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Bacteria commonly live in a spatially organized biofilm assemblage. The metabolic activity inside the biofilm leads to segmented physiological microenvironments. In nature, bacteria possess several pleomorphic forms to withstand certain ecological alterations. We hypothesized that pleomorphism also exists within the biofilm, which can be considered as the fundamental niche for bacteria. We report a distinct pattern of cell size variation throughout the biofilm of Bacillus subtilis. Cell size heterogeneity was observed in biofilm development, wherein the frequency of long cells is higher in outer regions, whereas lower in inner regions. Moreover, compared to planktonic cells, bacteria in the biofilm mode reduce their geometric ratio from 8.34 to 3.69 and 2.65 in the outer and inner regions, respectively. There were no significant differences observed in nutrient diffusion from the outer to the inner region, and more than 73 % of cells in the inner region were viable. However, the inner and middle regions were more acidic than the outer of the biofilm. Conclusively, growth rate-independent cell size reduction at low pH suggests that the resulting phenotype switching within biofilm was observed due to the pH gradient of neutral to acidic from the outer to the core of the biofilm. This gradient of H + ions concentration may create exploitative stress within the biofilm, which could favor specific pleomorphic cells to thrive in their specialized niches. By understanding the cell size variation in response to the local environment, we propose a model of biofilm formation by pleomorphic cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study marks the exploration into the production of ectoine, a valuable compound with significant potential as an antioxidant, osmoprotectant, anti-inflammatory agent, and stabilizer of cell membranes, proteins, and DNA integrity. Our focus centred on investigating the presence of ectoine and optimizing its production by the novel ectoine producer bacterial strain, Piscibacillus halophilus. For the optimization of ectoine production the effects of carbon and nitrogen sources, salt, pH, agitation and incubation period were optimized by one-factor-at-a-time. We started with an initial ectoine content of 46.92 mg/L, and through a series of optimization processes, we achieved a remarkable increase, resulting in an ectoine content of 1498.2 mg/L. The bacterial species P. halophilus achieved its highest ectoine production after 48 h of incubation, with conditions set at 10 % (w/v) salinity, pH of 7.50, and an agitation speed of 160 rpm. These precise conditions were found to be the most favourable for maximizing ectoine production by this strain. Besides, we have achieved successful purification of ectoine from the crude extract through a streamlined single-step process. This purification method has delivered an exceptional level of purity, surpassing 99.15 %, and an impressive yield of over 99 %. Importantly, we accomplished this using readily available and cost-effective strong acids (HCl) and strong bases (NaOH) to arrange pH gradients. The use of acid and base in the purification process of ectoine reflects an innovative and sustainable methodology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A novel bioreactor simulating human colonic conditions for in vitro cultivation of intestinal microbiota is presented. The PEristaltic mixed Tubular bioReactor (PETR) is modular designed and periodically kneaded to simulate intestinal peristalsis. The reactor is introduced, characterized from a bioprocess engineer's perspective and discussed in its ability to mimic colon conditions. PETR provides physiological temperature and appropriate anaerobic conditions, simulates intestinal peristalsis, and has a mean residence time of 32.8 ± 0.8 h comparable to the adult human colon. The single-tube design enables a time-constant and longitudinally progressive pH gradient from 5.5 to 7.0. Using a dialysis liquid containing high molecular weight polyethylene glycol, the integrated dialysis system efficiently absorbs short chain fatty acids (up to 60%) and water (on average 850 mL d -1 ). Cultivation of a typical gut bacterium (Bifidobacterium animalis) was performed to demonstrate the applicability for controlled microbiota cultivation. PETR is unique in combining simulation of the entire colon, peristaltic mixing, dialytic water and metabolite absorption, and a progressive pH gradient in a single-tube design. PETR is a further step to precise replication of colonic conditions in vitro for reliable and reproducible microbiota research, such as studying the effect of food compounds, prebiotics or probiotics, or the development and treatment of infections with enteric pathogens, but also for further medical applications such as drug delivery studies or to study the effect of drugs on and their degradation by the microbiota., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s -1 . This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Human activity is generating an excess of atmospheric CO 2 , resulting in what we know as ocean acidification, which produces changes in marine ecosystems. Until recently, most of the research in this area had been done under small-scale, laboratory conditions, using few variables, few species and few life cycle stages. These limitations raise questions about the reproducibility of the environment and about the importance of indirect effects and synergies in the final results of these experiments. One way to address these experimental problems is by conducting studies in situ, in natural areas where expected future pH conditions already occur, such as CO 2 vent systems. In the present work, we compile and discuss the latest research carried out in these natural laboratories, with the objective to summarize their advantages and disadvantages for research to improve these investigations so they can better help us understand how the oceans of the future will change., (Copyright © 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Chloride (Cl - ) is essential for O 2 evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P + Q A - ), rates of water oxidation steps (S-states), rate of proton evolution, and flash O 2 yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O 2 evolution rate (-20 %) and proton release (-36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S 2  → S 3  → S 0 ') slow with increasing light intensity and during O 2 /water exchange (S 0 ' → S 0  → S 1 ), while the non-protonogenic S 1  → S 2 transition is kinetically unaffected. As flash rate increases in Cl - cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br - . The Cl - advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H 3 O + Cl - in dry water channels vs. separated Br - and H + ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br - cultures for both proton evolution and less PSII charge recombination. In Br - cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br - with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels., Competing Interests: Declaration of competing interest G. Charles Dismukes reports financial support was provided by US Department of Energy. G. Charles Dismukes reports financial support was provided by National Science Foundation., (Copyright © 2023. Published by Elsevier B.V.)

