity in tumors and organs. To overcome these limitations, we propose a fast multislice CEST‐MRI sequence with high pH accuracy and spatial resolution. Methods: The sequence first uses a long saturation pulse to induce the steady‐state CEST contrast and a second short saturation pulse repeated after each image acquisition to compensate for signal losses based on an uneven irradiation scheme combined with a single‐shot rapid acquisition with refocusing echoes readout. Sequence sensitivity and accuracy in measuring pH was optimized by simulation and assessed by in vitro studies in pH‐varying phantoms. In vivo validation was performed in two applications by acquiring multislice pH images covering the whole tumors and kidneys after iopamidol injection. Results: Simulated and in vivo data showed comparable contrast efficiency and pH responsiveness by reducing saturation time. The experimental data from a homogeneous, pH‐varying, iopamidol‐containing phantom show that the sequence produced a uniform CEST contrast across slices and accurate values across slices in less than 10 minutes. In vivo measurements allowed us to quantify the 3D pH gradients of tumors and kidneys, with pH ranges comparable with the literature. Conclusion: The proposed fast multislice CEST‐MRI sequence allows volumetric acquisitions with good pH sensitivity, accuracy, and spatial resolution for several in vivo pH imaging applications. [ABSTRACT FROM AUTHOR]

PurposeIn this study, we aimed to use 3T magnetic resonance imaging (MRI), which is clinically available, to determine the extracellular pH (pHe) of liver tumors and prospectively evaluate the ability of chemical exchange saturation transfer (CEST) MRI to distinguish between benign and malignant liver tumors.MethodsDifferent radiofrequency irradiation schemes were assessed for ioversol-based pH measurements at 3T. CEST effects were quantified in vitro using the asymmetric magnetization transfer ratio (MTRasym) at 4.3 ppm from the corrected Z spectrum. Generalized ratiometric analysis was conducted by rationing resolved ioversol CEST effects at 4.3 ppm at a flip angle of 60 and 350°. Fifteen patients recently diagnosed with hepatic carcinoma and five patients diagnosed with hepatic hemangioma [1 male; mean age, 48.6 (range, 37–59) years] were assessed.ResultsBy conducting dual-power CEST MRI, the pH of solutions was determined to be 6.0–7.2 at 3T in vitro. In vivo, ioversol signal intensities in the tumor region showed that the extracellular pH in hepatic carcinoma was acidic(mean ± standard deviation, 6.66 ± 0.19), whereas the extracellular pH was more physiologically neutral in hemangioma (mean ± standard deviation, 7.34 ± 0.09).The lesion size was similar between CEST pH MRI and T2-weighted imaging.Conclusiondual-power CEST MRI can detect extracellular pH in human liver tumors and can provide molecular-level diagnostic tools for differentiating benign and malignant liver tumors at 3T.

Purpose: In this study, we aimed to use 3T magnetic resonance imaging (MRI), which is clinically available, to determine the extracellular pH (pHe) of liver tumors and prospectively evaluate the ability of chemical exchange saturation transfer (CEST) MRI to distinguish between benign and malignant liver tumors. Methods: Different radiofrequency irradiation schemes were assessed for ioversol-based pH measurements at 3T. CEST effects were quantified in vitro using the asymmetric magnetization transfer ratio (MTRasym) at 4.3 ppm from the corrected Z spectrum. Generalized ratiometric analysis was conducted by rationing resolved ioversol CEST effects at 4.3 ppm at a flip angle of 60 and 350°. Fifteen patients recently diagnosed with hepatic carcinoma and five patients diagnosed with hepatic hemangioma [1 male; mean age, 48.6 (range, 37–59) years] were assessed. Results: By conducting dual-power CEST MRI, the pH of solutions was determined to be 6.0–7.2 at 3T in vitro. In vivo, ioversol signal intensities in the tumor region showed that the extracellular pH in hepatic carcinoma was acidic(mean ± standard deviation, 6.66 ± 0.19), whereas the extracellular pH was more physiologically neutral in hemangioma (mean ± standard deviation, 7.34 ± 0.09).The lesion size was similar between CEST pH MRI and T2-weighted imaging. Conclusion: dual-power CEST MRI can detect extracellular pH in human liver tumors and can provide molecular-level diagnostic tools for differentiating benign and malignant liver tumors at 3T. [ABSTRACT FROM AUTHOR]

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.

