Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli.

Background Myocardial ischemia is a dynamic, complex process characterized by hyperkalemia, acidosis, and ATP depletion. While these three conditions alter cardiomyocyte electrophysiology, it is difficult to discern how much each one individually contributes to the resulting changes in action potential (AP). In this study, we test whether machine learning can deconvolute these distinct ischemic patterns within a single AP. Methods We developed a multi-target regression model trained on data generated by the Luo-Rudy (1991) computational model of a ventricular cardiomyocyte, simulating a wide range of ischemic conditions. The model was designed to predict two continuous variables: extracellular potassium concentration ([K + ]o) and intracellular pH (pHi). Results The model achieved high accuracy on a held-out test set, with mean squared errors (MSE) below 0.25 for [K + ]o and below 0.01 for pHi. To further generalize this model, we applied this trained model to a structurally distinct model, the Ten Tusscher (2006) framework. We were able to accurately predict [K + ]o and pHi from APs, demonstrating that the learned principles are robust. A feature importance analysis revealed that resting membrane potential (RMP) was the strongest predictor for [K + ]o, while action potential duration (APD) is most important for predicting pHi, underscoring these distinct cardiomyocyte electrophysiological patterns Conclusions Our approach can distinguish distinct ischemic drivers and has potential for in silico drug screening and mechanistic analysis.

Cystic fibrosis (CF) is caused by loss-of-function variants in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride and bicarbonate channel and affects multiple organs, with pancreatic involvement showing very high penetrance. In pancreatic ducts, CFTR drives secretion of alkaline, bicarbonate-rich fluid that maintains intraductal patency, neutralises gastric acid and permits safe delivery of digestive enzymes. Selective impairment of CFTR-dependent bicarbonate transport, even in the presence of residual chloride conductance, is strongly associated with exocrine pancreatic insufficiency, recurrent pancreatitis and cystic-fibrosis-related diabetes. These clinical manifestations are captured by pharmacodynamic anchors such as faecal elastase-1, steatorrhoea, pancreatitis burden and glycaemic control, providing clinically meaningful benchmarks for CFTR-targeted therapies. In this review, we summarise the principal mechanisms underlying pancreatic pathophysiology and the current approaches to clinical management. We then examine in vitro pancreatic duct models that are used to evaluate small molecules and emerging therapeutics targeting CFTR. These experimental systems include native tissue, primary cultures, organoids, co-cultures and microfluidic devices, each of which has its own advantages and limitations. Intact micro-perfused ducts provide the physiological benchmark for studying luminal pH control and bicarbonate (HCO3−) secretion. Primary pancreatic duct epithelial cells (PDECs) and pancreatic ductal organoids (PDO) preserve ductal identity, patient-specific genotype and key regulatory networks. Immortalised ductal cell lines grown on permeable supports enable scalable screening and structure activity analyses. Co-culture models and organ-on-chip devices incorporate inflammatory, stromal and endocrine components together with flow and shear and provide system-level readouts, including duct-islet communication. Across this complementary toolkit, we prioritise bicarbonate-relevant endpoints, including luminal and intracellular pH and direct measures of HCO3− flux, to improve alignment between in vitro pharmacology and clinical pancreatic outcomes. The systematic use of complementary models should facilitate the discovery of next-generation CFTR modulators and adjunctive strategies with the greatest potential to protect both exocrine and endocrine pancreatic function in people with CF.

A NOX2-independent mechanism of Hv1 channel activation promotes inflammatory cytokine release from BV-2 microglia via intracellular Ca2+ mobilisation.

Hypoxia and hypercapnia often accompany seawater warming and interactively alter marine ectotherm performance, potentially threatening their populations. To detail mechanistic responses, we investigated whole-animal physiology alongside cellular homeostasis in a species expected to be relatively robust to their impacts, the oyster Ostrea edulis. Acute warming alone (W) and combined with hypercapnia and hypoxia (deadly trio, DT) started at 18°C, increasing stepwise by 2°C per 48 hours until critical temperatures (34 °C). Mortality onset at a lower temperature under DT than W but rates equalized by 34°C. DT-exposed oysters' hemolymph PO2 began 29% lower at 18°C, but by 34°C was only slightly lower than in W oysters. In both groups, resting metabolic rate (RMR) and heart rate rose with warming. Hemolymph PO2 was stable until 26°C, whence it declined. DT elicited a higher heart rate, which began to fall after ∼32°C, while heart rate in W-exposed oysters continued rising. Relative increases in branchial metabolite levels of alanine and fumarate, profiled via 1H-NMR spectroscopy, indicated greater contributions of anaerobic metabolism in DT- than W-exposed oysters. Gill tissue showed higher levels of the mitochondrial stabilizer sirtuin-5 alongside higher antioxidative capacity under DT than W-exposed oysters, before declining at temperatures beyond 30°C. Muscle intracellular pH, gill heat shock protein 70 and metabolic profiles appeared unaffected by DT compared to warming. Our results suggest that DT places an additional energetic burden on the oyster, lowering the critical temperature. Nevertheless, tolerance patterns indicate resilience to DT, which may require a re-balancing of passive tolerance mechanisms, especially a probable emphasis on metabolic depression.

