Bacterial-induced pH shifts link individual cell physiology to macroscale collective behavior.

Bacteria have evolved a diverse array of signaling pathways that enable them to quickly respond to environmental changes. Understanding how these pathways reflect environmental conditions and produce an orchestrated response is an ongoing challenge. Herein, we present a role for collective modifications of environmental pH carried out by microbial colonies living on a surface. We show that by collectively adjusting the local pH value, Paenibacillus spp., specifically, regulate their swarming motility. Moreover, we show that such pH-dependent regulation can converge with the carbon repression pathway to down-regulate flagellin expression and inhibit swarming in the presence of glucose. Interestingly, our results demonstrate that the observed glucose-dependent swarming repression is not mediated by the glucose molecule per se, as commonly thought to occur in carbon repression pathways, but rather is governed by a decrease in pH due to glucose metabolism. In fact, modification of the environmental pH by neighboring bacterial species could override this glucose-dependent repression and induce swarming of Paenibacillus spp. away from a glucose-rich area. Our results suggest that bacteria can use local pH modulations to reflect nutrient availability and link individual bacterial physiology to macroscale collective behavior.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

A Reversible Proton Generator with On/Off Thermoswitch.

A reversible polymer photoacid with a thermal on/off switch at physiological temperature able to trigger a large pH modulation of its environment is prepared. Light is used to control the acidity of the solution. Additionally, the temperature could be used to modulate the photoacid efficiency, practically turning on and off the ability of the polymer to produce protons. The behavior of this thermoresponsive photoacid copolymer is the result of the combined action of the temperature-responsive N-isopropylacrylamide and of a reversible photoacid monomer based on a spiropyran derivative. The acidification of the aqueous medium is activated by irradiation at λ = 460 nm. The reverse reaction is achieved by removing the light stimuli or by exposing the solution to UV-light. Increasing the temperature above the lower critical solution temperature of the copolymer deactivates the photoacid and irradiation at λ = 460 nm does not lead to the generation of protons or to any detectable change in the pH value of the solution. Hence, the addition of N-isopropylacrylamide as a comonomer acts as a thermal on/off switch for the photoacid and the coupling of temperature-and light-responsiveness in the polyphotoacids yields a "thermophotoacid".

Study on Control of pH-Modulation Crystallization Utilizing Concept of Dissociation Equilibrium.

Crystallization by pH adjustment, as a type of reaction crystallization, is a solid-liquid separation method widely used in the area of pharmaceutical and pharmaceutical intermediate manufacturing. On the other hand, 3-alkenyl cephem compound is a typical zwitterionic pharmaceutical intermediate that possesses both an amino group and a carboxylic acid group. Such structure affords three main pH regions in solution and results in difficulties using crystallization by pH adjustment for isolation. As a consequence, 3-alkenyl cephem compound is usually crystallized at the point away from the solubility curve, causing unrestricted nucleation and flocculation behavior for the deposited particles which is difficult to filtrate. In this study, the pKa of 3-alkenyl cephem compound was intensively investigated to inhibit the nucleation. An optimal pH level point was also sought to make monodisperse particles. In particular, during crystallization by pH-modulation operation, the key point was identified to be the number of primary particles aggregated in the secondary particles. It was revealed that the increment number of primary particles led to the generation of larger monodisperse particles. This investigation, combined with solid-liquid equilibrium, enabled the acquisition of target species with good operability for filtration process. This present investigation becomes the prosperity in the zwitterion compound production that it has hardships to crystallize and filtrate.

pH modulation ameliorates the red blood cell storage lesion in a murine model of transfusion.