Purpose: In vivo 31P MRSI enables noninvasive mapping of absolute pH values via the pH‐dependent chemical shifts of inorganic phosphates (Pi). A particular challenge is the quantification of extracellular Pi with low SNR in vivo. The purpose of this study was to demonstrate feasibility of assessing both intra‐ and extracellular pH across the whole human brain via volumetric 31P MRSI at 7T. Methods: 3D 31P MRSI data sets of the brain were acquired from three healthy volunteers and three glioma patients. Low‐rank denoising was applied to enhance the SNR of 31P MRSI data sets that enables detection of extracellular Pi at high spatial resolutions. A robust two‐compartment quantification model for intra‐ and extracellular Pi signals was implemented. Results: In particular low‐rank denoising enabled volumetric mapping of intra‐ and extracellular pH in the human brain with voxel sizes of 5.7 mL. The average intra‐ and extracellular pH measured in white matter of healthy volunteers were 7.00 ± 0.00 and 7.33 ± 0.03, respectively. In tumor tissue of glioma patients, both the average intra‐ and extracellular pH increased to 7.12 ± 0.01 and 7.44 ± 0.01, respectively, compared to normal appearing tissue. Conclusion: Mapping of pH values via 31P MRSI at 7T using the proposed two‐compartment quantification model improves reliability of pH values obtained in vivo, and has the potential to provide novel insights into the pH heterogeneity of various tissues. [ABSTRACT FROM AUTHOR]

Extracellular acidification, playing a promoting role in the process of acute pancreatitis, has been reported to activate Cl− channels in several types of cells. However, whether extracellular acidification aggravates acute pancreatitis via activating Cl− channels remains unclear. Here, we investigated the effects of extracellular acidification on Cl− channels in rat pancreatic acinar AR42J cells using whole-cell patch-clamp recordings. We found that extracellular acidification induced a moderately outward-rectified Cl− current, with a selectivity sequence of I− > Br− ≥ Cl− > gluconate−, while intracellular acidification failed to induce the currents. The acid-sensitive currents were inhibited by Cl− channel blockers, 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate and 5-Nitro-2-(3-phenylpropylamino) benzoic acid. After ClC-3 was silenced by ClC-3 shRNA, the acid-sensitive Cl− currents were attenuated significantly, indicating that ClC-3 plays a vital role in the induction of acid-sensitive Cl− currents. Extracellular acid elevated the intracellular level of reactive oxygen species (ROS) significantly, prior to inducing Cl− currents. When ROS production was scavenged, the acid-sensitive Cl− currents were abolished. Whereas, the level of acid-induced ROS was unaffected with silence of ClC-3. Our findings above demonstrate that extracellular acidification induces a Cl− current in pancreatic acinar cells via promoting ROS generation, implying an underlying mechanism that extracellular acidification might aggravate acute pancreatitis through Cl− channels. • A chloride channel in AR42J cells was sensitive to extracellular acidification. • ClC-3 played a vital role in the induction of the acid-sensitive Cl− currents. • Reactive oxygen species involved in inducing the acid-sensitive Cl− currents. [ABSTRACT FROM AUTHOR]

Extracellular pH can indicate the variation in organelle function and cell state. It is important to measure extracellular pH (pHe) with a controllable distance. In this work, a potentiometric SECM dual-microelectrode was developed to monitor the pHe of MCF-7 cells under electrical stimulation. The distance between the dual-microelectrode and the cells was determined first with a gold microelectrode by recording the approaching curve, and the pH was determined using an open-circuit potential (OCP) technique with a polyaniline-modified Pt microelectrode. The pH microelectrode showed a response slope of 53.0 ± 0.4 mV/pH and good reversibility from pH 4 to pH 8, fast response within 10 s, and a potential drift of 1.13% for 3 h, and thus was employed to monitor the pHe of stimulated cells. The value of pHe decreased with the decrease in the distance to cells, likely due to the release of H+. With an increase in the stimulation potential or time, the pHe value decreased, as the cell membrane became more permeable, which was verified by fluorescence staining of calcein-AM/PI (propidium iodide). Based on these results, this method can be widely applied for determining the species released by biosystems at a controllable position. [ABSTRACT FROM AUTHOR]

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence. [ABSTRACT FROM AUTHOR]

