Proton Efflux Research Articles

Abstract 6034: Detection of potassium channel (KCN) gene expression, often multiple and pH sensitive, in cancer genomic landscapes, as low to mid level genes of interest, suggests potassium transmembrane flow may enable proton efflux in a Goldilocks manner

Abstract A reversed pH gradient across cell membranes underlies multiple malignant features, including invasiveness. Extracellular low pH activates metalloproteases and intracellular high pH activates ATP citrate lyase, inactivates caspases, etc. Hydrogen ions attracted to inner membrane fixed anions are putatively displaced by potassium ion flow through membrane channels. Altered expression of potassium channel (KCN) genes sensitive to pH in invasive brain tumors (Beckner, Proc AACR, A3039, 2022) and 2 mutated KCN genes (1 pH sensitive) in U87 glioblastoma cells (Clark MJ et al, PLOS Genet 2010, e1000832) led to this investigation. Published studies with altered expression or mutations in KCN genes qualifying them as genes of interest (GOI) were compared. Both study types included breast, lung, gastrointestinal, brain, bone marrow, liver, melanocytic malignancies, and 8 others were in either. At least 1 figure (heatmap, Venn diagram, phylogenetic tree, etc.) or table (not Supplemental) had 1 or more KCN genes in a group of GOI. Average numbers of GOI with 95% CI for 23 expression and 14 mutation studies were 38.5 ± 0.4 and 28.4 ± 0.4. No KCN GOI was further examined in the studies. Expression was according to tumor types, inhibitors, siRNAs, regulators of pathways, transcription factors, location, etc. Mutations were single nucleotide polymorphisms, insertions, deletions, fusions, etc. Tissue from surgery, organoids, xenografts, cell lines, stem cells, etc. were analyzed. Of 27 KCN GOI in expression studies, 2/3 were detected with other KCN GOI. For 12 mutated KCN genes there were no other KCN genes mutated in the same study. In expression studies, 8 (34.8%) had multiple KCN GOI, differing from mutation studies, p=0.015, Fishers Exact. In expression studies, 8 found pH sensitive KCN GOI including KCNK1 (2X), K3, K5, K6, K12, K15 (2X), and KCNJ16. In mutation studies, KCNK9 was the only one found. The most frequently mutated KCN subfamily was KCNJ, KCNJ5 (2X) and KCNJ12 (2X). All expression studies included at least 1 repeat GOI (KCN or other). APOE, RET, and RTN1 were each GOI 3 times. Additionally, 40 expressed GOI (including 6 KCN GOI) were found 2X. A small cell lung cancer study had 6 repeats (30%) in 20 GOI (ASCL1, BCL2, KCNA1, RET, SCN3A, and SOX2). KCN gene multiplicity, often pH sensitive members, among low to mid level GOI consistent with a Goldilocks' level of expression, and repeated association with cancer genes supports a pH related measured/regulated role for potassium channels in malignancy. Mainly inward (and some outward for recirculation) potassium transmembrane flows in tumor cells potentially compete with hydrogen ions attracted to inner membrane anions to promote proton efflux by displacement with subsequent reversed pH gradients that aid invasion, cell survival, etc. Citation Format: Marie E. Beckner. Detection of potassium channel (KCN) gene expression, often multiple and pH sensitive, in cancer genomic landscapes, as low to mid level genes of interest, suggests potassium transmembrane flow may enable proton efflux in a Goldilocks manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6034.

High cyclic electron transfer via the PGR5 pathway in the absence of photosynthetic control.

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO2 fixation into carbohydrates by the Calvin-Benson-Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b6f (cytb6f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH+). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO2 fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

Impact of Prenatal Exposure to Maternal Diabetes and High-Fat Diet on Postnatal Myocardial Ketone Body Metabolism in Rats.

Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. Using a rat model, we showed how fetal exposure to maternal diabetes causes cardiac disease through fuel-mediated mitochondrial dysfunction, and that a maternal high-fat diet (HFD) exaggerates the risk. Diabetic pregnancy increases circulating maternal ketones which can have a cardioprotective effect, but whether diabetes-mediated complex I dysfunction impairs myocardial metabolism of ketones postnatally remains unknown. The objective of this study was to determine whether neonatal rat cardiomyocytes (NRCM) from diabetes- and HFD-exposed offspring oxidize ketones as an alternative fuel source. To test our hypothesis, we developed a novel ketone stress test (KST) using extracellular flux analyses to compare real-time ß-hydroxybutyrate (βHOB) metabolism in NRCM. We also compared myocardial expression of genes responsible for ketone and lipid metabolism. NRCM had a dose-dependent increase in respiration with increasing concentrations of βHOB, demonstrating that both control and combination exposed NRCM can metabolize ketones postnatally. Ketone treatment also enhanced the glycolytic capacity of combination exposed NRCM with a dose-dependent increase in the glucose-mediated proton efflux rate (PER) from CO2 (aerobic glycolysis) alongside a decreased reliance on PER from lactate (anaerobic glycolysis). Expression of genes responsible for ketone body metabolism was higher in combination exposed males. Findings demonstrate that myocardial ketone body metabolism is preserved and improves fuel flexibility in NRCM from diabetes- and HFD-exposed offspring, which suggests that ketones might serve a protective role in neonatal cardiomyopathy due to maternal diabetes.

Chloroplast Inner Envelope Protein FtsH11 is Involved in the Adjustment of Assembly of Chloroplast ATP Synthase under Heat Stress.

The maintenance of a proton gradient across the thylakoid membrane is an integral aspect of photosynthesis that is mainly established by the splitting of water molecules in photosystem II and plastoquinol oxidation at the cytochrome complex, and it is necessary for the generation of ATP in the last step of photophosphorylation. Although environmental stresses, such as high temperatures, are known to disrupt this fundamental process, only a few studies have explored the molecular mechanisms underlying proton gradient regulation during stress. The present study identified a heat-sensitive mutant that displays aberrant photosynthesis at high temperatures. This mutation was mapped to AtFtsH11, which encodes an ATP-dependent AAA-family metalloprotease. We showed that AtFtsH11 localizes to the chloroplast inner envelope membrane and is capable of degrading the ATP synthase assembly factor BFA3 under heat stress. We posit that this function limits the amount of ATP synthase integrated into the thylakoid membrane to regulate proton efflux from the lumen to the stroma. Our data also suggest that AtFtsH11 is critical in stabilizing photosystem II and cytochrome complexes at high temperatures, and additional studies can further elucidate the specific molecular functions of this critical regulator of photosynthetic thermotolerance. This article is protected by copyright. All rights reserved.

Vertebrate OTOP1 is also an alkali-activated channel

Although alkaline sensation is critical for survival, alkali-activated receptors are yet to be identified in vertebrates. Here, we showed that the OTOP1 channel can be directly activated by extracellular alkali. Notably, OTOP1 biphasically mediated proton influx and efflux with extracellular acid and base stimulation, respectively. Mutations of K221 and R554 at the S5–S6 and S11–S12 linkers significantly reduced alkali affinity without affecting acid activation, suggesting that different domains are responsible for acid- and alkali-activation of OTOP1. The selectivity for H+ was significantly higher in OTOP1 activated by alkali than that by acid, further suggesting that the two activations might be independent gating processes. Given that the alkali-activation of OTOP1 and the required key residues were conserved in the six representative vertebrates, we cautiously propose that OTOP1 participates in alkaline sensation in vertebrates. Thus, our study identified OTOP1 as an alkali-activated channel.

Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.

Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway

BackgroundSalidroside (Sal), an active component from Rhodiola crenulata, has been confirmed to exert neuroprotective effects against hypoxia. However, its molecular mechanisms of intensifying mitochondrial function still largely unknown. In the present study, we aimed to explore the mechanisms by which Sal heightened mitochondrial function in CoCl2-induced HT22 hypoxic injury. MethodsThe hypoxic condition of HT22 cells was performed by CoCl2 stimulus. We then investigated the effects of Sal on the viability of hypoxic HT22 cells by cell counting kit-8. The contents of lactate dehydrogenase (LDH) release in cultured supernatant were detected by using commercial biochemical kit. Superoxide free radical scavenging activity, total antioxidant capacity assay kit with ferric reducing ability of plasma and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) methods were employed to detect the free radical scavenging ability and antioxidant capacity of Sal. Meanwhile, intracellular reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (MMP) were determined by corresponding specific labeled probes. Mitochondrial morphology was tested by Mito-tracker green with confocal microscopy. Hoechst 33342 and Annexin V-FITC/propidium iodide staining were also employed to evaluate the effect of Sal on cell apoptosis. Oxygen consumption rate (OCR), real-time ATP production and proton efflux rate were measured using a Seahorse analyzer. Additionally, the potential interactions of Sal with PI3K-AKT signaling pathway-related proteins were predicted and tested by molecular docking, molecular dynamics simulation (MDS) and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of p-PI3K, PI3K, p-AKT, AKT, p-JNK, JNK, p-p38 and p38 were estimated by western blot analysis. ResultsSal alleviated CoCl2-induced hypoxic injury in HT22 cells as evidenced by increased cell viability and decreased LDH release. In vitro antioxidant test confirmed that Sal had marvelous antioxidant abilities. The protected mitochondrial function by Sal treatment was illustrated by the decrease of ROS, Ca2+, mitochondrial fragment and the increase of MMP. In addition, Sal ameliorated the apoptosis of HT22 cells by decreasing Hoechst 33342 positive cells and the rate of apoptotic cells. Enhancement of energy metabolism in HT22 by Sal was demonstrated by increased OCR, real-time ATP generation and proton efflux rate. The molecular docking confirmed the potential binding of Sal to PI3K, AKT and CaMK II proteins with calculated binding energy of -1.32, -4.21 and -4.38 kcal/mol, respectively. The MDS test revealed the average hydrogen bond of complex Sal-PI3K and Sal-AKT were 0.79 and 4.46, respectively. The results of LSPR verified the potential binding of Sal to proteins PI3K, AKT and HIF-1α with affinity values of 5.20 × 10 − 3, 2.83 × 10 − 3 and 3.97 × 10 − 3 KD, respectively. Western blot analysis further argued that Sal consolidated the levels of p-PI3K and p-AKT. Meanwhile, Sal could downregulate the proteins expression of p-JNK and p-p38. ConclusionCollectively, our findings suggested that Sal can intensify mitochondrial function of CoCl2-simulated hypoxia injury in HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway. Sal is a potential agent for mitochondrial protection against hypoxia with the underlying molecular mechanisms of energy metabolism being further elucidated.

Triggering root proton efflux as an aluminum-detoxifying mechanism in cassava

Aluminum (Al) is a copious element in the earth's crust, typically causing high acidity in soil-plant systems. Much research has primarily investigated adverse impacts of Al on plants, little is known about its beneficial role in enhancing mineral nutrient availability and acquisition in cassava, which is a vital economic crop relevant to human health. Herein, we examined the effect of Al levels on proton and organic acid release from the roots of two cultivars under an acid-washed sand microcosm. Consequential effects of the Al were examined on the extractability of Al and selected nutrients (iron: Fe and phosphorus: P) in the rhizosphere and bulk sands and the nutrient uptake in the plant. The results demonstrated that the highest Al level significantly demoted fresh root weight (8.53 g) but promoted the proton release from roots (2.03 μmol h−1 g−1 fresh weight), compared to the control treatment (11.92 g and 0.40 μmol h−1 g−1 fresh weight). Water-extractable Al and Fe concentrations in the rhizosphere sand were higher by 188–276 % and 201–291 %, respectively, than bulk sand in the highest Al level. The moderate Al levels (<50 μmol Al L−1) also increased Fe accumulation in the plant, elaborating on the beneficial role of Al in enhancing Fe acquisition. The main organic anions (oxalate and tartrate) released from the roots were cultivar-dependent. Our study highlighted that moderate Al levels showed the benefits of Al in promoting proton release from roots, enhancing Fe availability in the rhizosphere zone, and Fe acquisition in the cassava plant.