BACKGROUND: Prolonged storage of packed red blood cells (pRBCs) induces a series of harmful biochemical and metabolic changes known as the RBC storage lesion. RBCs are currently stored in an acidic storage solution, but the effect of pH on the RBC storage lesion is unknown. We investigated the effect of modulation of storage pH on the RBC storage lesion and on erythrocyte survival after transfusion. METHODS: Murine pRBCs were stored in Additive Solution-3 (AS3) under standard conditions (pH, 5.8), acidic AS3 (pH, 4.5), or alkalinized AS3 (pH, 8.5). pRBC units were analyzed at the end of the storage period. Several components of the storage lesion were measured, including cell-free hemoglobin, microparticle production, phosphatidylserine externalization, lactate accumulation, and byproducts of lipid peroxidation. Carboxyfluorescein-labeled erythrocytes were transfused into healthy mice to determine cell survival. RESULTS: Compared with pRBCs stored in standard AS3, those stored in alkaline solution exhibited decreased hemolysis, phosphatidylserine externalization, microparticle production, and lipid peroxidation. Lactate levels were greater after storage in alkaline conditions, suggesting that these pRBCs remained more metabolically viable. Storage in acidic AS3 accelerated erythrocyte deterioration. Compared with standard AS3 storage, circulating half-life of cells was increased by alkaline storage but decreased in acidic conditions. CONCLUSIONS: Storage pH significantly affects the quality of stored RBCs and cell survival after transfusion. Current erythrocyte storage solutions may benefit from refinements in pH levels.

Zeolite-Loaded Hydrogels as Wound pH-Modulating Dressings for Diabetic Wound Healing.

Wound pH has emerged as a promising therapeutic target in diabetic foot ulcers (DFU). Here, we aimed to develop a microparticle-loaded hydrogel for pH modulation in wound fluid. In a screen of polymeric and inorganic microparticles, zeolites were identified as pH-modulating microparticles. Zeolites were encapsulated in a calcium cross-linked alginate hydrogel, a biocompatible matrix clinically used as a wound dressing. This hydrogel potently neutralized hydroxide ions in serum-containing simulated wound fluid. These findings encourage a further development of this pH-modulating device as a molecular therapeutic system for DFUs.

Wound pH-Modulating Strategies for Diabetic Wound Healing.

Significance: Chronic diabetic wounds on the lower extremities (diabetic foot ulcers, DFU) are one of the most prevalent and life-threatening complications of diabetes, responsible for significant loss of quality of life and cost to the health care system. Available pharmacologic treatments fail to achieve complete healing in many patients. Recent studies and investigational treatments have highlighted the potential of modulating wound pH in DFU. Recent Advances: Data from in vitro, preclinical, and clinical studies highlight the role of pH in the pathophysiology of DFU, and topical administration of pH-lowering agents have shown promise as a therapeutic strategy for diabetic wounds. In this critical review, we describe the role of pH in DFU pathophysiology and present selected low-molecular-weight and hydrogel-based pH-modulating systems for wound healing and infection control in diabetic wounds. Critical Issues: The molecular mechanisms leading to pH alterations in diabetic wounds are complex and may differ between in vitro models, animal models of diabetes, and the human pathophysiology. Wound pH-lowering bandages for DFU therapy must be tested in established animal models of diabetic wound healing and patients with diabetes to establish a comprehensive benefit-risk profile. Future Directions: As our understanding of the role of pH in the pathophysiology of diabetic wounds is deepening, new treatments for this therapeutic target are being developed and will be tested in preclinical and clinical studies. These therapeutic systems will establish a target product profile for pH-lowering treatments such as an optimal pH profile for each wound healing stage. Thus, controlling wound bed pH could become a powerful tool to accelerate chronic diabetic wound healing.

Simultaneous Urea and Phosphate Recovery from Synthetic Urine by Electrochemical Stabilization.