The stem cell fraction of a cell population is finely tuned by stimuli from the external microenvironment. Among these stimuli, a decrease of extracellular pH (pHe) may occur in a variety of physiological and pathological conditions, including hypoxia and inflammation. In this study, by using bone marrow stem cells and dental pulp stem cells, we provided evidence that extracellular acidosis endows the maintenance of stemness in mesenchymal cells. Indeed, continuous exposure for 21 d to low pHe (6.5-6.8) conditions impaired the osteogenic differentiation of both cell types. Moreover, the exposure to low pHe, for 1 and up to 7 d, induced the expression of stemness-related genes and proteins, drove cells to reside in the quiescent G0 alert state and enhanced their ability to form floating spheres. The pre-conditioning with extracellular acidosis for 7 d did not affect the differentiation potential of dental pulp stem cells since, when the cells were cultured again at physiological pHe, their multilineage potential was almost unmodified. Our data provided evidence of the role of extracellular acidosis as a modulator of the stemness of mesenchymal cells. This condition is commonly found both in systemic and local bone conditions, hence underlining the relevance of this phenomenon for a better comprehension of bone healing and regeneration.

The main challenges in developing effective anti-cancer therapies stem from the highly complex and heterogeneous nature of cancer, including the presence of multiple genetically-encoded and environmentally-induced cancer cell phenotypes within an individual. This diversity can make the development of successful treatments difficult as different phenotypes can have different responses to the same treatment. The lack of model-systems that can be used to simultaneously test the effect of therapies on multiple distinct phenotypic states further contributes to this problem. To mitigate these challenges, we suggest that in vitro model-systems that consist of several genetically-related but phenotypically distinct populations can be used as proxies for the several phenotypes (including adherent and circulating tumor cells) present in a patient with advanced disease. As proof of concept, we have developed such a model and showed that different phenotypes had different responses to the same challenge (i.e., a change in extracellular pH) both in terms of sensitivity and phenotypic plasticity. We suggest that similar model-systems could be developed and used when designing novel therapeutic strategies, to address the potential impact of phenotypic heterogeneity and plasticity of cancer on the development of successful therapies. Specifically, the effect of a therapy should be considered on more than one cancer cell phenotype (to increase its effectiveness), and both cell viability as well as changes in phenotypic state (to address potential plastic responses) should be evaluated. Although we are aware of the limitations of in vitro systems, we believe that the use of established cell lines that express multiple phenotypes can provide invaluable insights into the complex interplay between therapies and cancer's heterogeneous and plastic nature.

The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoarchitectures is at the heart of Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits pushed to the single molecule recognition. Within this frame, SERS-based nanosensors for localized pH measurements have been developed and employed for a wide range of applications. Nevertheless, to improve the performances of such nanosensors, many key issues concerning their assembling, calibration and stability, that could significantly impact on the outcome of the pH measurements, need to be clarified. Here, we report on the detailed characterization of a case study SERS-active pH nanosensor, based on the conjugation of gold nanoparticles with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA). We analyzed and optimized all the aspects of the synthesis procedure and of the operating conditions to preserve the sensor stability and provide the highest responsiveness to pH. Exploiting the dependence of the SERS spectrum on the protonation degree of the carboxylic group at the edge of the 4MBA molecules, we derived a calibration curve for the nanosensor. The extrapolated working point, i.e., the pH value corresponding to the highest sensitivity, falls at pH 5.6, which corresponds to the pKa value of the molecule confined at the nanoparticle surface. A shift of the pKa of 4MBA, observed on the molecules confined at the nanostructured interface respect to the bulk counterpart, unveils the opportunity to assembly a SERS-based pH nanosensor with the ability to select its working point in the sensitivity region of interest, by acting on the nanostructured surface on which the molecular probe is confined. As a proof-of-concept, the nanosensor was successfully employed to measure the extracellular pH of normal and cancer cells, demonstrating the capability to discriminate between them.