Dielectrophoresis (DEP), a precision nonlinear electrokinetic tool utilized within microfluidic devices, can induce bioparticle polarization that manifests as motion in the electric field; this phenomenon has been leveraged for phenotypic cellular and biomolecular detection, making DEP invaluable for diagnostic applications. As device operation times lengthen, reproducibility and precision decrease, which has been postulated to be caused by ion gradients within the supporting electrolyte medium. This research focuses on characterizing pH gradients above, at, and below the electrode charging frequency (0.2-1.4 times charging frequency) in an aqueous electrolyte solution in order to extend the parameter space for which microdevice-imposed artifacts on cells in clinical diagnostic devices have been characterized. The nonlinear alternating current (AC) electric fields (0.07 V pp /μm) required for DEP were generated via planar T-shaped and star-shaped microelectrodes overlaid by a 70 μm high microfluidic chamber. The experiments were designed to quantify pH changes temporally and spatially in the two microelectrode geometries. In parallel, a 50 nm hafnium oxide (HfO 2 ) thin film on the microelectrodes was tested to provide insights into the role of Faradaic surface reactions on the pH. Electric field simulations were conducted to provide insights into the gradient shape within the microelectrode geometries. Frequency dependence was also examined to ascertain ion electromigration effects above, at, and below the electrode charging frequency. The results revealed Faradaic reactions above, at, and below the electrode charging frequency. Comparison experiments further demonstrated that pH changes caused by Faradaic reactions increased inversely with frequency and were more pronounced in the star-shaped geometry. Finally, HfO 2 films demonstrated frequency-dependent properties, impeding Faradaic reactions.

Ocean acidification has been consistently evidenced to have profound and lasting impacts on marine species. Observations have shown seagrasses to be highly susceptible to future increased pCO 2 conditions, but the responses of early life stages as seedlings are poorly understood. This study aimed at evaluating how projected Mediterranean Sea acidification affects the survival, morphological and biochemical development of Posidonia oceanica seedlings through a long-term field experiment along a natural low pH gradient. Future ocean conditions seem to constrain the morphological development of seedlings. However, high pCO 2 exposures caused an initial increase in the degree of saturation of fatty acids in leaves and then improved the fatty acid adjustment increasing unsaturation levels in leaves (but not in seeds), suggesting a nutritional compound translocation. Results also suggested a P. oceanica structural components remodelling which may counteract the effects of ocean acidification but would not enhance seagrass seedling productivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN®. The basic pI of Fab and F(ab) 2 fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN® and papain digestion and the corresponding modifications were determined by LC-MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region., (© 2023. The Author(s).)

The reduction of carbon dioxide (CO 2 ) to high value-added multi-carbon compounds at the cathode is an emerging application of microbial electrosynthesis system (MES). In this study, a composite cathode consisting of hollow fiber membrane (HFM) and the carbon felt is designed to enhance the CO 2 mass transfer of the cathode. The result shows that the main products are acetate and butyrate without other substances. The electrochemical performance of the electrode is significantly improved after biofilm becomes matures. The composite cathode significantly reduces the "threshold" for the synthesis of butyrate. Moreover, CO 2 is dissolved and protons are consumed by synthesizing volatile fatty acids (VFAs) to maintain a stable pH inside the composite electrode. The synthesis mechanism of butyrate is that CO 2 is converted sequentially into acetate and butyrate. The microenvironment of the composite electrode enriches Firmicute. This composite electrode provides a novel strategy for regulating the microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨ m ). Chemiosmosis requires ion exchangers to remove Na + , K + , Ca 2+ , PO 4 3- , and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K + , the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H 2 O by osmosis. The effects of K + uptake through ligand-specific mK + channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K + influx is likely its effects on H + cycling and bioenergetics facilitated by mitochondrial (m) K + /H + exchange (mKHE), though the kinetics and consequences of K + efflux by KHE are not well described. We hypothesized that a major role of K + influx/efflux is stimulation of respiration via the influx of H + by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK + cycle to capture changes in mitochondrial volume, pH m , ΔΨ m , and respiration that would reflect a role for H + influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K + buffer at pH 6.9, or in a 140 mM Cs + buffer at pH 7.6 or 6.9 with added 10 mM K + , minimal Ca 2+ and free of Na + . O 2 content was measured by a Clark electrode, and pH m , and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpH m , and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpH m  = pH in -pH ext ) at pH ext 6.9> > 7.6, so that ΔΨ m was charged and maintained. BK Ca agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K + ionophore valinomycin depolarized ΔΨ m . We postulate that K + efflux-induced H + influx via KHE causes an inward H + leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpH m , the smaller component of the overall proton motive force, ΔμH + . Thus ΔpH m establishes and maintains the ΔΨ m required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K + influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Self-propelled droplets are of considerable interest as an appropriate model for understanding the self-propulsion of objects in the fields of nonequilibrium physics and nonlinear science. Several research groups have reported the monodirectional motion of droplets, that is, chemotaxis, using stimuli-responsive materials. However, the precise control of chemotaxis remains challenging from the perspective of synthetic chemistry because chemotactic motion is primarily induced by the consumption of reactive oil or surfactants. Herein, we report a chemical system containing pH-responsive fumaric acid derivatives, in which the oil droplet exhibited positive chemotaxis over a wide pH range-from basic to acidic conditions. From the measurements of the interfacial tension between the oil and aqueous phases, it was deduced that the positive chemotaxis was due to heterogeneity in the interfacial tension of the droplet surface, which was accompanied by the production of surface-active compounds in the pH gradient in a linear-type channel.

