Intracellular pH Values Research Articles

Phosphorus (P) magnetic resonance spectroscopy for evaluation of brain tissue metabolism and measuring non-invasive pH. A study involving 23 volunteers. Part I

Evaluation of brain metabolism is an important part in examination of brain lesions. Phosphorus magnetic resonance spectroscopy opens up great opportunities for studying the energy metabolism and allows noninvasive examination of metabolic processes occurring both in healthy and in pathologic brain tissue by obtaining a spectrum of phosphorus-containing metabolites involved in the turnover of cell membrane phospholipids. The technique presented in this paper was used to conduct 31P MR spectroscopy and to estimate the ratio between the peaks of the main metabolites and intracellular pH of the healthy brain tissue of 23 volunteers in the age group under 30 years old in clinical settings. Based on the recorded stable phosphorus spectra of metabolites of the healthy brain tissue, the value of intracellular pH (6.963±0.044) and the ratio of the main PME/PDE peaks (1.17±0.20) were calculated. The database was created to subsequently analyze the metabolic changes in brain tissue spectra in norm and in pathology, as well as the intracellular pH variations that have diagnostic and prognostic value.

Fluorescence-SERS dual-signal probes for pH sensing in live cells

The activity of many cells is closely related to the pH of their internal environment, and the increase of intracellular pH value is a common feature of many drug-resistant tumors. The intracellular pH needs to be determined as the research basis when studying the intracellular transport protein and ion concentration changes. Nanomaterials are now widely used as the effective imaging and drug delivery vehicles, but it remains to be studied as a sensor for intracellular environment. Based on this, we have constructed a nanoprobe with fluorescence and surface-enhanced Raman scattering (SERS) dual-signal for sensing intracellular pH. The principle of this strategy is to construct fluorescence-SERS dual-signal nanoprobes by modifying pH-responsive fluorescent probes and SERS reporter molecules on the surface of gold nanorods (AuNRs) with the core-shell structure. The fluorescence-SERS dual response of the nanoprobe to pH can be achieved by investigating the fluorescence and SERS spectra of nanoprobes at different pH. Moreover, by incubating the nanoprobes into different cells, different double-signal response results can be obtained, thereby achieving intracellular pH sensing. The nanoprobe has dual responsiveness to fluorescence and SERS, which makes up for many of the deficiencies of single-signal probes, and realizes high sensitivity, accuracy and stability of intracellular pH detection. It is expected to be widely applied in the fields of medicine, chemistry and biology.

Carboxylate-Containing Two-Photon Probe for the Simultaneous Detection of Extra- and Intracellular pH Values in Colon Cancer Tissue.

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p Ka values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.

The Lysine 299 Residue Endows the Multisubunit Mrp1 Antiporter with Dominant Roles in Na+ Resistance and pH Homeostasis in Corynebacterium glutamicum.

Corynebacterium glutamicum is generally regarded as a moderately salt- and alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+ resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the Δmrp1 Δmrp2 double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+ antiporters of C. glutamicum were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed, C. glutamicum might employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+ resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136 Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+ level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation during C. glutamicum stress acclimatization.IMPORTANCE The capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially useful Corynebacterium glutamicum Although C. glutamicum exhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns of C. glutamicum putative Na+/H+ antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na+ levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na+/H+ antiporters in sodium antiport and pH regulation.

Enhanced Targeted Delivery of Doxorubicin Based on Acid Induced Charge Reversal and Combinational Stimuli‐Responsive Nanocarrier

To elevate the efficacy and to attenuate the adverse effects remain a tough challenge in exploring anticancer drug delivery systems. A nanocarrier characteristic of acid induced charge reversal and acid/redox/magnetic combinational stimuli‐responsiveness is designed. This system exhibits weaker repulsion between the carrier and the cancer cell membrane, resulting in enhanced uptake. After nanocarriers enter the cell, the system exhibits negative‐to‐positive charge reversal in response to the low intracellular pH value. Moreover, after the uptake, the carrier will be further dissociated by glutathione in cytoplasm via the reduction of the disulfide bond, leading to the rapid release of the encapsulated DOX into the nuclei. In vitro release study expresses the sequent stimulus release profile. The antitumor activity tested in CT26 tumor‐bearing mice reveals efficient therapeutic efficacy and as low adverse effect as PBS. H&E staining and immunohistochemical analysis of tumor tissues as well as pathological test of tissue sections of vital organs confirm the high antitumor activity and low tissue toxicity. Thus, the belief that this system has a good prospect in the field of cancer clinical chemotherapy.

Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone.

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10–20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

Manipulating extracellular tumour pH: an effective target for cancer therapy.

The pH in tumour cells and the tumour microenvironment has played important roles in cancer development and treatment. It was thought that both the extracellular and intracellular pH values in tumours are acidic and lower than in normal cells. However, recent progress in the measurement of pH in tumour tissue has disclosed that the intracellular pH (pHi) of cancer cells is neutral or even mildly alkaline compared to normal tissue cells. This review article has summarized the recent advancement in the measurement pHi and extracellular pH (pHe) in cancer cells, and the effect of pHi and pHe on proliferation, migration and biological functions of cancer cells. This paper has also elaborated recent treatment strategies to manipulate pHi and pHe for cancer treatment. Based on the recent progress in pHi and pHe manipulation in cancer treatment, we have proposed potential nanoparticle-based strategies to manipulate pHi and pHe to effectively treat cancer.

Real-time tracing the changes in the intracellular pH value during apoptosis by near-infrared ratiometric fluorescence imaging.

We developed a near-infrared ratiometric fluorescent nanoprobe with single-excitation dual-emission. The proposed nanoprobe was applied for real-time monitoring the changes in the pH value during hydrogen peroxide-induced apoptosis by ratio imaging. The pH value of HeLa cells changed from 6.6 to 5.6 after H2O2 (100 μM) stimulation for 40 min.

Ratiometric photoluminescence sensing based on Ti3C2 MXene quantum dots as an intracellular pH sensor.

Intracellular pH sensing is of importance and can be used as an indicator for monitoring the evolution of various diseases and the health of cells. Here, we developed a new class of surface-functionalized MXene quantum dots (QDs), Ti3C2, by the sonication cutting and hydrothermal approach and further explored their intracellular pH sensing. The functionalized Ti3C2 QDs exhibit bright excitation-dependent blue photoluminescence (PL) originating from the size effect and surface defects. Meanwhile, Ti3C2 QDs demonstrate a high PL response induced by the deprotonation of the surface defects. Furthermore, combining the highly pH sensitive Ti3C2 QDs with the pH insensitive [Ru(dpp)3]Cl2, we developed a ratiometric pH sensor to quantitatively monitor the intracellular pH values. These novel MXene quantum dots can serve as a promising platform for developing practical fluorescent nanosensors.

Linking CREB function with altered metabolism in murine fibroblast-based model cell lines.

The cAMP-responsive element binding protein CREB is frequently overexpressed and activated in tumors of distinct histology, leading to enhanced proliferation, migration, invasion and angiogenesis as well as reduced apoptosis. The de-regulated expression of CREB might be linked with transcriptional as well as post-transcriptional regulation mechanisms. We show here that altered CREB expression levels and function are associated with changes in the cellular metabolism. Using comparative proteome-based analysis an altered expression pattern of proteins involved in the cellular metabolism in particular in glycolysis was found upon CREB down-regulation in HER-2/neu-transfected cell lines. This was associated with diminished expression levels of the glucose transporter 1, reduced glucose uptake and reduced glycolytic activity in HER-2/neu-transfected cells with down-regulated CREB when compared to HER-2/neu+ cells. Furthermore, hypoxia-induced CREB activity resulted in changes of the metabolism in HER-2/neu transfected cells. Low pH values in the supernatant of HER-2/neu transformants were restored by CREB down-regulation, but further decreased by hypoxia. The altered intracellular pH values were associated with a distinct expression of lactate dehydrogenase, and its substrate lactate. Moreover, enhanced phosphorylation of CREB on residue Ser133 was accompanied by a down-regulation of pERK and an up-regulation of pAKT. CREB promotes the detoxification of ROS by catalase, therefore protecting the mitochondrial activity under oxidative stress. These data suggest that there might exists a link between CREB function and the altered metabolism in HER-2/neu-transformed cells. Thus, targeting these altered metabolic pathways might represent an attractive therapeutic approach at least for the treatment of patients with HER-2/neu overexpressing tumors.

PH-dependent general base catalyzed activation rather than isocyanate liberation may explain the superior anticancer efficacy of laromustine compared to related 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine prodrugs.