Urine is a widely available renewable source of nitrogen and phosphorous. The nitrogen in urine is present in the form of urea, which is rapidly hydrolyzed to ammonia and carbonic acid by the urease enzymes occurring in nature. In order to efficiently recover urea, the inhibition of urease must be done, usually by increasing the pH value above 11. This method, however, usually is based on external chemical dosing, limiting the sustainability of the process. In this work, the simultaneous recovery of urea and phosphorous from synthetic urine was aimed at by means of electrochemical pH modulation. Electrochemical cells were constructed and used for urea stabilization from synthetic urine by the in situ formation of OH- ions at the cathode. In addition, phosphorous precipitation with divalent cations (Ca2+, Mg2+) in the course of pH elevation was studied. Electrochemical cells equipped with commercial (Fumasep FKE) and developmental (PSEBS SU) cation exchange membranes (CEM) were used in this study to carry out urea stabilization and simultaneous P-recovery at an applied current density of 60 A m-2. The urea was successfully stabilized for a long time (more than 1 month at room temperature and nearly two months at 4 °C) at a pH of 11.5. In addition, >82% P-recovery could be achieved in the form of precipitate, which was identified as amorphous calcium magnesium phosphate (CMP) by using transmission electron microscopy (TEM).

pH modulation in adhesive cells with a protonic biotransducer.

Protons (H+) are essential for most physiological functions in organelles and cells. In this study, we have demonstrated a sulfonated polyaniline (SPA) biotransducer that can modulate the intracellular pH in C6 cells with an applied potential, which is directly coupled with H+ to facilitate engineering interactions with physiological processes in the cells. To modulate the pH in the intracellular fluid, we improved the performance of SPA biotransducer by coating of a carbon nanotube (CNT) supportive layer that provides high H+ selectivity in the solution and also high H+ capacity in the hybrid SPA electrode. The intracellular pH modulation was succeeded by applying a potential difference of less than ±0.6 V. pH modulation in the cells is effected by using the biotransducer, which drives the activity of plasma membrane potential and the flow of molecules through the permeable membrane between cells and culture medium, whereas the poly (3,4-ethylenedioxythiophene) (PEDOT)-based biotransducer, which does not have H+ selectivity, was insufficient for modulation. Furthermore, the protonic biotransducer can control the increase/decrease in mitochondria membrane potential, reactive oxygen species and intracellular Ca2+ concentration. Therefore, the protonic biotransducer provides a new perspective to transfer a H+ signal into the cells for modulating the functions.

Basic Amino Acids Modulated Neutral-pH PEDOT:PSS for Stable Blue Perovskite Light-Emitting Diodes.

State-of-the-art external quantum efficiencies (EQEs) have exceeded 20% for near-infrared, red, and green perovskite light-emitting diodes (PeLEDs) so far. Nevertheless, the cutting-edge blue counterparts demonstrate an inferior device performance, which impedes the commercialization and industrialization of PeLEDs in ultrahigh-definition displays. As the most popular hole transport layer, poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) bears the acidic and hygroscopic drawbacks, which deteriorates the device efficiency and long-term stability of blue PeLEDs. In this work, the basic amino acids with zwitterionic characteristics are proposed to modulate the pH of PEDOT:PSS, which are arginine, lysine, and histidine. It is found that they play a triple function to the blue perovskite films: modulating the acidity of PEDOT:PSS, controlling the crystalline process, and passivating the defects at the PEDOT:PSS/perovskite interface. As a result, the utilization of neutral PEDOT:PSS leads to a significant enhancement in stability and photoluminescence quantum yield. Eventually, the pure-blue PeLEDs achieve a record EQE of 5.6% with the emission peak at 467 nm. This research proves that the interfacial engineering of hole transport layers is a reliable strategy to enhance the device efficiency and operation stability of blue PeLEDs.

Removal of protozoan (oo)cysts and bacteria during microalgae harvesting: Outcomes from a lab-scale experiment.

The efficiency of microalgae harvesting on the removal of Giardia spp. cysts, Cryptosporidium spp. oocysts, total coliforms, Escherichia coli, Enterococcus spp. and Clostridium spp. was assessed in lab-scale experiments (Jartest and Flotatest) using effluent from a flat panel photobioreactor used for Chlorella sorokiniana cultivation. Three harvesting methods were evaluated: (1) flocculation induced by pH modulation followed by sedimentation (pH-SED), (2) flocculation induced by pH modulation followed by dissolved air flotation (pH-DAF), and (3) coagulation using an organic coagulant (Tanfloc SG) followed by dissolved air flotation (Coag-DAF). The results indicated that the three harvesting methods were efficient in removing protozoan (oo)cysts and bacteria, achieving percentages of removal higher than 97% for all the analyzed pathogens. Among the three methods, pH-SED showed the best removal performance: 99.60% (2.5 log) for Giardia spp. cysts, 100% (>6.3 log) for total coliforms, 100% (>4.6 log) for Escherichia coli, 100% (>5.8 log) for Enterococcus spp. and 99.96% (3.6 log) for Clostridium spp. Clostridium spp. seemed to be more tolerant to the harvesting methods than the other groups of bacteria analyzed in the study, and its presence was positively correlated to the presence of Giardia spp. cysts.