Ceriporia lacerata HG2011 enhances P mobilization and wheat agronomic performance irrespective of P fertilization levels

To identify soil phosphorus (P) mobilization and wheat agronomic performance in response to the P mobilizer Ceriporia lacerata HG2011 could provide a new strategy for improving fertilizer P efficiency in wheat cultivation. Liquid culture showed that C. lacerata HG2011 converted Ca3 (PO4 )2 , FePO4 , AlPO4 , phytate, lecithin and ribonucleic acid into soluble inorganic P, which was stimulated by ammonium and urea but less influenced by P supply. In the incubation experiment, this fungus colonized on wheat roots, and mobilized P in the soils regardless of Olsen P levels. The efflux of protons, organic acids and phosphatase could be involved in insoluble P mobilization. In the greenhouse pot experiment, C. lacerata HG2011 increased soil Olsen P under different P fertilization levels, improved wheat P uptake by 15.39%-28.70%, P fertilizer use efficiency by 4.26%-13.04% and grain yield by 12.24%-22.39%. Ceriporia lacerata HG2011 was able to colonize on wheat roots, mobilize P in soils and improve wheat agronomic performance irrespective of P fertilization levels. Ceriporia lacerata HG2011 could be used to enhance the quality of compost or as a bio-fertilizer for P mobilization in modern sustainable agriculture.

Abstract P3056: Influence Of Maternal Diabetes And High Fat Diet On Myocardial Ketone Body Metabolism In The Offspring

Alteration in myocardial substrate utilization and energy metabolism is a well-known contributor to the pathogenesis of heart disease. Recent studies suggest the failing heart may benefit from ketone bodies as an energy source. Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. We showed fetal exposure to excess circulating fuels, especially to the combination of maternal high fat diet (HFD) and diabetes, exaggerates CVD risk through fuel-mediated mitochondrial dysfunction including complex I dysfunction. Diabetic pregnancy increases ketone body exposure in utero, but whether prenatal exposures impair myocardial metabolism of ketones postnatally remains unknown. The objective of this study was to determine whether neonatal rat cardiomyocytes (NRCM) exposed to maternal diabetes and HFD in utero can increase ketone oxidation as a preferred fuel source over glycolysis. To test our hypothesis, we developed a ketone stress test (KST) or modified extracellular flux analyses to compare real-time ß-hydroxy butyrate (ßOHB) metabolism in NRCM and myocardial expression of genes responsible for ketone and lipid metabolism. While control and combination exposed NRCM had similar basal respiration, the presence of ketone (4.5 mM) increased maximal respiration, spare respiratory capacity, and ATP-limited glycolytic rate in the combination exposed group. Interestingly, baseline proton efflux rate (PER) from lactate (anaerobic glycolysis) decreased while PER from CO 2 (aerobic glycolysis) increased in response to ketones in the combination exposed group. Expression of genes responsible for ketone body metabolism ( Hmgcs2 and Bdh1 ) was not different. The study uses a novel KST to analyze real-time ketone body metabolism and demonstrates myocardial ketone body metabolism is preserved and may be protective in the NRCM exposed to maternal overnutrition.

Model-based driving mechanism analysis for butyric acid production in Clostridium tyrobutyricum