The tumor microenvironment (TME) plays a central role in cancer progression and metastasis. A key feature of the TME is extracellular acidity, which promotes disease progression, immune evasion, and drug resistance. Tumor acidity is increasingly recognized as a critical factor in cancer development and a negative prognostic indicator. Here, we demonstrate that the membrane glycoprotein dysadherin promotes colorectal cancer (CRC) malignancy by modulating TME acidity. Comprehensive bioinformatics and pathological analyses of CRC patient samples revealed that increased tumor acidity is a hallmark of CRC progression and strongly correlates with high expression of dysadherin. Functional studies confirmed that dysadherin enhances malignant traits, particularly under acidic conditions. Mechanistically, dysadherin activates the integrin/FAK/STAT3 signaling pathway, leading to the upregulation of carbonic anhydrase 9 (CA9). CA9 facilitates proton export, contributing to extracellular acidification while maintaining intracellular pH homeostasis, thereby enabling cancer cells to survive and thrive in acidic environments. In a murine liver metastasis model, dysadherin deletion impaired cellular adaptation to the acidic TME and markedly attenuated metastatic colonization, whereas restoring CA9 expression effectively rescued metastatic potential. Overall, our findings identify the dysadherin/CA9 axis as a potential therapeutic target in CRC and provide new insights into how tumors exploit acidosis to drive malignant development and progression.

Regulatory RNAs are crucial for transcriptional and posttranscriptional regulation in bacteria and hold significant potential as tools for gene expression in synthetic biology. In our previous study, YhfH in the Bacillus thuringiensis BMB171 strain was identified as a regulatory RNA that functions as an antisense RNA to modulate the expression of LipR, a transcriptional regulator involved in intracellular pH regulation in glucose-rich environments. This study further reveals that YhfH, predominantly expressed during the stationary phase, acts as a small RNA (sRNA) to regulate the expression of the pyrimidine biosynthetic operon, thereby influencing the bacterial growth in pyrimidine-limited media. Additionally, YhfH RNA serves as a messenger RNA (mRNA) encoding a peptide, YhfH-P, which inhibits its own transcription. Moreover, YhfH-P represses the expression of the ilv-leuoperon, which is primarily responsible for branched-chain amino acid synthesis, by binding to its promoter. Collectively, YhfH RNA exhibits versatile functions as an sRNA, antisense RNA, and mRNA, thus acting as a "tri-function" RNA. The discovery of YhfH significantly expands our understanding of RNA regulatory potential, and its mechanism of action could provide valuable insights for designing streamlined genetic circuits with diverse regulatory functions in synthetic biology.

Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is shown to be both essential and fully reversible. Structural transitions involve an ensemble of gated states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.

Reduced ATP-to-phosphocreatine ratios in neuropsychiatric post-COVID condition: Evidence from 31P magnetic resonance spectroscopy.

Intracellular pH regulates β-catenin with low pHi increasing adhesion and signaling functions.

Bacterial carbonic anhydrases (CAs) are essential for intracellular pH regulation, bicarbonate homeostasis, and energy metabolism, making them attractive antimicrobial targets. Here, building on evidence that acetazolamide (AZA) delivered via hyaluronic acid-palmitate (HA-PA) nanocarriers impairs Escherichia coli growth and its glucose uptake, we investigated the physiological roles of β- and γ-class CAs using sulphonamide inhibitors with distinct selectivity encapsulated in HA-PA nanomicelles to ensure intracellular delivery. AZA, a potent dual β/γ-CA inhibitor, ethoxzolamide (EZA), a selective β-CA inhibitor, and hydrochlorothiazide (HCT), a weaker inhibitor of both classes, were tested for effects on bacterial physiology. The nanoparticles reduced growth in a dose- and class-dependent manner, with AZA exerting the strongest activity, EZA intermediate inhibition, and HCT only modest effects at higher concentrations. Early metabolic responses assessed via intracellular ATP after three hours of exposure revealed an unexpected and reproducible ATP increase for all inhibitors relative to untreated cells, suggesting reduced ATP consumption in bicarbonate-dependent pathways. These findings provide indirect yet compelling evidence that β- and γ-class CAs influence bacterial energy homeostasis and support the rationale for CA inhibition as an antimicrobial strategy, while highlighting HA-PA carriers as effective systems for delivering CA inhibitors intracellularly and enhancing their functional activity in bacterial cells.

