pH Dependence Research Articles

UV/H2O2 produced degradation of 2,4-D and 4-CPA

2,4-dichlorophenoxyacetic (2,4-D) acid and 4-chlorophenoxyacetic acid (4-CPA), as widely utilized organochlorine pesticides, are normally detected in wastewater and atmosphere. Their harmfulness to the ecosystem cannot be ignored. UV/H2O2 is one of the most conventional advanced oxidation processes, which has remarkable advantages for the removal of refractory organic pollutants in wastewater. However, the reaction mechanisms and pathways of pollutants with reactive radicals are still unclear. In this study, the degradation efficacy and mechanisms during the photodegradation of 2,4-D and 4-CPA in UV/H2O2 processes were investigated by experiments and density functional theory calculations. The results showed that 2,4-D was more likely to be degraded by UV/H2O2 treatment than UV photolysis alone. In contrast, 4-CPA can be degraded effectively and rapidly under UV irradiation alone. By LC-MS analysis, the degradation products of 2,4-D and 4-CPA in the UV/H2O2 processes were identified and they were produced by hydroxylation and photolysis. Therefore, we speculated a competition mechanism presented between direct photolysis and indirect photolysis in the UV/H2O2 degradation of 2,4-D and 4-CPA. The OH-initiated reaction mechanisms of 2,4-D and 4-CPA in aqueous environments were investigated using quantum chemical methods SMD/M06–2X/6–311++G (3df, 2p)//SMD/M06–2X/6-311 + G (d,p). The pH dependence of the reaction mechanisms and rate constants in their degradation process induced by ·OH was also investigated. The calculation results showed that the ionic forms (2,4-D- and 4-CPA-) were more prone to react with ·OH in comparison to the corresponding molecular forms (2,4-D and 4-CPA). The total rate constants of 2,4-D and 4-CPA with OH increased with the pH from 1 to 10, and decreased with continued pH increase. The toxicities of 2,4-D, 4-CPA and their products were assessed by the QSAR-based toxicity evaluation software. The results showed that several products were more toxic than their parent compounds, such as 2,4-dichlorophenol, 3,5-dichlorocatechol, 4-chlorophenol and hydroquinone. This work can contribute to clarifying the environmental risks of 2,4-D and 4-CPA and offer a complete understanding for the photodegradation behavior of 2,4-D and 4-CPA in aqueous ecosystems.

A remarkable peroxidase-like behavior of the catalase KatA from the pathogenic bacteria Helicobacter pylori: The oxidation reaction with formate as substrate and the stabilization of an [Fe(IV) = O Trp•] intermediate assessed by multifrequency EPR spectroscopy

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9–285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e− oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H+/2e− oxidation in a cyt P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO2 to regulate the pH in the acidic environment.

Trace cisplatin adsorption by thiol-functionalized sponge (TFS) and Sn/SnO2-coated TFS: Adsorption study and mechanism investigation

To remove trace cisplatin from aqueous solution, commercial sponges were functionalized by esterification with 3-mercaptopropionic acid, followed by reduction with Na2S·9H2O or SnCl2·2H2O. The resulting thiol-functionalized sponges (TFSs), TFS_1 and TFS_2, were tested for the removal of cisplatin (235 μg L−1) achieving maximum removal of 95.5 ± 0.8% and 99.5 ± 0.1% respectively, which were significantly higher than the non-functionalized counterpart. The successful grafting of thiol groups, verified through FTIR, elemental analysis, SEM-EDS, and XPS characterization, facilitated Pt−S complexation during adsorption. The aqua-derivatives of cisplatin, formed through hydration, complexed with thiol sites through ligand displacement. Additionally, the presence of Sn/SnO2 coating on TFS_2 further enhanced the adsorption process. The rapid adsorption process conformed to pseudo-second-order kinetic model, involving both diffusion and chemisorption. While the Langmuir isotherm model generally described the monolayer adsorption behavior of cisplatin, the aggregation of Sn/SnO2 onto TFS_2 at 343 K introduced surface heterogeneity, rendering the Freundlich model a better fit for the adsorption isotherm. Differential pH dependence and the evaluation of mean free energy, derived from the Dubinin-Radushkevich isotherm model, indicated that cisplatin adsorption onto TFS_1 involved physisorption, including electrostatic attraction, while chemisorption predominated for TFS_2. Increasing the temperature notably promoted adsorption by facilitating the thermal-favored formation of Pt−S bonds.

Breaking the pH Limitation of Nanozymes: Mechanisms, Methods, and Applications.

Although nanozymes have drawn great attention over the past decade, the activities of peroxidase-like, oxidase-like, and catalase-like nanozymes are often pH dependent with elusive mechanism, which largely restricts their application. Therefore, a systematical discussion on the pH-related catalytic mechanisms of nanozymes together with the methods to overcome this limitation is in need. In this review, various nanozymes exhibiting pH-dependent catalytic activities are collected and the root causes for their pH dependence are comprehensively analyzed. Subsequently, regulatory concepts including catalytic environment reconstruction and direct catalytic activity improvement to break this pH restriction are summarized. Moreover, applications of pH-independent nanozymes in sensing, disease therapy, and pollutant degradation are overviewed. Finally, current challenges and future opportunities on the development of pH-independent nanozymes are suggested. It is anticipated that this review will promote the further design of pH-independent nanozymes and broaden their application range with higher efficiency.

