Single-cell intracellular pH dynamics regulate the cell cycle by timing the G1 exit and G2 transition.

Transient changes in intracellular pH (pHi) regulate normal cell behaviors, but roles for spatiotemporal pHi dynamics in single-cell behaviors remain unclear. Here, we mapped single-cell spatiotemporal pHi dynamics during mammalian cell cycle progression both with and without cell cycle synchronization. We found that single-cell pHi is dynamic throughout the cell cycle: pHi decreases at G1/S, increases in mid-S, decreases at late S, increases at G2/M and rapidly decreases during mitosis. Importantly, although pHi is highly dynamic in dividing cells, non-dividing cells have attenuated pHi dynamics. Using two independent pHi manipulation methods, we found that low pHi inhibits completion of S phase whereas high pHi promotes both S/G2 and G2/M transitions. Our data also suggest that low pHi cues G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. Furthermore, dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work reveals that spatiotemporal pHi dynamics are necessary for cell cycle progression at multiple phase transitions in single human cells.

MDSCs sneak CSCs out of (immuno)surveillance.

Cancer stem cells (CSCs) are known for their superior tumor-initiating and tumor-repopulating potential, partly reflecting their pronounced ability to evade immune recognition. Liu and colleagues recently identified a new aldehyde dehydrogenase (ALDH)-dependent mechanism whereby triple-negative breast CSCs evade immunosurveillance upon recruitment of myeloid-derived suppressor cells.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells.

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

The role of soluble adenylyl cyclase in sensing and regulating intracellular pH.

Soluble adenylyl cyclase (sAC) differs from transmembrane adenylyl cyclases (tmAC) in many aspects. In particular, the activity of sAC is not regulated by G-proteins but by the prevailing bicarbonate concentrations inside cells. Therefore, sAC serves as an exquisite intracellular pH sensor, with the capacity to translate pH changes into the regulation of localization and/or activity of cellular proteins involved in pH homeostasis. In this review, we provide an overview of literature describing the regulation of sAC activity by bicarbonate, pinpointing the importance of compartmentalization of intracellular cAMP signaling cascades. In addition, examples of processes involving proton and bicarbonate transport in different cell types, in which sAC plays an important regulatory role, were described in detail.

Naphthalimide-based, Single-Chromophore, Emission Ratiometric Fluorescent Sensor for Tracking Intracellular pH.

We report a novel, reversible, cell-permeable, pH-sensor, TRapH. TRapH afforded a pH-sensitive ratiometric emission response in the pH range ~3-6, enabling imaging and quantification of pH in living cells. The biological-applicability of TRapH was illustrated via live-tracking of intracellular pH dynamics in living mammalian cells induced by a synthetic H+-transporter.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

Transmembrane pH gradient imaging in rodent glioma models.

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.

Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pHi), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 µM H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pHi decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Comparative physiology reveals heat stress disrupts acid-base homeostasis independent of symbiotic state in the model cnidarian Exaiptasia diaphana.

Climate change threatens the survival of symbiotic cnidarians by causing photosymbiosis breakdown in a process known as bleaching. Direct effects of temperature on cnidarian host physiology remain difficult to describe because heatwaves depress symbiont performance, leading to host stress and starvation. The symbiotic sea anemone Exaiptasia diaphana provides an opportune system to disentangle direct versus indirect heat effects on the host, as it can survive indefinitely without symbionts. We tested the hypothesis that heat directly impairs cnidarian physiology by comparing symbiotic and aposymbiotic individuals of two laboratory subpopulations of a commonly used clonal strain of E. diaphana, CC7. We exposed anemones to a range of temperatures (ambient, +2°C, +4°C and +6°C) for 15-18 days, then measured their symbiont population densities, autotrophic carbon assimilation and translocation, photosynthesis, respiration and host intracellular pH (pHi). Symbiotic anemones from the two subpopulations differed in size and symbiont density and exhibited distinct heat stress responses, highlighting the importance of acclimation to different laboratory conditions. Specifically, the cohort with higher initial symbiont densities experienced dose-dependent symbiont loss with increasing temperature and a corresponding decline in host photosynthate accumulation. In contrast, the cohort with lower initial symbiont densities did not lose symbionts or assimilate less photosynthate when heated, similar to the response of aposymbiotic anemones. However, anemone pHi decreased at higher temperatures regardless of cohort, symbiont presence or photosynthate translocation, indicating that heat consistently disrupts cnidarian acid-base homeostasis independent of symbiotic status or mutualism breakdown. Thus, pH regulation may be a critical vulnerability for cnidarians in a changing climate.

An otopetrin family proton channel promotes cellular acid efflux critical for biomineralization in a marine calcifier.

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.

Neutralization of Intracellular pH Homeostasis to Inhibit Osteoclasts Based on a Spatiotemporally Selective Delivery System.

Osteoporosis is a global disease caused by abnormal overactivation of osteoclasts. The acidic environment in sealing zone of osteoclasts with H+ pumped from cytoplasm is critical to the maturation of osteoclasts. Therefore, reducing the intracellular H+ concentration can reduce the H+ secretion of osteoclasts from the source. In our study, we developed a novel nanovesicle which encapsulates Na2HPO4 with a liposome hybridizes with preosteoclast membrane (Na2HPO4@Lipo-pOCm). These nanovesicles release Na2HPO4 into the preosteoclast by targeting preosteoclasts and membrane fusion, reducing the intracellular H+ concentration, and achieve biological cascade regulation of osteoclasts through simple pH regulation. In vitro and in vivo experiments confirmed that these nanovesicles reduce mitochondrial membrane potential by decreasing intracellular H+ concentration, thereby reducing the ROS in osteoclasts as well as the expression of the upstream transcription factor FOXM1 of Acp5. In short, this nanovesicle can significantly inhibit the osteoclasts and ameliorate osteoporosis caused by OVX.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain dicarboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al., 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore-gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study.