BackgroundButyric acid, an essential C4 platform chemical, is widely used in food, pharmaceutical, and animal feed industries. Clostridium tyrobutyricum is the most promising microorganism for industrial bio-butyrate production. However, the metabolic driving mechanism for butyrate synthesis was still not profoundly studied.ResultsThis study reports a first-generation genome-scale model (GEM) for C. tyrobutyricum, which provides a comprehensive and systematic analysis for the butyrate synthesis driving mechanisms. Based on the analysis in silico, an energy conversion system, which couples the proton efflux with butyryl-CoA transformation by two redox loops of ferredoxin, could be the main driving force for butyrate synthesis. For verifying the driving mechanism, a hydrogenase (HydA) expression was perturbed by inducible regulation and knockout. The results showed that HydA deficiency significantly improved the intracellular NADH/NAD+ rate, decreased acetate accumulation (63.6% in serum bottle and 58.1% in bioreactor), and improved the yield of butyrate (26.3% in serum bottle and 34.5% in bioreactor). It was in line with the expectation based on the energy conversion coupling driving mechanism.ConclusionsThis work show that the first-generation GEM and coupling metabolic analysis effectively promoted in-depth understanding of the metabolic driving mechanism in C. tyrobutyricum and provided a new insight for tuning metabolic flux direction in Clostridium chassis cells.

Abstract 3039: Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment

Abstract Glial buffering of K+ prevents seizures, hence gliomas in patients on anticonvulsants may harbor potassium related anomalies. Cell membrane potentials are very sensitive to K+ and may link to tumor acidosis when they become more positive than the equilibrium potential for H+ ions (Vaupel, P, et al., Cancer Res, 1989). Kir5.1(KCNJ16) heterodimerizes with Kir4.1(KCNJ10), a major glial potassium channel, to stop K+ conductance in acidosis. Trek1&amp;2(KCNK2&amp;10) are also pH sensitive K+ channels. Glioblastomas (more malignant and invasive) versus oligodendrogliomas showed decreased KCNJ16 in patients on anticonvulsants (Beckner, ME, Proc AACR, A2319, 2021). However, loss of genomic integrity in tumors and potential variation between assays prompted house keeping gene (HKG) normalization to lessen confounding factors. REMBRANDT (12/31/2020, Georgetown Database of Cancer) had 220 glioblastomas and 67 oligodendrogliomas with microarray gene expression via medians of each reporter (1-8 per gene) in Adobe Flash readouts (ended 1/1/21). A subgroup, 75 glioblastomas and 24 oligodendroglioma patients, took anticonvulsants. Comparison of genes of interest (GOI) initially had t tests between types of gliomas in the subgroup. Here, differences between glioma types for each GOI reporter were compared to differences for HKGs (PPIA, RPLP0, YWHAZ, and B2M). In gliomas with anticonvulsants, paired 2-tailed t tests for a null difference of median reporters between tumor types were less sensitive than new comparisons of oligodendroglioma minus glioblastoma (OMG) differences for GOIs versus OMG differences in HKGs. For KCNJ16, the t test, p = 0.015, for a decrease in glioblastomas (versus oligodendrogliomas) in initial studies improved to p ≤ 0.0001 by using HKGs in comparisons. The KCNK2&amp;10 decrease in glioblastomas became significant, p = 0.047. Among other glycolytic and acidosis related genes, CA12, GLO1&amp;2, PFKFB1&amp;3, showed stronger trends, with p values from 0.08 to 0.13 (decreases and increase) in comparisons using HKGs. Conclusion: HKG normalization presumably corrects for loss of genomic integrity, assay variations, etc. in comparisons using microarrays of different tumors. The pH-sensitive K+ channel genes studied here were expressed less in glioblastomas. The resulting pH-insensitive K+ distributions in glioblastomas putatively shift their cell membrane potentials to less negative values which are sufficient to allow passive H+ efflux that causes extracellular acidosis with accompanying activation of invasion. Acknowledgement: The data utilized in this study were provided by the Georgetown Database of Cancer (G-DOC), a project of the Georgetown Lombardi Comprehensive Cancer Center designed to provide translational research tools to the scientific community. Citation Format: Marie E. Beckner. Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3039.