An overarching challenge in the development of supramolecular sensor systems is to enhance their sensitivity, which commonly involves the synthesis of refined receptors with increased affinity to the analyte. We show that a dramatic sensitivity increase by 1-2 orders of magnitude can be achieved by encapsulating supramolecular chemosensors inside liposomes and exposing them to a pH gradient across the lipid bilayer membrane. This causes an imbalance of the influx and efflux rates of basic and acidic analytes leading to a significantly increased concentration of the analyte in the liposome interior. The utility of our liposome-enhanced sensors was demonstrated with various host-dye reporter pairs and sensing mechanisms, and we could easily increase the sensitivity towards multiple biologically relevant analytes, including the neurotransmitters serotonin and tryptamine., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Post-translational modifications ("PTMs") in monoclonal antibodies (mAbs) contribute to charge variant distribution, which will affect biological efficacy and safety. For the characterization of mAbs, charge variants are used as a critical quality attributes for product quality, stability consistency and effectiveness. Charge variants in mAbs are characterized by a time-consuming and a multistep process starting from cation/anion exchange chromatography, acidic/basic fractions collection and subsequent reverse phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis. Hence, an alternative characterization approach that would be highly selective for ion exchange chromatography-based charge variant analysis, which is compatible with on-line MS detection, is needed in the biopharma industry. Against this backdrop, multiple studies are being conducted to develop a simple straight on-line charge variant analysis method. In this regard, we apply the current study, which aims to develop a charge variant analytical method, based on volatile buffers with low ionic strength that can be used for on-line MS detection of charge variants of mAbs. This would enable the detection on "PTMs" using low ionic strength mobile phase compatible with MS. Hence, fruitful data can be obtained with a single chromatography run without any test sample preparation, eliminating the need for multiple steps of analysis, time-consuming process and multiple sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the characterization of charge-related PTMs on the intact protein stage. In this regard, this study is about development of a method having combination of chromatography and volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native form. The method is qualified considering pharmacopeia guidelines because the ultimate aim is to transfer this method for Quality Control (QC) release testing of a monoclonal antibody, which is critical for batch release and the regulatory point of view. Acidic and basic variants have been separated with high resolution peak profile. Furthermore, there was no matrix interference and good separation selectivity in terms of specificity was obtained using this method. The experimental data suggested for the linearity of the method are 2.4 mg/mL to 3.6 mg/mL with % RSD below 2.0%. Additionally, Limit of Quantitation is found to be 0.15 mg/mL, which is 5% of loading amount. Consistently, the data show that the method is precise under the same operating conditions with a short time interval. Overall a simple, accurate, robust and precise pH gradient cation exchange chromatography method was developed and qualified for the characterization of a therapeutic native mAb. Additionally, this method can be used to claim a biosimilar product profile of an in-house product compare to an innovator.

The phenomenon of pH excursion was demonstrated for pH gradient elution of monoclonal antibodies (mAbs) on strong cation exchange resins under high overloading conditions. The mAbs differed in molecular structure and isoelectric point, and the resins in matrix structure and ligand density. In all cases, elution of the proteins was accompanied with distortion of the concentration, pH and conductometric profiles. To elucidate that phenomenon, titration curves were generated for liquid solutions of the proteins as well as for suspensions of the resins with the proteins adsorbed on their surface. The course of the curves was found to be affected by the presence of the proteins both in liquid and adsorbed phases. The effect enhanced with increase in the initial pH of the binding buffer and in the protein concentration. To quantify that phenomenon, a mechanistic model was used, which accounted for the protein dissociation in both phases. The model reproduced the titration curves and the observed trends in changes of their courses. The simulation results indicated that the pattern of pH transitions recorded for different mAbs on the resins mostly depended on their adsorption affinity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cancer cells frequently exhibit an acidic extracellular microenvironment, where inversion of the transmembrane pH gradient is associated with tumor proliferation and metastasis. To elucidate a new therapeutic target against cancer, the current study aimed to determine the mechanism by which the dipeptidyl peptidase-4 inhibitor anagliptin regulates the cellular pH gradient and concomitant extracellular acidosis during cancer progression. A total of 5x10 5 CT-26 cells (resuspended in phosphate buffer saline) were injected subcutaneously in the right flank of male BALB/c mice (weighing 25-28 g). The tumor samples were harvested, and lactate was detected using a lactate assay kit. Immunohistochemistry was used to detect the Ki67 and PCNA. MTT assay and flow cytometric were used to detect cell viability. Intracellular pH was detected by fluorescence pH indicator. The results revealed that anagliptin effectively reduced tumor growth, but did not affect the body weight of treated mice. Anagliptin reduced the accumulation of lactate in tumor sample. Treatment with anagliptin stimulated the apoptosis of CT-26 cells. And lactate excretion inhibition is accompanied by an increase in extracellular pH (pHe) after treatment with anagliptin. Furthermore, anagliptin induced intracellular acidification and reversed the low pHe gradient via monocarboxylate transporter-4 (MCT-4)-mediated lactate excretion. Additionally, anagliptin reversed the aberrant transmembrane extracellular/intracellular pH gradient by suppressing MCT-4-mediated lactate excretion, while also reducing mitochondrial membrane potential and inducing apoptosis. These data revealed a novel function of anagliptin in regulating lactate excretion from cancer cells, suggesting that anagliptin may be used as a potential treatment for cancer., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Li et al.)