The pathogenesis of stress-related disorders involves aberrant glucocorticoid secretion, and decreased pH and increased lactate in the brain are common phenotypes in several psychiatric disorders. Mice treated with glucocorticoids develop these phenotypes, but it is unclear how glucocorticoids affect brain pH. Therefore, we investigated the effect of corticosterone (CORT), the main glucocorticoid in rodents, on extracellular pH and lactate release in cultured astrocytes, which are the main glial cells that produce lactate in the brain. CORT treatment for one week decreased the extracellular pH and increased the extracellular lactate level via glucocorticoid receptors. CORT also increased the intracellular pyruvate level and upregulated pyruvate dehydrogenase kinase 4 (PDK4), while PDK4 overexpression increased extracellular lactate and decreased the extracellular pH. Furthermore, PDK4 inhibition suppressed the increase in extracellular lactate and the decrease in extracellular pH induced by CORT. These results suggest that increased lactate release via accumulation of intracellular pyruvate in astrocytes by chronic glucocorticoid exposure contributes to decreased brain pH.

The main challenges in developing effective anti-cancer therapies stem from the highly complex and heterogeneous nature of cancer, including the presence of multiple genetically-encoded and environmentally-induced cancer cell phenotypes within an individual. This diversity can make the development of successful treatments difficult as different phenotypes can have different responses to the same treatment. The lack of model-systems that can be used to simultaneously test the effect of therapies on multiple distinct phenotypic states further contributes to this problem. To mitigate these challenges, we suggest that in vitro model-systems that consist of several genetically-related but phenotypically distinct populations can be used as proxies for the several phenotypes (including adherent and circulating tumor cells) present in a patient with advanced disease. As proof of concept, we have developed such a model and showed that different phenotypes had different responses to the same challenge (i.e., a change in extracellular pH) both in terms of sensitivity and phenotypic plasticity. We suggest that similar model-systems could be developed and used when designing novel therapeutic strategies, to address the potential impact of phenotypic heterogeneity and plasticity of cancer on the development of successful therapies. Specifically, the effect of a therapy should be considered on more than one cancer cell phenotype (to increase its effectiveness), and both cell viability as well as changes in phenotypic state (to address potential plastic responses) should be evaluated. Although we are aware of the limitations of in vitro systems, we believe that the use of established cell lines that express multiple phenotypes can provide invaluable insights into the complex interplay between therapies and cancer's heterogeneous and plastic nature. [ABSTRACT FROM AUTHOR]

Measurements made in the blood, part of the extracellular compartment, are used in the clinical assessment of acid-base disorders; however, intracellular events determine much of the metabolic importance of these disorders. Intracellular and interstitial compartment acid-base balance is complex and varies in different tissues. This review considers the determination of extracellular pH in the context of ion transport processes at the interface of cells and the interstitial fluid, and between epithelial cells lining the transcellular contents of the gastrointestinal and urinary tracts that open to the external environment. A further consideration is the role of these membrane transporters in the generation of acid-base disorders and the associated disruption of electrolyte balance. This review suggests a process of internal and external balance for pH regulation similar to that of potassium, and considers the role of secretory gastrointestinal epithelia and renal epithelia with respect to normal pH homeostasis and clinical disorders. Electroneutrality of electrolytes in the extracellular fluid is a fundamental feature of reciprocal changes in Cl- or non-Cl- anions and HCO3-. Normal mechanisms for protecting cell pH and producing normal gastrointestinal and renal secretions in healthy states also may result in disease when abnormal. In a similar manner, organic anions such as ketoacid anions and lactate, normally transported as fuels between organs, result in acid-base disturbances in disease. Understanding the genomic basis of these transporters may contribute to specific treatments. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding the CFTR chloride channel produce cystic fibrosis (CF). CF patients are more susceptible to bacterial infections in lungs. The most accepted hypothesis sustains that a reduction in the airway surface liquid (ASL) volume favor infections. Alternatively, it was postulated that a reduced HCO3− transport through CFTR leads to a decreased ASL pH, favoring bacterial colonization. The issue is controversial, since recent data from cultured primary cells and CF children showed normal pH values in the ASL. We have reported previously a decreased mitochondrial Complex I (mCx-I) activity in cultured cells with impaired CFTR activity. Thus, we hypothesized that the reduced mCx-I activity could lead to increased lactic acid production (Warburg-like effect) and reduced extracellular pH (pHe). In agreement with this idea, we report here that cells with impaired CFTR function (intestinal Caco-2/pRS26, transfected with an shRNA-CFTR, and lung IB3-1 CF cells) have a decreased pHe. These cells showed increased lactate dehydrogenase (LDH) activity, LDH-A expression, and lactate secretion. Similar effects were reproduced in control cells stimulated with recombinant IL-1β. The c-Src and JNK inhibitors PP2 and SP600125 were able to increase the pHe, although the differences between control and CFTR-impaired cells were not fully compensated. Noteworthy, the LDH inhibitor oxamate completely restored the pHe of the intestinal Caco-2/pRS26 cells and have a significant effect in lung IB3-1 cells; therefore, an increased lactic acid secretion seems to be the key factor that determine a reduced pHe in these epithelial cells. [ABSTRACT FROM AUTHOR]