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+ /H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4 Cl prepulse methodology; and (2) by combining the NH4 Cl prepulse with zoniporide. 31 P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-13 C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13 C NMR spectroscopy was used to monitor hyperpolarized [1-13 C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH4 Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4 Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4 Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-13 C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

ClC-K2 Cl- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pHi ) measurements in split-opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell-specific ClC-K2-/- mice to demonstrate that ClC-K2 inhibition enables to distinguish signals from A- and B-intercalated cells. We show that ClC-K2 Cl- channel is expressed on the basolateral side of intercalated cells, where it governs Cl- -dependent H+ /HCO3- transport. ClC-K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC-K2-/- mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1-positive A-type and decreased pHi in pendrin-positive B-type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A-type and decreased proton transport in B-type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of intercalated cells in split-opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A- and B-type in response to systemic acid/base stimuli.

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density.

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Ratiometric Sensing of Intracellular pH Based on Dual Emissive Carbon Dots.

Accurate monitoring of intracellular pH in living cells is critical for developing a better understanding of cellular activities. In the current study, label-free carbon dots (p-CDs), which were fabricated using a straightforward one-pot solvothermal treatment of p-phenylenediamine and urea, were employed to create a new ratiometric pH nanosensor. Under single-wavelength excitation (λex = 500 nm), the p-CDs gave dual emission bands at 525 and 623 nm. The fluorescent intensity ratio (I525/I623) was linearly related to pH over the range 4.0 to 8.8 in buffer solutions, indicating that the ratiometric fluorescence nanoprobe may be useful for pH sensing. In pH measurements, the p-CDs also demonstrated outstanding selectivity, reversibility, and photostability. Owing to the advantages outlined above, the nanoprobe was used to monitor the pH of HeLa cells effectively. The label-free CD-based ratiometric nanoprobe features comparatively easy manufacturing and longer excitation and emission wavelengths than the majority of previously reported CD-based ratiometric pH sensors, which is ultimately beneficial for applications in biological imaging.

Recent advances in the design of intracellular pH sensing nanoprobes based on organic and inorganic materials.

In the biological system, the intracellular pH (pHi) plays an important role in regulating diverse physiological activities, including enzymatic action, ion transport, cell proliferation, metabolism, and programmed cell death. The monitoring of pH inside living cells is also crucial for studying cellular events such as phagocytosis, endocytosis, and receptor-ligand internalization. Furthermore, some organelles, viz., endosomes and lysosomes, have intracompartmental pH, which is critical for maintaining the stability of protein structure and function. The dysfunction and abnormal pH regulation can result in terminal diseases such as cancer, Alzheimer, and so forth. Therefore, the accuracy of intracellular pH measurement is always the top priority and demands cutting-edge research and analysis. Such techniques, such as Raman spectroscopy and fluorescence imaging, preferably use nanotechnology due to their remarkable advantages, such as a non-invasive approach and providing accuracy, repeatability, and reproducibility. In the past decades, there have been numerous attempts to design and construct non-invasive organic and inorganic materials-based nanoprobes for pHi sensing. For Raman-based techniques, metal nanostructures such as Au/Ag/Cu nanoparticles are utilized to enhance the signal intensity. As for the fluorescence-based studies, the organic-based small molecules, such as dyes, show higher sensitivity toward pH. However, they possess several drawbacks, including high photobleaching rate, and autofluorescence background signals. To this end, there are alternative nanomaterials proposed, including semiconductor quantum dots (QDs), carbon QDs, upconversion nanoparticles, and so forth. Moreover, the fluorescence technique allows for ratiometric measurement of pHi, which as a result, offers a reliable calibration curve. This timely review will critically examine the current progression in the existing nanoprobes. In addition, based on our knowledge and available research findings, we provide a brief future outlook that may advance the state-of-the-art methodologies for pHi sensing.

Changes in Activity of the Plasma Membrane H+-ATPase as a Link Between Formation of Electrical Signals and Induction of Photosynthetic Responses in Higher Plants.

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.

Low CO2 partial pressure steers CHO cells into a defective metabolic state.

PURPOSE: The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines. METHODS AND RESULTS: The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells. CONCLUSION: Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control.

Live Streaming of a Single Cell's Life over a Local pH-Monitoring Nanowire Waveguide.

Spatiotemporal pH monitoring of single living cells across rigid cell and organelle membranes has been challenging, despite its significance in understanding cellular heterogeneity. Here, we developed a mechanically robust yet tolerably thin nanowire waveguide that enables in situ monitoring of pH dynamics at desired cellular compartments via direct optical communication. By chemically labeling fluorescein at one end of a poly(vinylbenzyl azide) nanowire, we continuously monitored pH variations of different compartments inside a living cell, successfully observing organelle-exclusive pH homeostasis and stimuli-selective pH regulations. Importantly, it was demonstrated for the first time that, during the mammalian cell cycle, the nucleus displays pH homeostasis in interphase but a tidal pH curve in the mitotic phase, implying the existence of independent pH-regulating activities by the nuclear envelope. The rapid and accurate local pH-reporting capability of our nanowire waveguide would be highly valuable for investigating cellular behaviors under diverse biological situations in living cells.