The electrocatalytic nitrate-to-ammonia reduction reaction route (NARR) is one of the emerging routes toward green ammonia synthesis, and its conversion efficiency is controlled mainly by the hydrogenation selectivity. This study proposed a likely NARR route feasible and effective even in a neutral condition. Its high catalytic selectivity and efficiency were achieved by a switch of the sulfate solution to the phosphate buffer solution (PBS), while conditions of NO 3 - concentration, pH, and applied potential were maintained unchanged. Specifically, the faradaic efficiencies toward NH 3 (FE NH 3 ) in Na 2 SO 4 were as low as 9.8, 19.8, and 11.4 % versus remarkably jumping to 82.8, 90.5, and 89.5 % in PBS under -0.75, -1.0, and -1.25 V, respectively. The corresponding faradaic efficiencies toward NO 2 - (FE NO 2 - ), 77.0, 69.2, and 73.7 % in Na 2 SO 4 . This insight was further revealed by the visualization of the pH gradient near the electrode surface during NARR and confirmed by density functional theory calculations; PBS notably facilitated the proton transport and active mitigation over the proton transfer barrier. The use of PBS resulted in a maximal partial current density toward NH 2 - to NH 3 . This insight was further revealed by the visualization of the pH gradient near the electrode surface during NARR and confirmed by density functional theory calculations; PBS notably facilitated the proton transport and active mitigation over the proton transfer barrier. The use of PBS resulted in a maximal partial current density toward NH 3 (J NH 3 ) and NH 3 formation rate (r NH 3 ) up to 133.5 mA cm -2 and 1.74×10 -7  mol s -1  cm -2 in 1.0 m KNO 3 at -1.25 V., (© 2022 Wiley-VCH GmbH.)

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A procedure for adjusting the content of charge variants of monoclonal antibody by ion exchange chromatography has been developed. The band splitting phenomenon was utilized to split the protein load into two parts, i.e., the flowthrough and bound fractions, which were either enriched or depleted with some of variants. The phenomenon was triggered by thermodynamic effects resulting from oversaturation of the resin binding sites at high column loadings as well as from kinetic effects arising from limited rates of mass transport. Cation exchange chromatography (CEX) and anion exchange chromatography (AEX) separations were examined, with the reverse order of the variant elution: acidic, main, basic in CEX, and basic, main, acidic in AEX, and the corresponding reverse enrichment tendency in the collected fractions. The separations were performed by pH gradient, whose course was simplified to two stages: isocratic loading and washing at mild pH to load and partly elute the protein, followed by a rapid pH change towards non-binding conditions to desorb the remains of the protein load. To improve yield of the operation, possibility of recycling of waste fractions was considered. To predict the process performance, a dynamic model was developed, which accounted for both adsorption kinetics and thermodynamics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Preventing methane evolution is a key issue to guarantee stable hydrogen production in microbial electrolysis cell (MEC). In this study, low-cost hydrophilic porous materials, such as non-woven cloth (NWC) and polyvinylidenedifluoride (PVDF), were investigated as alternatives to proton exchange membrane (PEM) in MEC. The MEC with a NWC (NWC-MEC) improved the current density and hydrogen production rate (HPR) of 262.5±10 A m -3 and 2.5±0.2 m 3 m -3 d -1 , respectively, due to its lower pH gradient (0.37) and ion transport resistance (0.9±0.1 mΩ m 2 ). Hydrogen production in NWC-MEC (from 2.5 to 2.1 m 3 m -3 d -1 ) and PVDF-MEC (from 2.2 to 2.0 m 3 m -3 d -1 ) showed more stable performance compared to PEM-MECs (from 2.2 to 1.6 m 3 m -3 d -1 ) during 30 days of operation. Moreover, results of anodic microbial community analysis indicate that the growth of methanogens of NWC-MEC and PVDF-MEC was effectively inhibited in 30 days., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution., Competing Interests: Declaration of Competing Interest The authors affiliated to GoSilico declare that the software ChromX used in this study is a commercial product of GoSilico. The presented research does not depend on the usage of ChromX, the model can be implemented in any kind of modeling software., (Copyright © 2021. Published by Elsevier B.V.)

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera ® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera ® and Reditux™ displayed differences at the molecular level. MabThera ® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera ® . This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Numerous artificial micro- and nanomotors, as well as various swimmers have been inspired by living organisms that are able to move in a coordinated manner. Their cooperation has also gained a lot of attention because the resulting clusters are able to adapt to changes in their environment and to perform complex tasks. However, mimicking such a collective behavior remains a challenge. In the present work, magnesium microparticles are used as chemotactic swimmers with pronounced collective features, allowing the gradual formation of macroscopic agglomerates. The formed clusters act like a single swimmer able to follow pH gradients. This dynamic behavior can be used to spot localized corrosion events in a straightforward way. The autonomous docking of the swimmers to the corrosion site leads to the formation of a local protection layer, thus increasing corrosion resistance and triggering partial self-healing., (© 2021 Wiley-VCH GmbH.)

