Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO3− transport. Without CFTR, airway epithelial HCO3− secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.
Lactic acid at sufficiently acidic pH is a potent microbicide, and lactic acid produced by vaginal lactobacilli may help protect against reproductive tract infections. However, previous observations likely underestimated healthy vaginal acidity and total lactate concentration since they failed to exclude women without a lactobacillus-dominated vaginal microbiota, and also did not account for the high carbon dioxide, low oxygen environment of the vagina. Fifty-six women with low (0-3) Nugent scores (indicating a lactobacillus-dominated vaginal microbiota) and no symptoms of reproductive tract disease or infection, provided a total of 64 cervicovaginal fluid samples using a collection method that avoided the need for sample dilution and rigorously minimized aerobic exposure. The pH of samples was measured by microelectrode immediately after collection and under a physiological vaginal concentration of CO2. Commercial enzymatic assays of total lactate and total acetate concentrations were validated for use in CVF, and compared to the more usual HPLC method. The average pH of the CVF samples was 3.5 ± 0.3 (mean ± SD), range 2.8-4.2, and the average total lactate was 1.0% ± 0.2% w/v; this is a five-fold higher average hydrogen ion concentration (lower pH) and a fivefold higher total lactate concentration than in the prior literature. The microbicidal form of lactic acid (protonated lactic acid) was therefore eleven-fold more concentrated, and a markedly more potent microbicide, than indicated by prior research. This suggests that when lactobacilli dominate the vaginal microbiota, women have significantly more lactic acid-mediated protection against infections than currently believed. Our results invite further evaluations of the prophylactic and therapeutic actions of vaginal lactic acid, whether provided in situ by endogenous lactobacilli, by probiotic lactobacilli, or by products that reinforce vaginal lactic acid.
Cells maintain intracellular pH (pHi) within a narrow range (7.1–7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H+-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.
Objectives: Saliva contains a variety of host defense factors. It influences calculus formation and periodontal disease. Different studies have been done to find exact correlation of salivary biomarkers with periodontal disease. With a multitude of biomarkers and complexities in their determination, the salivary pH may be tried to be used as a quick chairside test. The aim of this study was to analyze the pH of saliva and determine its relevance to the severity of periodontal disease. Study Design: The study population consisted of 300 patients. They were divided into three groups of 100 patients each: Group A had clinically healthy gingiva, Group B who had generalized chronic gingivitis and Group C who had generalized chronic periodontitis. The randomized unstimulated saliva from each patient was collected and pH was tested. Data was analyzed statistically using analysis of variance technique. Results: The salivary pH was more alkaline for patients with generalized chronic gingivitis as compared with the control group (P = 0.001) whereas patients with generalized chronic periodontitis had more acidic pH as compared with the control group (P = 0.001). Conclusion: These results indicate a significant change in the pH depending on the severity of the periodontal condition. The salivary pH shows significant changes and thus relevance to the severity of periodontal disease. Salivary pH may thus be used as a quick chairside diagnostic biomarker.
Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC-MS/MS gradients. From 14 HpH fractions, we routinely identified over 30,000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404).