Abstract 1302: Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy

Abstract While immune checkpoint blockade (ICB) treatments such as nivolumab and ipilimumab have spelled hope for many cancer patients, not all patients benefit from this form of therapy. One mechanism by which tumors suppress the anti-tumor T cell activity that results from ICB is through decreasing extracellular tumor pH (pHe), a function inherent to tumor cells due to the Warburg effect. We thus hypothesize that increasing pHe prior to ICB therapy potentiates the T cell-mediated anti-tumor effect of ICB treatment. To undergo this study, we first screened a panel of six inhibitors termed pH sensitizers, and found that esomeprazole significantly reduced the proton efflux rate (PER) of 4T1, but not B16-F10 cells. Furthermore, esomeprazole did not significantly reduce cell viability of either 4T1 or B16-F10 cells, and did not significantly inhibit T cell activity. Moving forward, our next objectives are to investigate the effects of esomeprazole and the combination of esomeprazole and ICB on tumor-bearing mice. Assessment of the tumor growth in response to esomeprazole and ICB and ex vivo interrogation of the immune cell infiltrate into the tumors will elucidate the efficacy of the treatment combination in terms of tumor control and anti-tumor immunity. Modification of pHe by esomeprazole will be examined by acidoCEST MRI, which will also provide insights as to the ideal pHe parameters for optimal ICB therapy in patients. Citation Format: Renee Chin, Jorge de la Cerda, William Schuler, Mark D. Pagel. Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1302.

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals.

Alkalinization of sperm cytosol is essential for plasma membrane hyperpolarization, hyperactivation of motility, and acrosomal exocytosis during sperm capacitation in mammals. The plasma membrane of sperm cells contains different ion channels implicated in the increase of internal pH (pHi) by favoring either bicarbonate entrance or proton efflux. Bicarbonate transporters belong to the solute carrier families 4 (SLC4) and 26 (SLC26) and are currently grouped into Na+/HCO3− transporters and Cl−/HCO3− exchangers. Na+/HCO3− transporters are reported to be essential for the initial and fast entrance of HCO3− that triggers sperm capacitation, whereas Cl−/HCO3− exchangers are responsible for the sustained HCO3− entrance which orchestrates the sequence of changes associated with sperm capacitation. Proton efflux is required for the fast alkalinization of capacitated sperm cells and the activation of pH-dependent proteins; according to the species, this transport can be mediated by Na+/H+ exchangers (NHE) belonging to the SLC9 family and/or voltage-gated proton channels (HVCN1). Herein, we discuss the involvement of each of these channels in sperm capacitation and the acrosome reaction.

Triiron Tetrairon Phosphate (Fe7(PO4)6) Nanomaterials Enhanced Flavonoid Accumulation in Tomato Fruits.

Flavonoids contribute to fruit sensorial and nutritional quality. They are also highly beneficial for human health and can effectively prevent several chronic diseases. There is increasing interest in developing alternative food sources rich in flavonoids, and nano-enabled agriculture provides the prospect for solving this action. In this study, triiron tetrairon phosphate (Fe7(PO4)6) nanomaterials (NMs) were synthesized and amended in soils to enhance flavonoids accumulation in tomato fruits. 50 mg kg−1 of Fe7(PO4)6 NMs was the optimal dose based on its outstanding performance on promoting tomato fruit flavonoids accumulation. After entering tomato roots, Fe7(PO4)6 NMs promoted auxin (IAA) level by 70.75 and 164.21% over Fe-EDTA and control, and then up-regulated the expression of genes related to PM H+ ATPase, leading to root proton ef-flux at 5.87 pmol cm−2 s−1 and rhizosphere acidification. More Mg, Fe, and Mn were thus taken up into plants. Subsequently, photosynthate was synthesized, and transported into fruits more rapidly to increase flavonoid synthesis potential. The metabolomic and transcriptomic profile in fruits further revealed that Fe7(PO4)6 NMs regulated sucrose metabolism, shi-kimic acid pathway, phenylalanine synthesis, and finally enhanced flavonoid biosynthesis. This study implies the potential of NMs to improve fruit quality by enhancing flavonoids synthesis and accumulation.