Mitochondrial Ca 2+ handling is accomplished by balancing Ca 2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca 2+ buffering in the matrix and Ca 2+ efflux mainly via Ca 2+ ion exchangers, such as the Na + /Ca 2+ exchanger (NCLX) and the Ca 2+ /H + exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca 2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca 2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca 2+ load via repetitive application of a finite CaCl 2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca 2+ efflux at different matrix Ca 2+ loads was revealed by inhibiting Ca 2+ uptake or reuptake with Ru360 after increasing number of CaCl 2 boluses. In Na + -free experimental buffer and with Ca 2+ uptake inhibited, the rate of Ca 2+ efflux and steady-state free matrix Ca 2+ [mCa 2+ ] ss increased as the number of administered CaCl 2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca 2+ buffering while maintaining a constant [mCa 2+ ] ss , decreased the rate of Ca 2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca 2+ buffering rate and decreased [mCa 2+ ] ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca 2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca 2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca 2+ efflux. The results indicate that Ca 2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca 2+ . A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca 2+ buffering system and the matrix free Ca 2+ concentration., (Copyright © 2020 Natarajan, Glait, Mishra, Stowe, Camara and Kwok.)

There is considerable interest in developing drugs and probes targeted to mitochondria in order to understand and treat the many pathologies associated with mitochondrial dysfunction. The large membrane potential, negative inside, across the mitochondrial inner membrane enables delivery of molecules conjugated to lipophilic phosphonium cations to the organelle. Due to their combination of charge and hydrophobicity, quaternary triarylphosphonium cations rapidly cross biological membranes without the requirement for a carrier. Their extent of uptake is determined by the magnitude of the mitochondrial membrane potential, as described by the Nernst equation. To further enhance this uptake here we explored whether incorporation of a carboxylic acid into a quaternary triarylphosphonium cation would enhance its mitochondrial uptake in response to both the membrane potential and the mitochondrial pH gradient (alkaline inside). Accumulation of arylpropionic acid derivatives depended on both the membrane potential and the pH gradient. However, acetic or benzoic derivatives did not accumulate, due to their lowered pK a . Surprisingly, despite not being taken up by mitochondria, the phenylacetic or phenylbenzoic derivatives were not retained within mitochondria when generated within the mitochondrial matrix by hydrolysis of their cognate esters. Computational studies, supported by crystallography, showed that these molecules passed through the hydrophobic core of mitochondrial inner membrane as a neutral dimer. This finding extends our understanding of the mechanisms of membrane permeation of lipophilic cations and suggests future strategies to enhance drug and probe delivery to mitochondria., (Copyright © 2020 Pala, Senn, Caldwell, Prime, Warrington, Bright, Prag, Wilson, Murphy and Hartley.)

In ionexchange chromatography, the pH gradient mode becomes more and more popular today for the analysis of therapeutic proteins as this mode can provide higher or alternative selectivity to the commonly used salt gradient mode. Ideally, a linear pH response is expected when performing linear gradients. However up to now, only a very few buffer systems have been developed and are commercially available which can perform nearly linear pH responses when flowing through a given column. It is also known that a selected buffer system (mobile phase) can work well on one column but can fail on other column. The goal of this study was to practically evaluate the effects that ionexchange columns (weak and strong exchangers) might have on effluent pH, when performing linear pH gradient separations of therapeutic monoclonal antibodies. To attain this objective, the pH was monitored on-line at the column outlet using a specific setup. To make comprehensive observations of the phenomenon, four different mobile phase conditions and five cation exchange columns (weak and strong exchangers) were employed. The obtained pH responses were systematically compared to responses measured in the absence of the columns. From this work, it has become clear that both the column and mobile phase can have significant effects on pH gradient chromatography and that their combination must be considered when developing a new method. Phase systems (column + mobile phase) providing linear pH responses are indeed the most suitable for separating mAbs with different isoelectric points and, with them, it is possible to elute mAbs across wide retention time ranges and with high selectivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The Agrio-Caviahue is a natural stream-lake system composed of the Upper Agrio River (UAR) -Lake Caviahue-Lower Agrio River (LAR). The system has a broad pH gradient from extremely acid in the hot spring to almost neutral pH only 60 km downstream the source, and varies as a function of the distance. The aim of this study was to analyze the dynamics of the dissolved organic matter (DOM) of this extreme system. For that matter, an absorbance and fluorescence-based characterization was performed on natural water samples and hydrophobic resin DOM extracts during different seasons in nine sampling stations. Between August and December, the hydrological connectivity is maximum due to precipitation and snowmelt, respectively. During these months, the stream that exits the lake governs the chemical characteristics downstream mainly in the period of high connectivity. In contrast, in the dry season when the flow of LAR is minimum, the two major affluents of this river influence its pH and DOM features, and deliver inputs of humified DOM from the wetlands where the tributaries are born. DOM was characterized by having low absorbance. The PARAFAC analysis of the fluorescent DOM (FDOM) validated three fluorescent components in the natural samples (with humic and non-humic features), two of which were also recorded in the extracts, meaning that no additional hydrophobic components were missed under the limit of detection of the fluorometer. The unique features of Agrio-Caviahue DOM resemble those found in DOM from Yellowstone hot springs, both acid and alkaline., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

At species' range edges, individuals often face novel environmental conditions that may limit range expansion until populations adapt. The potential to adapt depends on genetic variation upon which selection can act. However, populations at species' range edges are often genetically depauperate. One mechanism increasing genetic variation is reshuffling existing variation through sex. Sex, however, can potentially limit adaptation by breaking up existing beneficial allele combinations (recombination load). The gene swamping hypothesis predicts this is specifically the case when populations expand along an abiotic gradient and asymmetric dispersal leads to numerous maladapted dispersers from the range core swamping the range edge. We used the ciliate Tetrahymena thermophila as a model for testing the gene swamping hypothesis. We performed replicated range expansions in landscapes with or without a pH-gradient, while simultaneously manipulating the occurrence of gene flow and sexual versus asexual reproduction. We show that sex accelerated evolution of local adaptation in the absence of gene flow, but hindered it in the presence of gene flow. However, sex affected adaptation independently of the pH-gradient, indicating that both abiotic gradients and the biotic gradient in population density lead to gene swamping. Overall, our results show that gene swamping alters adaptation in life-history strategies.