An accurate map of intracellular organelle pH is crucial for comprehending cellular metabolism and organellar functions. However, a unified intracellular pH spectrum using a single probe is still lacking. Here, we developed a novel quantum entanglement-enhanced pH- sensitive probe called SITE-pHorin (single excitation and two emissions pH sensor protein), which features a wide pH-sensitive range and ratiometric quantitative measurement capabilities. We subsequently measured the pH of various organelles and their subcompartments, including mitochondrial subspaces, Golgi stacks, endoplasmic reticulum (ER), lysosomes, peroxisomes, and endosomes in COS-7 cells. For the long-standing debate on the pH of the mitochondrial compartments, we measured the pH of the mitochondrial cristae (mito-cristae) as 6.60±0.40, the pH of the mitochondrial intermembrane space (mito-IMS) as 6.95±0.30, and the pH of the two populations of the mitochondrial matrix (mito-matrix) at approximately 7.20±0.27 and 7.50±0.16, respectively. Notably, the pH of the lysosome exhibited a single, narrow Gaussian distribution centered at 4.79±0.17, which is consistent with an optimal lysosomal acidic pH between 4.5 and 5.0. Furthermore, quantum chemistry computations revealed that both the deprotonation of the residue Y182 and the discrete curvature of the deformed benzene ring in the chromophore are necessary for the quantum entanglement mechanism of SITE-pHorin. Intriguingly, our findings reveal an accurate pH gradient (0.6-0.9 pH units) between the mitochondrial cristae and the mitochondrial matrix, suggesting that prior knowledge about ΔpH (0.4-0.6) and the mitochondrial proton motive force (pmf) is underestimated.

Modulating tumor acidity with hydroxyethyl starch-based nanoparticles by targeting CA9 to eliminate cancer stem cells and overcome immunosuppression.

Tumor cells evolve adaptive mechanisms to function optimally within an acidic microenvironment. This adaptation is driven by the enhanced metabolic activity of tumors, which frequently results in acidosis and hypoxia. Carbonic anhydrase enzymes orchestrate cell homeostasis and affect cancer cell fate. Advances in structure-based design have enabled the identification of selective carbonic anhydrase XII inhibitors. The study aimed to evaluate the anti-proliferative activity of sulfonamide derivatives and elucidate the plausible mode of action. MTT assay was conducted to evaluate cytotoxicity of acetazolamide, parent C3 and its derivatives C4 and C6 against breast cancer cell lines MCF-7, MDA-MB-231 and prostate cancer cells PC3. Further studies for C3, C4 and C6 compounds were conducted against MCF-7. Enzyme-linked immunosorbent assay of carbonic anhydrases IX and XII and fluorescent intracellular pH assay were conducted. Investigated compounds and acetazolamide reduced proliferation of all tested cells after 72 h. C3, C4, and C6 compounds reduced carbonic anhydrase XII concentrations. C3 and C6 did not appear significant reduction in CAIX in MCF-7. Conversely, C4 displayed statistically significant increase. Cells exposed to C3, C4, and C6 revealed a decrease in intracellular pH levels. In conclusion, all three compounds exhibited antiproliferative activities characterized by modulation of tumor pHi/pHe.

The development of nanoprobes targeting single neurons is crucial for elucidating the intrinsic mechanisms of the nervous system. However, limitations of existing nanoprobes in terms of structural design, functional expansion, and tip exposure methods significantly hinder accurate analysis and in-depth research on single-neuron behaviors. Here, we develop a nanoscale tip-processing strategy based on atmospheric plasma jet branch self-focusing (APJBSF) for achieving in situ integration of outer surface sensing, inner self-reference and inner delivery functions in the effective working area of the nanoprobe tip, enabling the three functions to collaboratively analyze single-neuron behaviors. For the outer sensing channel, protective layers with nanoscale thickness are selectively removed using the ABJBSF technique. This process allows for the controlled exposure levels of functional tip surfaces at nanoscale dimensions, while simultaneously broadening the range of applicable protective coatings for nanoprobes, which facilitates reliable recordings at intracellular specific sites. For the inner self-reference and delivery channels, intracellular delivery experiments indicate that the in situ reference enhances the accuracy and stability of intracellular recordings and the triune in situ integrated nanoprobe (TIINP) enables accurate monitoring of intracellular pH changes induced by in situ delivery. The proposed TIINP structure and the tip-processing technique for nanoprobes provide powerful tools for enhancing the functionality and reliability of single-neuron analysis.