Serially pH-Modulated Hydrogels Based on Boronate Ester and Polydopamine Linkages for Local Cancer Therapy.

Elaborately and serially pH-modulated hydrogels possessing optimized viscoelastic natures for short gelation time and single syringe injection were designed for peritumoral injection of an anticancer agent. Boronate ester bonds between phenylboronic acid (PBA) (installed in HA-PBA (HP)) and dopamine (included in HA-dopamine (HD)) along with self-polymerization of dopamine (via interactions between HD conjugates) were introduced as the main cross-linking strategies of a hyaluronic acid (HA) hydrogel. Considering pKa values (8.0-9.5) of PBA and dopamine, the pH of each polymer dispersion was controlled elaborately for injection through a single syringe, and the final pH was tuned nearby the physiological pH (pH 7.8). The shear-thinning behavior, self-healing property, and single syringe injectability of a designed hydrogel cross-linked nearby physiological pH may provide its convenient application to peritumoral injection and prolonged retention in local cancer therapy. Erlotinib (ERT) was encapsulated in a microsphere (MS), and it was further embedded in an HP/HD-based hydrogel for sustained and locoregional delivery. A rheologically tuned hydrogel containing an ERT MS exhibited superior tumor-suppressive efficiencies compared to the other groups in A549 tumor-bearing mice. A designed injectable hydrogel through a single syringe system may be efficiently applied to local cancer therapy with lower toxicities to healthy organs.

Developing pH-Modulated Spray Dried Amorphous Solid Dispersion of Candesartan Cilexetil with Enhanced In Vitro and In Vivo Performance.

Candesartan cilexetil (CC), a prodrug and highly effective antihypertensive agent, is a poorly soluble (BCS Class II) drug with limited bioavailability. Here, we attempted to improve CC's bioavailability by formulating several CC-loaded amorphous solid dispersions with a hydrophilic carrier (PVPK30) and pH modifier (sodium carbonate) using the spray drying technique. Solubility, in vitro dissolution, and moisture content tests were used for screening the optimized formulation. We identified an optimized formulation of CC/PVPK30/SC, which at the ratio of 1:0.5:1 (w/w/w) exhibited a 30,000-fold increase in solubility and a more than 9-fold enhancement in dissolution compared to pure CC. Solid-state characterization revealed that in pH-modulated CC amorphous solid dispersion (CCSDpM), CC's crystallinity was altered to an amorphous state with the absence of undesirable interactions. Stability studies also showed that the optimized formulation was stable with good drug content and drug release under accelerated conditions of up to 4 weeks and real-time stability conditions of up to 12 weeks. Furthermore, pharmacokinetic parameters, such as AUC and Cmax of candesartan, had a 4.45-fold and 7.42-fold improvement, respectively, in CCSDpM-treated rats compared to those in the CC-treated rats. Thus, these results suggest that CCSDpM is highly effective for increasing oral absorption. The application of these techniques can be a viable strategy to improve a drug's bioavailability.

Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Application of carbonic anhydrase inhibitors to increase the penetration of doxorubicin and its liposomal formulation into 2D and 3D triple negative breast cancer cell cultures.