Laromustine (also known as cloretazine, onrigin, VNP40101M, 101M) is a prodrug of 90CE, a short-lived chloroethylating agent with anticancer activity. The short half-life of 90CE necessitates the use of latentiated prodrug forms for in vivo treatments. Alkylaminocarbonyl-based prodrugs such as laromustine exhibit significantly superior in vivo activity in several murine tumor models compared to analogs utilizing acyl, and alkoxycarbonyl latentiating groups. The alkylaminocarbonyl prodrugs possess two exclusive characteristics: (i) They are primarily unmasked by spontaneous base catalyzed elimination; and (ii) they liberate a reactive carbamoylating species. Previous speculations as to the therapeutic superiority of laromustine have focused upon the inhibition of enzymes by carbamoylation. We have investigated the therapeutic interactions of analogs with segregated chloroethylating and carbamoylating activities (singly and in combination) in the in vivo murine L1210 leukemia model. The combined treatment with chloroethylating and carbamoylating prodrugs failed to result in any synergism and produced a reduction in the therapeutic efficacy compared to the chloroethylating prodrug alone. Evidence supporting an alternative explanation for the superior tumor selectivity of laromustine is presented that is centered upon the high pH sensitivity of its base catalyzed activation, and the more alkaline intracellular pH values commonly found within tumor cells.

A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution.We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pKa∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pKa values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress.

A pH-responsive glycolipid-like nanocarrier for optimising the time-dependent distribution of free chemical drugs in focal cells

Though Drug delivery systems have achieved accumulation at tumor sites via passive targeting and active targeting, the therapeutic effects are far from perfect. The unsatisfactory results are mainly due to limited drug release from the nanocarriers at tumor sites, while the pharmacological activities of the drug are attributed to the concentration of the free drug and the time maintained at the pharmacological targets. A pH-responsive chitosan based glycolipid-like nanocarrier (CSO-FBA-SA) was fabricated by conjugating stearyl alcohol (SA) to chitosan oligosaccharide (CSO) with the linkage of 4-formylbenzoic acid (FBA). FBA was a kind of aromatic aldehyde carbonyl compounds, which can form the benzoic-imine bond. In the presence of a Schiff’s base structure, the carrier showed improved properties and could be quickly degraded in an acidic environment. In order to explore the process and mechanism of the nanocarriers in focal cells, the method for determining the intracellular concentration of released free doxorubicin was established, and the time-dependent change of the DOX-loaded micelles was revealed. The sight of drug release was also obtained with CLSM. The cytotoxicity of the CSO-FBA-SA/DOX against human breast cancer MCF-7 cells increased by 2.75-fold and 3.77-fold in comparison with the CSO-SA/DOX and DOX, respectively. Furthermore, the CSO-FBA-SA/DOX showed a 2.12-fold higher cytotoxicity against the MCF-7 cells than that treated against human ovarian cancer SKOV-3 cells with lower intracellular pH value, which indicated that the cellular inhibition positively correlated with the intracellular pH value. High tumor accumulation and fast drug release of the CSO-FBA-SA/DOX in tumor was responsible for the remarkable tumor growth inhibitory effect. Moreover, the CSO-FBA-SA/DOX could selectively respond to the acidic environment and release DOX in tumor only, which had relatively minimal cytotoxicity towards normal tissues. The results showed that this newly developed glycolipid-like nanocarrier could act as a potential vector for delivering the drug effectively with a low systemic toxicity.

Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl- channel.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short-lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP-binding site 1 formed by the nucleotide-binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl- channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pHi ) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open-time constant (τo ) of wild-type CFTR. By contrast, the fast closed-time constant (τcf ), which describes the short-lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo , τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating.

A review: the trend of progress about pH probes in cell application in recent years.

Intracellular pH values are some of the most important factors that govern biological processes and the acid-base homeostasis in cells, body fluids and organs sustains the normal operations of the body. Subcellular organelles including the acidic lysosomes and the alkalescent mitochondria undergo various processes such as intracellular digestion, ATP production and apoptosis. Due to their precise imaging capabilities, fluorescent probes have attracted great attention for the illustration of pH modulated processes. Furthermore, based on the unique acidic extracellular environment of acidic lysosomes, fluorescent probes can specifically be activated in cancer cells or tumors. In this review, recently reported lysosome and mitochondria specific pH imaging probes as well as pH-activatable cancer cell-targetable probes have been discussed.