BackgroundTunneling nanotubes (TNTs) are hollow tubular connections between animal cells that are membrane‐enclosed and based on F‐actin. They carry a range of biological cargo, including proteins involved in neurodegenerative diseases like Tau and mutant huntingtin (mHTT). Nonetheless, the molecular mechanisms underlying their formation, especially in the brain, remain ambiguous. Ras homolog abundant in the striatum (Rhes) is associated with Huntington's disease and tauopathy. We discovered that Rhes causes TNT‐like protrusions and facilitates the transfer of mHTT across neuronal cells in culture and the brain.MethodUsing cell culture models, Co‐immunoprecipitation assays, High resolution microscopy and mass spectrometry, we investigated the role of Rhes in TNT formation and cargo transfer. Pharmacological and knockout approaches were used to assess the impact of SLC4A7 loss on TNT formation and mHTT/Tau transfer in cellular models and the intact striatum.ResultsWe discovered that Rhes causes TNT‐like protrusions and facilitates the transfer of mHTT across neuronal cells in culture and the brain. The preliminary findings indicated that Rhes can transmit Tau between cultivated cells. Nonetheless, the processes remain unclear. Employing an unbiased mass spectrometry technique, we identified solute carrier family four member 7 (SLC4A7), a regulator of intracellular pH, as a novel regulator of Rhes‐mediated tunneling nanotubes (TNTs). Rhes binds directly to SLC4A7 and alters intracellular pH. Pharmacological and knockout investigations indicate that the loss of SLC4A7 reduces TNTs and inhibits the dissemination of mHTT in cellular models and within the intact striatum. The preliminary findings indicate that Rhes can enhance TNT formation in microglial cultures.ConclusionsRhes plays a crucial role in regulating the production of TNT and the dissemination of Tau and mHTT throughout the brain through neurons and microglia. Targeting Rhes presents a therapeutic possibility for Huntington's disease and tauopathies.

CO2 diffusion across plasma membranes depends on both membrane CO2 permeability ( ) and the transmembrane CO2 concentration gradient (Δ[CO2]) - Fick's law. Human aquaporin-5 (hAQP5) enhances CO2 diffusion by increasing , whereas carbonic anhydrases (CAs) do so by enhancing CO2 consumption/production and thus Δ[CO2]. Here we systematically assess functional interactions among a gas channel and intra-/extracellular CAs. On Day 1 we inject Xenopus oocytes with cRNA encoding hAQP5 (control: H2O). On Day 4 we inject hCAII protein in 'Tris' buffer (control: 'Tris'). We assess CO2 fluxes by introducing extracellular 1.5% CO2/10 mM HCO3 - and using microelectrodes to measure (1) the maximal increase of extracellular surface pH (ΔpHS), (2) the maximal rate of pHS relaxation (dpHS/dt)Max and (3) the maximal rate of intracellular pH decrease (dpHi/dt)Max. By itself hCAII minimally increases ΔpHS - measured on the side of the membrane opposite to the added cytosolic CA (CAi) - even at our highest doses (100ng/oocyte). However hAQP5 alone triples ΔpHS, an effect further doubled by increasing hCAII. By itself bovine erythrocyte CA (bCA) in the extracellular fluid doubles (dpHi/dt)Max magnitude - measured on the side of the membrane opposite to the added extracellular CA (CAo) - an effect further doubled by hAQP5. Our pH measurements (1) confirm synergy between CAo and CAi; establish synergy between hAQP5 and both (2) CAo and (3) CAi; and (4) show that the ability of CAi to enhance ΔpHS is a useful tool for assessing the CO2 permeability of membrane proteins (e.g. hAQP5). KEY POINTS: According to Fick's law transmembrane CO2 flux ( ) is the product of membrane permeability ( ) and transmembrane concentration gradient (Δ[CO2]): = ×Δ[CO2]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances , and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance (Δ[CO2]) by consuming accumulated or replenishing lost CO2. We now examine interactions among #1-#3. We assess CO2 fluxes - produced by addition/removal of extracellular CO2/HCO3 - - using microelectrodes to monitor extracellular surface pH (pHS) and intracellular pH (pHi) of Xenopus oocytes heterologously expressing hAQP5, injected with human CAII (hCAII), and/or exposed to extracellular bovine CA (bCA). Enhancing effects on CO2 fluxes are synergistic among hAQP5, hCAII and bCA, any of which can become rate-limiting, depending on the status of the other two. CO2/HCO3 - addition transiently increases pHS (ΔpHS), hCAII augments ΔpHS (ΔΔpHS) and hAQP5 enhances ΔΔpHS (ΔΔΔpHS) - a novel tool to assess potential CO2 channels.