In this study, the use of electrochemistry in cell biology is extended by showing that electrochemically generated microscale pH gradients can be used to gain new insights into the regulation of cytosolic pH of normal and cancer cells. The developed procedure involves positioning a carbon fiber microelectrode into the extracellular space of adherently growing cells and setting its potential to values suitable for electrooxidation or electroreduction of water. While the electrooxidation of water decreases the pH of the solution surrounding the microelectrode (because it produces hydronium ions), the electroreduction of water increases the pH of the same solution (because it produces hydroxide ions). Fluorescence microscopy is then used to observe the impact of the electrochemically generated microscale pH gradient on the cytosolic pH of cells loaded with a fluorescent pH sensor. The obtained results indicate that electrochemically induced acid stress affects the cytosolic pH of normal cells significantly faster than that of cancer cells while electrochemically induced alkaline stress appears to have very limited impact on the cytosolic pH of both cell types. In comparison to classic experiments concerning the regulation of cytosolic pH using perfusion chambers, the developed, electrochemistry‐based, approach has the advantages of a better spatial and temporal resolution and elimination of the flow‐induced shear stress. Changing cell surroundings by electrolysis: the use of electrochemistry in cell biology is extended by showing that electrochemically generated microscale pH gradients can be used to gain new insights into the regulation of cytosolic pH of normal and cancer cells. [ABSTRACT FROM AUTHOR]

Abstract Protons are potent modulators of GABA A receptors (GABA A Rs) and α 1 Phe64 residue was implicated in their pH sensitivity. Recently, we have demonstrated that this residue is involved in flipping transitions which precede channel opening. We thus re-addressed the mechanism of GABA A R modulation by protons by considering the gating scheme extended by flipping. The impact of pH changes was examined on currents mediated by wild-type α 1 β 2 γ 2 receptors or by their α 1 Phe64Leu or α 1 Phe64Cys mutants and elicited by saturating concentrations of full (GABA) or partial (piperidine-4-sulfonic acid) agonists. To describe the impact of extracellular pH on receptor gating, we combined macroscopic analysis of currents elicited by rapid agonist applications with single-channel studies. Acidification (pH 6.0) increased current amplitudes (in the case of leucine mutants effect was stronger when P4S was used) and decreased the rate and the extent of desensitization whereas alkalization (pH 8.0) had the opposite but weaker effect. Deactivation kinetics for wild-type receptors was slowed down by acidification while in the case of mutants this effect was observed upon alkalization. Moreover, α 1 Phe64 mutations enhanced GABA A R sensitivity to alkaline pH. Single-channel analysis revealed that acidification prolonged burst durations and affected shut but not open time distributions. Model simulations for macroscopic and single-channel activity indicated a novel mechanism in which protons primarily affected opening and desensitization rates but not flipping/unflipping. This evidence for the impact of protons on the receptor gating together with previously demonstrated effect on the agonist binding, point to a complex effect of extracellular pH on GABA A R macromolecule. Highlights • Extracellular pH changes affect open probability of GABA A receptor. • Protons affect GABA A R by altering primarily desensitization and opening rates. • Flipping transitions appear not to be affected by pH changes. • α 1 Phe64 mutations enhance receptor sensitivity to alkalization. [ABSTRACT FROM AUTHOR]