The aim of our study was to assess the influence of two carbonic anhydrase (CA) inhibitors (methazolamide (MTZ)) and U-104 on weakly basic anticancer drug doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) delivery into monolayer-cultured 4T1 murine breast cancer cells (2D cultures) and tumor spheroids (3D cultures) at pH 6.0 and 7.4. The effect of compounds on cell viability was evaluated by MTT assay. Spheroids were formed using 3D Bioprinting method. The penetration of DOX and PLD into cells and spheroids was evaluated using fluorescence microscopy. Both MTZ and U-104 increased the DOX (5 µM) and PLD (concentration corresponding to 5 µM DOX) penetration into monolayer-cultured cells at acidic conditions but did not enhance drug delivery at physiological pH. Pretreatment with U-104 inhibitors also increased DOX and PLD delivery into tumor spheroids. Thus, U-104 may be worthy of further studies as possible transport modulator of weakly basic drugs.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

Calcium carbonate nanoparticles stimulate tumor metabolic reprogramming and modulate tumor metastasis.

AIM: CaCO3 nanoparticles (nano-CaCO3) can neutralize the acidic pHe of solid tumors, but the lack of intrinsic imaging signal precludes noninvasive monitoring of pH-perturbation in tumor microenvironment. We aim to develop a theranostic version of nano-CaCO3 to noninvasively monitor pH modulation and subsequent tumor response. MATERIALS & METHODS: We synthesized ferromagnetic core coated with CaCO3 (magnetite CaCO3). Magnetic resonance imaging (MRI) was used to determine the biodistribution and pH modulation using murine fibrosarcoma and breast cancer models. RESULTS: Magnetite CaCO3-MRI imaging showed that nano-CaCO3 rapidly raised tumor pHe, followed by excessive tumor-associated acid production after its clearance. Continuous nano-CaCO3 infusion could inhibit metastasis. CONCLUSION: Nano-CaCO3 exposure induces tumor metabolic reprogramming that could account for the failure of previous intermittent pH-modulation strategies to achieve sustainable therapeutic effect.

Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine: Synthesis, spectroscopic and WGA binding studies.

We introduce here a new fluorescent derivative of 1-thio-ß-N-acetylglucosamine linked to a pyrene system through a triazolylpentyl spacer, designed to self-assemble into a multivalent glycocluster. The synthesis was achieved by efficient CuAAC click reaction between a pyrene functionalized with an azide group and a suitable alkynyl thiomonosaccharide. Spectroscopic studies by fluorometry indicated that the self-assembly in aqueous medium is modulated by concentration and pH changes, the latter due to the presence of the amino group close to the π system. Circular dichroism experiments revealed a moderate positive signal, suggesting that the pyrene-thioGlcNAc conjugate can aggregate into a chiral supramolecular assembly. The sugar moiety showed to specifically and reversibly interact with the wheat germ agglutinin, a fact that was demonstrated by turbidity assay. SEM microscopy of a lyophilized solution at pH 10 revealed a fibrillar morphology compatible with the presence of tubular micelles, whereas crystalline and amorphous solids are formed at lower pHs.

Novel approaches for designing drugs that interfere with pH regulation.

INTRODUCTION: In all living species, pH regulation is a tightly controlled process, with a plethora of proteins involved in its regulation. These include sodium-proton exchangers, carbonic anhydrases, anion exchangers, bicarbonate transporters/cotransporters, H+-ATPases, and monocarboxylate transporters. All of them play crucial roles in acid-base balancing, both in eukaryotic as well as in prokaryotic organisms, making them interesting drug targets for the management of pathological events (in)directly involved in pH regulation. Areas covered: Interfering with pH regulation for the treatment of tumors and microbial infections is the main focus of this review, with particular attention paid to inhibitors targeting the above-mentioned proteins. The latest advances in each field id reviewed. Expert opinion: Interfering with the pH regulation of tumor cells is a validated approach to tackle primary tumors and metastases growth. Carbonic anhydrases are the most investigated proteins of those aforementioned, with several inhibitors in clinical development. Recent advances in the characterization of proteins involved in pH homeostasis of various pathogens evidenced their crucial role in the survival and virulence of bacterial, fungal, and protozoan microorganisms. Some encouraging results shed light on the possibility to target such proteins for obtaining new anti-infectives, overcoming the extensive drug resistance problems of clinically used drugs.