The arginine-dependent acid resistance (Adi) system is a vital component that enables Escherichia coli and other enterobacteria to withstand the extreme acidity in the human gastrointestinal tract. It consists of the proton-consuming decarboxylation of arginine, catalyzed by AdiA, and the uptake of arginine, as well as the excretion of the more alkaline agmatine, catalyzed by the antiporter AdiC. The corresponding genes adiA and adiC are induced in E. coli under acidic conditions (pH < 5.5), a process that is tightly regulated by the AraC/XylS transcriptional activator AdiY. Here, we show that the pH-sensing mechanism of AdiY functions through the protonation of two histidines (His34 and His60) in the N-terminal domain. Replacing these histidine residues with alanine, glutamine, or aspartate abolishes the pH-dependent activation of AdiY, both in vivo, as demonstrated by promoter-reporter assays, and in vitro, as indicated by the loss of DNA-binding activity detected by surface plasmon resonance spectroscopy. Biochemical analyses of purified wild-type AdiY using size-exclusion chromatography and intrinsic tryptophan fluorescence revealed a pronounced and reversible pH-dependent conformational change that does not occur in the pH-sensing-deficient AdiY variant. A model is proposed in which AdiY forms a monomer at physiological pH. At a lower intracellular pH, the protonation of histidine in AdiY causes a conformational change that leads to the binding of AdiY as a tetramer to the DNA. This work elucidates the molecular mechanism of a one-component signal transduction system that combines both sensory and responsive functions.IMPORTANCEThroughout their life, Escherichia coli and other bacteria may encounter acidic environments, for example, when passing through the human stomach. Their chances of survival under these conditions depend on the number and efficiency of acid resistance systems. Although many acid resistance mechanisms have been extensively studied, the molecular mechanism by which bacteria sense low pH is not yet fully understood. This study demonstrates that the transcription factor AdiY acts as a direct pH sensor by using two histidines to detect intracellular acidification in E. coli. When these histidines become protonated, AdiY changes its conformation and activates genes that support cell survival under acid stress. These findings not only reveal a new way in which bacteria can perceive extremely low pH environments but also provide the basis for the development of AdiY as a pH reporter.

Heterogeneity within clonal cell populations remains a critical bottleneck within bioprocess engineering, notably by undermining bioproduction yields. Efforts to mitigate its impact have, however, been hampered by technological difficulties quantifying metabolism at the single-cell level. Here, we propose a framework based on single-cell biosensor analysis that enables robust characterisation of cell's metabolic states, leveraging it to detect and isolate isogeneic heterogeneity in response to environmental perturbations and within microbial cell factories. We identify acute and gradual glucose depletion to induce differentiation of metabolically distinct subpopulations and reveal these subpopulations to exhibit differential production capabilities, with lower intracellular pH subpopulations exhibiting enhanced product accumulation within violacein-producing strains but reduced yields within lycopene-producing strains. Lastly, we highlight galactose cultivation as a method to modulate subpopulation dynamics towards higher-producing lycopene phenotypes. Altogether, our research provides insights into subpopulation differentiation and establishes promising avenues for the engineering of more robust and higher-producing strains.

Nanometal-based therapies face challenges arising from the overexpression of proton efflux transporters in cancer cells, which acidifies the extracellular tumor microenvironment (TME) while preserving a relatively neutral intracellular pH, thereby compromising therapeutic efficacy and fostering an immunosuppressive TME. Here, we integrate the proton pump inhibitor pantoprazole (PTZ) with manganese ferrite nanoparticles (MFNs) within an acidity-responsive polymer for enhanced ferroptosis and cGAS-STING activation mediated immunotherapy. This assembly (PTZ/MFNAs) facilitates tumor accumulation through the enhanced permeability and retention effect while initially restricting the release of metal ions. Upon reaching the tumor site, PTZ release increases intracellular acidity, which further triggers assembly disintegration, accelerates the release of iron and manganese ions, and neutralizes the extracellular microenvironment to alleviate immunosuppression. The released manganese ions synergistically collaborate with iron ions to amplify reactive oxygen species (ROS) generation for ferroptosis while activating the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway, stimulating innate immunity. This potentiation of innate immunity, coupled with the reversal of TME immunosuppression, collectively and effectively inhibits tumor growth and metastasis. Therefore, the PTZ/MFNAs co-delivery system represents a promising pH-modulation strategy to enhance iron/manganese ions-mediated ferroptosis and cGAS-STING activation-induced immunotherapy.