Hematogenous tumor metastasis begins with the invasion and spread of primary tumor cells in the local tissue leading to intravasation. We hypothesized that tumor cells might actively migrate toward intratumor vessels with the extracellular metabolic gradient acting as a guiding cue. Here, we determined in vitro whether the extracellular gradient of pH can act as a cue for directional migration in MDA-MB-231 cells. Cell migration was determined by the wound-healing assay under gradients of extracellular pH (~0.2 units/mm) and oxygen concentration (~6% O 2 /mm) that were produced by a microfluidic device, gap cover glass (GCG). Without GCG, the migration of cells was spatially homogeneous; the same number of cells migrated to the rectangular wound space from the left and right boundaries. In contrast, when GCG generated pH/O 2 gradients across the wound space, the number of cells migrating to the wound space from the boundary with higher pH/O 2 values was considerably decreased, indicating a preferential movement of cells toward the region of higher pH/O 2 in the gradient. The addition of hepes in the extracellular medium abolished both the extracellular pH gradient and the directional cell migration under GCG. We conclude that relatively small gradients of pH in the extracellular medium compared to those found in Na + /H + exchanger-driven cell migration were sufficient to guide MDA-MB-231 cells. The directional cell migration as guided by the metabolic gradient could effectively elevate the probability of intravasation and, ultimately, hematogenous metastasis.

Paper-based analytical device (PAD) has received more and more attention in the field of point-of-care test (POCT) due to its low cost, portability and simple operation. Sensitivity and selectivity are two important aspects in clinical diagnostic analysis. However, low sensitivity of a PAD limits its wider application for POCT. Here we introduced a PAD that can clean and enrich the protein content from salty media with both electric field (E) and pH gradient (double E/pH gradients), with which 100-fold enrichment effect could be achieved within 70 s as demonstrated by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) from artificial urine media. With post staining of the protein stacking band with bromophenol blue (BPB), selective colorimetric detection of human serum albumin (HSA) was achieved simply with smartphone camera in the clinically significant range of 10-300 mg‧L -1 (R 2  = 0.99) with a limit of detection (LOD) of 4.9 mg‧L -1 . Detection of microalbuminuria (MAU) of diabetic patients was demonstrated with this method without difference (ɑ = 0.01) to that by the clinical method (immunoturbidimetry). This work demonstrated the potential of this PAD method in online sample pretreatment and detection of target component from complex physiological samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-β receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterization. Here, we report the development of a novel pH gradient-based anion-exchange chromatography method, which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterization of proteoforms at the intact protein level under native conditions. The analysis of four different proteins demonstrates outstanding chromatographic selectivity, while the mass spectra obtained are of excellent quality enabling the identification of proteoforms, including near isobaric variants, spanning 4 orders of magnitude in abundance. An in-depth analysis of ovalbumin from chicken egg white yields the identification and relative quantification of more than 150 different proteoforms, including fragmented and dimeric forms. More than 20 different ovalbumin charge variants together with their glycoform distributions are identified and quantified, many of which have not been reported previously.

In this second part of the series, recently commercialized cation exchanger stationary phases were systematically investigated for their capabilities to separate therapeutic monoclonal antibodies. It was demonstrated that the different combinations of stationary and mobile phases result in diverse retention, selectivity and efficiency. Hence, the whole phase system (combination of stationary and mobile phase) should be considered when developing a method. In addition, retention behavior is mAb dependent and should be individually optimized. Another interesting observation was that in cation exchange chromatographic separations of large proteins, the particle size of the columns probably impacts retention rather than efficiency, due to the non-porous particle structure - and therefore the higher specific surface area of smaller particles -. Particle size influences the specific surface area and total porosity. Therefore, columns packed with larger particles showed lower retention (when the ion exchanger group was the same e.g. strong exchanger sulfonic group) while no link was observed between efficiency and particle size. The retention, efficiency and selectivity of the studied columns were quite different and strongly dependent on the elution mode (i.e. salt gradient, pH gradient or combined salt/pH gradient mode). The columns can be considered to be complementary, suggesting that it is useful to have more than one type of column on hand while developing new charge variant assays. Moreover, this work shows that it is especially attractive to make use of short, narrow bore ion exchange columns that offer the possibility to perform 4-6 min long separations of both intact and partially digested antibodies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Silk sericin is a globular protein whose resistance against fouling is important for applications in biomaterials and water-purification membranes. Here it is shown how sericin generates a water-exclusion zone that may facilitate antifouling behavior. Negatively charged microspheres were used to mimic the surface charge and hydrophobic domains in bacteria. Immersed in water, regenerated silk sericin formed a 100-µm-sized exclusion zone (for micron-size foulants), along with a proton gradient with a decrease of >2 pH-units. Thus, when in contact with sericin, water molecules near the surface restructure to form a physical exclusionary barrier that might prevent biofouling. The decreased pH turns the aqueous medium unviable for neutrophilic bacteria. Therefore, resistance to biofouling seems explainable, among other factors, on the basis of water-exclusionary phenomena. Furthermore, sericin may play a role in triggering the fibroin assembly process by lowering the pH to the required value.

A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10 mm × 600 μm × 50 μm) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4-9 and pH 6.7-7.7 of 10 mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10 h rehydration, 5-14 h IEF and ca 10 h stain-imaging, the IEF array had the following merits: 25 min rehydration for sample loading, 4 min IEF, and 2 min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60 ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA 1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis., (Copyright © 2019. Published by Elsevier B.V.)

Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl. This two buffer system provides an equivalent or slightly better separation than the standard, mobile phases for therapeutic mAbs. It was also demonstrated that working with salt-mediated pH gradients, allows to extend the possibilities in method development, since the concentration of salt in the mobile phase has a significant impact on selectivity. Using HPLC modeling software (Drylab), it was possible to successfully develop CEX methods for authentic mAb samples within only 6 h, by optimizing the gradient steepness and salt concentration in the B eluent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Energy partitioning and plant growth are mediated in part by a type I H + -pumping pyrophosphatase (H + -PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H + -PPase from Arabidopsis also functions in "reverse mode" to synthesize PP i using the transmembrane H + gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon P i application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H + -PPase mutant lines ( vhp1-1 and fugu5-1 ) lacking the classical PP i -induced outward currents related to H + pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H + -PPase. Current amplitudes related to reverse-mode H + transport depended on the membrane potential, cytosolic P i concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H + -PPase (AVP1) demonstrated P i -dependent PP i synthase activity in the presence of a pH gradient. Our work establishes that a plant H + -PPase can operate as a PP i synthase beyond its canonical role in vacuolar acidification and cytosolic PP i scavenging. We propose that the PP i synthase activity of H + -PPase contributes to a cascade of events that energize plant growth., (© 2019 Scholz-Starke et al.)

Glyphosate is currently the most widely used herbicide in the world, yet screening of environmental waters for this chemical is limited by the need for specialized derivatization and measurement methods that can be tedious and time-consuming. In this work, we present a novel method for the detection and quantification at trace levels of glyphosate and aminomethylphosphonic acid (AMPA) in environmental water samples. The detection and quantification of the analytes was performed by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Chromatographic separation was achieved with an ion-exchange column and a pH-gradient elution of a solution of ammonium hydroxide and ammonium acetate. The limit of detection for glyphosate and AMPA was 0.25 μg L -1 and the limit of quantification was 0.5 μg L -1 with a 20-μL injection. The method was used to investigate the levels of glyphosate and AMPA in surface water samples from the Yarra River catchment area and urban constructed stormwater wetlands. The results indicate that at the time of sampling, no glyphosate or AMPA was present in the samples from the Yarra River catchment area (n = 10). However, glyphosate was detected above the limit of quantification in 33% of the wetland samples (n = 12), with concentrations ranging from 1.95 to 2.96 μg L -1 . Similarly, AMPA was quantified in 83% of the wetland samples, with concentrations ranging from 0.55 to 2.42 μg L -1 . To our knowledge, this is the first report of a pH-gradient LC-MS/MS method for glyphosate and AMPA analysis at ultratrace levels, with minimal sample processing, avoiding costly, time-consuming derivatization and preconcentration steps. Graphical abstract ᅟ.

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.

This study presents the first longitudinal measurement of the intracellular/extracellular pH gradient in a rat glioma model using noninvasive magnetic resonance imaging. The acid-base balance in the brain is tightly controlled by endogenous buffers. Tumors often express a positive pH gradient (pH i - pH e ) compared with normal tissue that expresses a negative gradient. Alkaline pH i in tumor cells increases activity of several enzymes that drive cellular proliferation. In contrast, acidic pH e is established because of increased lactic acid production and subsequent active transport of protons out of the cell. pH i was mapped using chemical exchange saturation transfer, whereas regional pH e was determined using hyperpolarized 13 C bicarbonate magnetic resonance spectroscopic imaging. pH i and pH e were measured at days 8, 12, and 15 postimplantation of C6 glioma cells into rat brains. Measurements were made in tumors and compared to brain tissue without tumor. Overall, average pH gradient in the tumor changed from -0.02 ± 0.12 to 0.10 ± 0.21 and then 0.19 ± 0.16. Conversely, the pH gradient of contralateral brain tissue changed from -0.45 ± 0.16 to -0.25 ± 0.21 and then -0.34 ± 0.25 (average pH ± 1 SD) Spatial heterogeneity of tumor pH gradient was apparent at later time points and may be useful to predict local areas of treatment resistance. Overall, the intracellular/extracellular pH gradients in this rat glioma model were noninvasively measured to a precision of ∼0.1 pH units at 3 time points. Because most therapeutic agents are weak acids or bases, a priori knowledge of the pH gradient may help guide choice of therapeutic agent for precision medicine., Competing Interests: Conflict of Interest: None reported.

Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pH 5.3 to pH 2.7 with 2 mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10-250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (p < 0.001) decreased in CCRF-CEM (3.68 vs 1.23 ng/mg protein), SK-N-BE(2) (1.98 vs 1.31 ng/mg protein) and CHLA-20 (2.06 vs 0.90 ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human activity is generating an excess of atmospheric CO 2 , resulting in what we know as ocean acidification, which produces changes in marine ecosystems. Until recently, most of the research in this area had been done under small-scale, laboratory conditions, using few variables, few species and few life cycle stages. These limitations raise questions about the reproducibility of the environment and about the importance of indirect effects and synergies in the final results of these experiments. One way to address these experimental problems is by conducting studies in situ, in natural areas where expected future pH conditions already occur, such as CO 2 vent systems. In the present work, we compile and discuss the latest research carried out in these natural laboratories, with the objective to summarize their advantages and disadvantages for research to improve these investigations so they can better help us understand how the oceans of the future will change., (© 2018 Elsevier Ltd. All rights reserved.)