Abstract At the intersection of the newly emerging fields of optoacoustic imaging and theranostic nanomedicine, promising clinical progress can be made in dismal prognosis of ovarian cancer. An acidic pH targeted wormhole mesoporous silica nanoparticle (V7-RUBY) was developed to serve as a novel tumor specific theranostic nanoparticle detectable using multispectral optoacoustic tomographic (MSOT) imaging. We report the synthesis of a small, < 40 nm, biocompatible asymmetric wormhole pore mesoporous silica core particle that has both large loading capacity and favorable release kinetics combined with tumor-specific targeting and gatekeeping. V7-RUBY exploits the acidic tumor microenvironment for tumor-specific targeting and tumor-specific release. In vitro, treatment with V7-RUBY containing either paclitaxel or carboplatin resulted in increased cell death at pH 6.6 in comparison to drug alone (p < 0.0001). In orthotopic ovarian xenograft mouse models, V7-RUBY containing IR780 was specifically detected within the tumor 7X and 4X higher than the liver and >10X higher than in the kidney using both multispectral optoacoustic tomography (MSOT) imaging with secondary confirmation using near infrared fluorescence imaging (p < 0.0004). The V7-RUBY system carrying a cargo of either contrast agent or an anti-neoplastic drug has the potential to become a theranostic nanoparticle which can improve both diagnosis and treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Budding at the tumor invasive front has been correlated with the malignant properties of many cancers. Malic enzyme 1 (ME1) promotes the Warburg effect in cancer cells and induces epithelial–mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC). Therefore, we investigated the role of ME1 in tumor budding in OSCC. Tumor budding was measured in 96 human OSCCs by immunostaining for an epithelial marker (AE1/AE3), and its expression was compared with that of ME1. A significant correlation was observed between tumor budding and ME1 expression. The correlation increased with the progression of cancer. In human OSCC cells, lactate secretion decreased when lactate fermentation was suppressed by knockdown of ME1 and lactate dehydrogenase A or inhibition of pyruvate dehydrogenase (PDH) kinase. Furthermore, the extracellular pH increased, and the EMT phenotype was suppressed. In contrast, when oxidative phosphorylation was suppressed by PDH knockdown, lactate secretion increased, extracellular pH decreased, and the EMT phenotype was promoted. Induction of chemical hypoxia in OSCC cells by CoCl2 treatment resulted in increased ME1 expression along with HIF1α expression and promotion of the EMT phenotype. Hypoxic conditions also increased matrix metalloproteinases expression and decreased mitochondrial membrane potential, mitochondrial oxidative stress, and extracellular pH. Furthermore, the hypoxic treatment resulted in the activation of Yes-associated protein (YAP), which was abolished by ME1 knockdown. These findings suggest that cancer cells at the tumor front in hypoxic environments increase their lactate secretion by switching their energy metabolism from oxidative phosphorylation to glycolysis owing to ME1 overexpression, decrease in extracellular pH, and YAP activation. These alterations enhance EMT and the subsequent tumor budding. Tumor budding and ME1 expression are thus considered useful markers of OSCC malignancy, and ME1 is expected to be a relevant target for molecular therapy.

Modulation of pH is crucial to maintaining the chemical homeostasis of biological environments. The irregular metabolic pathways exhibited by cancer cells result in the production of acidic byproducts that are excreted and accumulate in the extracellular tumor microenvironment, reducing the pH. As a consequence of the lower pH in tumors, cancer cells increase the expression of metastatic phenotypes and chemotherapeutic resistance. A significant limitation in current cancer therapies is the inability to locally deliver chemotherapeutics, leading to significant damage to healthy cells in systemic administration. To overcome these challenges, we present an injectable chitosan-poly(ethylene glycol) hydrogel that is dual-loaded with doxorubicin and sodium bicarbonate providing alkaline buffering of extracellular acidity and simultaneous chemotherapeutic delivery to increase chemotherapeutic efficacy. We conducted in vitro studies of weak base chemotherapeutic and alkaline buffer release from the hydrogel. The release of doxorubicin from hydrogels increased in a low-pH environment and was dependent on the encapsulated sodium bicarbonate concentration. We investigated the influence of pH on the doxorubicin efficacy and viability of MCF-7 and MDA-MB-231 breast cancer cell lines. The results show a 2- to 3-fold increase in IC 50 values from neutral pH to low pH, showing decreased cancer cell viability at neutral pH as compared to acidic pH. The IC 50 results were shown to correlate with a decrease in intracellular uptake of doxorubicin at low pH. The proposed hydrogels were confirmed to be nontoxic to healthy MCF-10A mammary epithelial cells. Rheological studies were performed to verify that the dual-loaded hydrogels were injectable. The mechanical and release properties of the hydrogels were maintained after extended storage. The chemotherapeutic activity of doxorubicin was evaluated in the presence of the proposed pH-regulating hydrogels. The findings suggest a promising nontoxic, biodegradable hydrogel buffer delivery system that can achieve two simultaneous important goals of local acidosis neutralization and chemotherapeutic release.