Synergistic antibacterial activity of tetrandrine combined with colistin against MCR-mediated colistin-resistant Salmonella

It has been recognized that colistin resistance is a growing problem that seriously impairs the clinical efficacy of colistin against bacterial infections. One strategy that has been proven to have therapeutic effect is to overcome the widespread emergence of antibiotic-resistant pathogens by combining existing antibiotics with promising non-antibiotic agents. In this work, antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of the combination. The molecular mechanisms of tetrandrine in combination with colistin were analyzed using fluorometric assay and Real-time PCR. To predict possible interactions between tetrandrine and MCR-1, molecular docking assay was taken. Finally, we evaluated the in vivo efficacy of tetrandrine in combination with colistin against MCR-positive Salmonella. Overall, the combination of tetrandrine and colistin showed significant synergistic activity. In-depth mechanistic analysis showed that the combination of tetrandrine with colistin enhances the membrane-damaging ability of colistin, undermines the functions of proton motive force (PMF) and efflux pumps in MCR-positive bacteria. The results of molecular docking and RT-PCR analyses showed that tetrandrine not only affects the expression of mcr-1 but is also an effective MCR-1 inhibitor. Compared with colistin monotherapy, the combination of tetrandrine with colistin significantly reduced the bacterial load in vivo. Our findings demonstrated that tetrandrine serves as a potential colistin adjuvant against MCR-positive Salmonella.

Bicalutamide may enhance kidney injury in diabetes by concomitantly damaging energy production from OXPHOS and glycolysis

Bicalutamide (Bic), frequently used in androgen-deprivation therapy for treating prostate cancer, was demonstrated to induce multiple apoptosis and fibrosis pathways and mitochondrial dysfunction in renal mesangial cells. Whether Bic also damages the glycolytic pathway has never been cited. To investigate this, we performed an in vitro model study with mesangial cells, and at the same time, collected data from an in vivo experiment. Bic induced hypoxia-inducible factor (HIF)-1 which upregulates phosphorylated-5′-AMP-activated protein kinase (p-AMPK) and severely suppresses the rate of adenosine triphosphate (ATP) production in both the oxidative phosphorylation and glycolysis pathways. Bic suppressed the oxygen consumption rate, extracellular acidification rate, and mitochondrial proton efflux rate, downregulated in vivo but upregulated in vitro glucose transporter (GLUT)-1, reduced glucose uptake, inhibited key glycolytic enzymes, including phosphofructokinase (PFK), pyruvate kinase (PK), and pyruvate dehydrogenase (PDH), and upregulated hexokinase II (HKII) and lactic dehydrogenase A (LDHA). In vivo, Bic downregulated renal cubilin levels, thereby disrupting the glomerular reabsorption function. Conclusively, Bic can damage bioenergenesis from both mitochondria and glycolysis. It was suggested that long-term administration of Bic can initiate renal damage depending on the duration and dose of treatment, which requires cautious follow-up.

Physiological mechanism of the response to Cr(VI) in the aerobic denitrifying ectomycorrhizal fungus Pisolithus sp.1

Pisolithus sp. 1 (P sp. 1) is an ectomycorrhizal fungus (EMF) with a strong Cr(VI) tolerance and reduction ability. The noninvasive microttest technique (NMT), real-time quantitative PCR (qRT–PCR), and the three-dimensional excitation-emission matrix (3D-EEM) were used to deeply explore the physiological mechanism of the P sp. 1 response to Cr(VI) and investigate the relationship between Cr(VI) reduction and denitrification in P sp. Cr(VI) induced the strongest elevations in nitrate reductase (NR) activity and NO production in the mycelia after treatment with Cr(VI) for 48 h under aerobic conditions. The NR inhibitor tungstate significantly inhibited Cr(VI) reduction, proton efflux and the expression of the NR gene (niaD) and NiR gene (niiA). In addition, NO was generated via NR-regulated denitrification. Combined treatments with Cr(VI) and the NO scavenger carboxy-PTIO (cPTIO) significantly increased O2-, H2O2 and MDA contents and reduced SDH, CAT, GSH, GR and GSNOR activity. Therefore, the NR-driven aerobic denitrifying process requires protons, and the generated NO reduces the oxidative stress effect of Cr(VI) on mycelia by reducing ROS accumulation and lipid peroxidation, enhancing mycelial and CAT activity, and promoting GSH recycling and regeneration. Psp.1 can also secrete humic acid-like and protein-like substances to combine with Cr(III) in a culture system.