It is generally accepted that the gastric mucus layer provides a protective barrier between the lumen and the mucosa, shielding the mucosa from acid and digestive enzymes and preventing autodigestion of the stomach epithelium. However, the precise mechanisms that contribute to this protective function are still up for debate. In particular, it is not clear what physical processes are responsible for transporting hydrogen protons, secreted within the gastric pits, across the mucus layer to the lumen without acidifying the environment adjacent to the epithelium. One hypothesis is that hydrogen may be bound to the mucin polymers themselves as they are convected away from the mucosal surface and eventually degraded in the stomach lumen. It is also not clear what mechanisms prevent hydrogen from diffusing back toward the mucosal surface, thereby lowering the local pH. In this work we investigate a physics-based model of ion transport within the mucosal layer based on a Nernst-Planck-like equation. Analysis of this model shows that the mechanism of transporting protons bound to the mucus gel is capable of reproducing the trans-mucus pH gradients reported in the literature. Furthermore, when coupled with ion exchange at the epithelial surface, our analysis shows that bicarbonate secretion alone is capable of neutralizing the epithelial pH, even in the face of enormous diffusive gradients of hydrogen. Maintenance of the pH gradient is found to be robust to a wide array of perturbations in both physiological and phenomenological model parameters, suggesting a robust physiological control mechanism. NEW & NOTEWORTHY This work combines modeling techniques based on physical principles, as well as novel numerical simulations to test the plausibility of one hypothesized mechanism for proton transport across the gastric mucus layer. Results show that this mechanism is able to maintain the extreme pH gradient seen in in vivo experiments and suggests a highly robust regulation mechanism to maintain this gradient in the face of dynamic lumen composition., (Copyright © 2017 the American Physiological Society.)

Energy-transfer reactions are the key for living open systems, biological chemical networking, and the development of life-inspired nanoscale machineries. It is a challenge to find simple reliable synthetic chemical networks providing a localization of the time-dependent flux of matter. In this paper, we look to photocatalytic reaction on TiO 2 from different angles, focusing on proton generation and introducing a reliable, minimal-reagent-consuming, stable inorganic light-promoted proton pump. Localized illumination was applied to a TiO 2 surface in solution for reversible spatially controlled "inorganic photoproton" isometric cycling, the lateral separation of water-splitting reactions. The proton flux is pumped during the irradiation of the surface of TiO 2 and dynamically maintained at the irradiated surface area in the absence of any membrane or predetermined material structure. Moreover, we spatially predetermine a transient acidic pH value on the TiO 2 surface in the irradiated area with the feedback-driven generation of a base as deactivator. Importantly we describe how to effectively monitor the spatial localization of the process by the in situ scanning ion-selective electrode technique (SIET) measurements for pH and the scanning vibrating electrode technique (SVET) for local photoelectrochemical studies without additional pH-sensitive dye markers. This work shows the great potential for time- and space-resolved water-splitting reactions for following the investigation of pH-stimulated processes in open systems with their flexible localization on a surface.

The mitochondrial electrochemical gradient (Δp), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide ( · O 2 - ) and H 2 O 2 overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced · O 2 - and H 2 O 2 production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased · O 2 - and H 2 O 2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial · O 2 - and H 2 O 2 generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced · O 2 - and H 2 O 2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K + ionophore used to dissipate ΔΨ, drastically diminished · O 2 - and H 2 O 2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also contributed to · O 2 - generation by normal mitochondria and that IR mediated ΔΨ impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ΔpH restricts electron flow, controls a limited amount of · O 2 - generation, and results in a more reduced myocardium; however, IR causes ΔpH impairment and prompts a more oxidized myocardium.

Chemiosmotic coupling - the harnessing of electrochemical ion gradients across membranes to drive metabolism - is as universally conserved as the genetic code. As argued previously in these pages, such deep conservation suggests that ion gradients arose early in evolution, and might have played a role in the origin of life. Alkaline hydrothermal vents harbour pH gradients of similar polarity and magnitude to those employed by modern cells, one of many properties that make them attractive models for life's origin. Their congruence with the physiology of anaerobic autotrophs that use the acetyl CoA pathway to fix CO 2 gives the alkaline vent model broad appeal to biologists. Recently, however, a paper by Baz Jackson criticized the hypothesis, concluding that natural pH gradients were unlikely to have played any role in the origin of life. Unfortunately, Jackson mainly criticized his own interpretations of the theory, not what the literature says. This counterpoint is intended to set the record straight., (© 2017 WILEY Periodicals, Inc.)

The present study signifies the development of a unique, optimized procedure for both selective determination and separation of different flavonoid nutraceuticals from edible plants. Totally ten different flavonoids were determined (HPLC-DAD) and isolated from five different plants using the developed process with a remarkable purity of 91-98% and recovery of 88-95%. Box-Behnken experimental design model yielded a optimized amount of 40.36 mg/g of AI extract (Pinostrobin) and 28.95 mg/g of AI extract (Baicalein) with a high correlation coefficient (0.98-0.99), indicating a good fit between the second order regression model and the experimental observations. The final purity of compounds through optimized process is 97.1% (Pinostrobin) and 93.5% (Baicalein) respectively. The optimized yields depicted a total recovery of 92% for pinostrobin, and 89% for Baicalein respectively. Thus, the developed process worked as a potential alternative which when statistically optimized results in a remarkable recovery of flavonoids from various plants. Being an environmentally friendly protocol the process could be useful in industrial separation of commercially important flavonoids widely applied in food industry.