Using Nanoliposomes To Construct a FRET-Based Ratiometric Fluorescent Probe for Sensing Intracellular pH Values.

Nanoliposomes were first used to encapsulate two fluorophores, fluorescein and cresyl violet, to construct a FRET-based ratiometric fluorescent probe, for sensing intracellular pH values. The morphology, stability, interference factors, biocompatibility, and performance were investigated in detail. The fluorophores-encapsulated nanoliposomes (FEN) probe can be uptaken by cells within 30 min, which is faster than any other nanosensor, and cell viability was not significantly changed upon treatment with the FEN probe for 24 h, showing the low cytotoxicity and good biocompatibility of the probe. Quantitative determinations of intracellular pH of MCF-7 cells and the pH fluctuations associated with inflammation have been successfully performed with our probe. This work demonstrated that nanoliposomes could be served as an emerging approach for constructing useful fluorescent nanosensors with low toxicity.

Growth-related changes of phosphorus metabolites in VX-2 carcinoma implanted into rabbit thigh muscle: in vivo 31P MR spectroscopy

The purpose of this study was to evaluate the usefulness of in vivo 31P magnetic resonance spectroscopy (MRS) for monitoring changes in growth-related phosphate metabolite concentration and intracellular pH value in rabbit thigh muscle implanted with VX-2 carcinoma. The time-course magnetic resonance imaging (MRI) and in vivo 31P MRS were examined weekly in the course of 10 weeks following the onset of a VX-2 carcinoma implantation. The spectra were quantitatively analyzed to obtain vital information on the time course variation of the phosphorus metabolites and intracellular pH value according to the tumor growth. Elevation in the concentrations of phosphormonoesters (PME), inorganic phosphate (Pi), and phosphodiesters (PDE) was observed over the time course of 3-4 weeks after the implantation of VX2 carcinoma, while the rest of the metabolites, PCr and ATP tended to be constant. The concentration changes of PME, Pi, and PDE were positively correlated with the volumes of tumor necrosis. The intracellular pH values decreased with the time course of tumor growth and the volumes of tumor necrosis. In vivo 31P MRS is capable of non-invasive monitoring of intracellular pH values as well as the concentration changes of phosphate metabolites during tumor growth.

An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells

It is crucial to monitor intracellular pH values and their fluctuation since the organelles of cells have different pH distribution. Herein we construct a new small molecule fluorescent probe HBT-O for monitoring the subtle pH values within the scope of neutral to acid in living cells. The probe exhibited good water solubility, a marked turquoise to olivine emission color change in response to pH, and tremendous fluorescence hypochromatic shift of ∼50nm (1718cm−1) as well as the increased fluorescence intensity when the pH value changed from neutral to acid. Thus, the probe HBT-O can distinguish the subtle changes in the range of normal pH values from neutral to acid with significant fluorescence changes. These properties can be attributed to the intramolecular charge transfer (ICT) process of the probe upon protonation in buffer solutions at varied pH values. Moreover, the probe was reversible and nearly non-toxic for living cells. Then the probe was successfully used to detect pH fluctuation in living cells by exhibiting different fluorescence colors and intensity. These findings demonstrate that the probe will find useful applications in biology and biomedical research.

Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44.

Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process.

The effect of neurostimulation on the intracellular concentrations of proton-containing metabolites and macroergic phosphates in the brain cortex upon schizophrenia according to the data from 1H and 31P magnetic resonance spectroscopy

The effect of neuroactivation on the concentrations of direct (phosphocreatine, PCr; adenosine triphosphate, ATP) and indirect (N-acetylaspartate, NAA) participants of energy metabolism and the intracellular pH values in the activated brain loci in health and upon early-stage schizophrenia is considered. Single-voxel 1H and 2D 31P magnetic resonance spectroscopy methods were used. The neuroactivation areas were localized according to the data from functional magnetic resonance imaging (fMRI). The NAA level after a short stimulus in the premotor cortex in schizophrenic patients remained unchanged, while the health group showed a reversible decrease in this parameter even 12 s after stimulus presentation. The long video stimulation has no effect on the contents of PCr and ATP in the visual cortex upon schizophrenia, while the analogous action on the health brain causes a decrease in the PCr level with the ATP level being constant. In the absence of neurostimulation, all measured indices were identical in both schizophrenic patients and control group.