Changing lanes: seasonal differences in cellular metabolism of adipocytes in grizzly bears (Ursus arctos horribilis).

Obesity is among the most prevalent of health conditions in humans leading to a multitude of metabolic pathologies such as type 2 diabetes and hyperglycemia. However, there are many wild animals that have large seasonal cycles of fat accumulation and loss that do not result in the health consequences observed in obese humans. One example is the grizzly bear (Ursus arctos horribilis) that can have body fat content > 40% that is then used as the energy source for hibernation. Previous in vitro studies found that hibernation season adipocytes exhibit insulin resistance and increased lipolysis. Yet, other aspects of cellular metabolism were not addressed, leaving this in vitro model incomplete. Thus, the current studies were performed to determine if the cellular energetic phenotype-measured via metabolic flux-of hibernating bears was retained in cultured adipocytes and to what extent that was due to serum or intrinsic cellular factors. Extracellular acidification rate and oxygen consumption rate were used to calculate proton efflux rate and total ATP defined as both ATP from glycolysis and from mitochondrial respiration. Hibernation adipocytes treated with hibernation serum produced less ATP and exhibited lower maximal respiration and glycolysis ratesthan active season adipocytes. These effects were reversed with serum from the opposite season. Insulin had little influence on total ATP production and lipolysis in both hibernation and active serum-treated adipocytes. Together, these results suggest that the metabolic suppression occurring in hibernation adipocytes are downstream of insulin signaling and likely due to a combined reduction in mitochondria number and/or function and glycolytic processes. Future elucidation of the serum components and the cellular mechanisms that enable alterations in mitochondrial function could provide a novel avenue for the development of treatments for human metabolic diseases.

Mitochondrion-targeted supramolecular "nano-boat" simultaneously inhibiting dual energy metabolism for tumor selective and synergistic chemo-radiotherapy.

Rationale: Tumor energy metabolism has been a well-appreciated target of cancer therapy; however, the metabolism change of cancer cells between oxidative phosphorylation and glycolysis poses a challenge to the above. In this study, we constructed an innovative mitochondrion-targeted supramolecular “nano-boat” based on peptide self-assembly for tumor combined chemo-radiotherapy by simultaneously inhibiting the dual energy metabolism.Methods: A lipophilic self-assembled peptide and a positively charged cyclen were integrated to fabricate a brand new mitochondrion-targeted nano-platform for the first time. The indices of mitochondrial dysfunction including mitochondrial membrane potential, apoptosis proteins expression and ultrastructure change were evaluated using a JC-1 probe, western blotting and biological transmission electron microscopy, respectively. Energy metabolism assays were conducted on a Seahorse XF24 system by detecting the oxygen consumption rate and the glycolytic proton efflux rate. The radio-sensitization effect was investigated by colony formation, the comet assay, and γ-H2AX staining.Results: The supramolecular “nano-boat” could selectively kill cancer cells by much higher enrichment and reactive oxygen species generation than those in normal cells. In the cancer cells treated with the supramolecular “nano-boat”, the dysfunctional morphological changes of the mitochondrial ultrastructure including swelling and pyknosis were evidently observed, and the endogenous mitochondrial apoptosis pathway was effectively triggered by abundant of cytochrome C leaking out. Concurrently, the dual metabolic pathways of glycolysis and oxidative phosphorylation were severely inhibited. More importantly, the supramolecular “nano-boat” displayed an excellent radio-sensitization effect with a sensitization enhancement ratio value as high as 2.58, and hence, in vivo efficiently combining radiotherapy yielded an enhanced chemo-radiotherapy effect.Conclusion: Our study demonstrated that the rationally designed peptide-based “nano-boat” could efficiently induce cancer cell apoptosis by the energy metabolism inhibition involving multiple pathways, which may provide the motivation for designing novel and universal mitochondria-targeted drug delivery systems for cancer therapy.