Assessment of the physicochemical, functional and structural characteristics of a defatted flour from guamuchil (Pithecellobium dulce (Roxb.) seeds

Guamuchil tree is native of México and its fruits, which belong to Leguminosae family, contain seeds that are wasted. In this work, the physicochemical, functional and structural characterization of a defatted flour obtained from guamuchil seeds (DGuSF) was carried out. DGuSF had a low moisture (5.44 %) and lipid (2.91 %) contents and high protein (33.18 %) and carbohydrate (55.54 %) contents. Functional properties were highly dependence of pH, showing the highest values of 4.71 g H2O/g, 50 %, 84 %, 56 %, and 79 % for water binding capacity (WBCa), emulsifying capacity (EmA), emulsifying stability (EmS), foaming capacity (FoC), and foaming stability (FoS), respectively, at a value of 10. In the case of least gelling concentration (LGCo) the better value of 8 % was at pH 4, being obtained at this same condition the highest apparent viscosity (ηa) of 290.8 Pa·s. Values of 72.3 % and 40.5 % for in vitro protein enzymatic digestibility (IVPED) and in vitro antioxidant activity (IVVA), respectively, were also obtained for DGuSF, while their protein fractions presented molecular masses in the scale of ∼13–150 kDa. According to these results, guamuchil seeds could represent an alternative for the elaboration of DGuSF, to be applied as a food constituent because of its good compositional and functional characteristics.

Identification of a Catalytic Lysine Residue Conserved Among GHKL ATPases: MutL, GyrB, and MORC

DNA mismatch repair endonuclease MutL is a member of GHKL ATPase superfamily. Mutations of MutL homologs are causative of a hereditary cancer, Lynch syndrome. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although involvement of a basic residue had not been conceived in the catalytic mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is catalyzed by a residue with an alkaline pKa. Analyses of mutant aqMutLs showed that Lys79 is the catalytic residue, and the corresponding residues were confirmed to be critical for activities of human MutL homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations. Furthermore, it was confirmed that the catalytic lysine residue is conserved among DNA gyrases and microrchidia ATPases, other members of GHKL ATPases, indicating that the catalytic mechanism proposed here is applicable to these members of the superfamily.

Towards rational design in electrochemical denitrification by analyzing pH-dependence

Abstract A small fraction of NOx (<1%) always exists in CO2 feedstock (e.g. exhausted gas), which can significantly reduce the efficiency of CO2 electroreduction by ∼30%. Hence, electrochemical denitrification is the precondition of CO2 electroreduction. The pH effect is a key factor, which can be used to tune the selectivity between N2 and N2O production in electrochemical denitrification. However, there are extremely large controversy for many years on the origin of pH dependence in electrocatalysis. To this end, we present a new scheme to accurately model the pH dependence of electrochemical mechanism. An extremely small pH variation from pH 12.7 to pH 14 can be accurately reproduced for N2O production. More importantly, the obviously different pH dependence of N2 production, compared to N2O, can be attributed to a cascade path. In other words, the N2 was produced from the secondary conversion of the as-produced N2O molecule (the major product), instead of the original reactant NO. This is further supported by more than 35 experiments over varying catalysts (Fe, Ni, Pd, Cu, Co, Pt and Ag), partial pressures (20%, 50% and 100%) and potentials (from −0.2 to 0.2 V vs. reversible hydrogen electrode). All in all, these insights herein overturn our long-lasting views in NO electroreduction and inspire the rational design should steer from catalyst engineering to reactor optimization.

Amide proton transfer-weighted imaging with a short acquisition time based on a self B0 correction using the turbo spin echo-Dixon method: A phantom study

PurposeConventional amide proton transfer (APT)-weighted imaging requires a chemical exchange saturation transfer (CEST) sequence with multiple saturation frequency offsets and a B0 correction sequence, plus a long acquisition time that can be reduced by applying the conventional method using CEST images with seven radiation pulses (i.e., the seven-points method). For a further reduction of acquisition times, we propose fast two-dimensional (2D) APT-weighted imaging based on a self B0 correction using the turbo spin echo (TSE)-Dixon method. We conducted a phantom study to investigate the accuracy of TSE-Dixon APT-weighted imaging. MethodsWe prepared two types of phantoms with six samples for a concentrationdependent evaluation and a pH-dependent evaluation. APT-weighted images were acquired by the conventional, seven-points, and TSE-Dixon methods. Linear regression analyses assessed the dependence between each method's APT signal intensities (SIs) and the concentration or pH. We performed a one-way analysis of variance with Tukey's honestly significant difference post hoc test to compare the APT SIs among the three methods. The agreement of the APT SIs between the conventional and seven-points or TSE-Dixon methods was assessed by a Bland- Altman plot analysis. ResultsThe APT SIs of all three acquisition methods showed positive concentration dependence and pH dependence. No significant differences were observed in the APT SIs between the conventional and TSE-Dixon methods at each concentration. The Bland-Altman plot analyses showed that the APT SIs measured with the seven-points method resulted in 0.42% bias and narrow 95% limits of agreement (LOA) (0.93%–0.09%) compared to the conventional method. The APT SIs measured using the TSE-Dixon method showed 0.14% bias and similar 95% LOA (−0.33% to 0.61%) compared with the seven-points method. The APT SIs of all three methods showed positive pH dependence. At each pH, no significant differences in the APT SIs were observed among the methods. Bland-Altman plot analyses showed that the APT SIs measured with the seven-points method resulted in low bias (0.03%) and narrow 95% LOA (−0.30% to 0.36%) compared to the conventional method. The APT SIs measured by the TSE-Dixon method showed slightly larger bias (0.29%) and similar 95% LOA (from −0.15% to 0.72%) compared to those measured by the seven-points method. ConclusionThese results demonstrated that our proposed method has the same concentration dependence and pH dependence as the conventional method and the seven-points method. We thus expect that APT-weighted imaging with less influence of motion can be obtained in clinical examinations.

A Review on Biochar as an Adsorbent for Pb(II) Removal from Water

Heavy metal contamination in drinking water is a growing concern due to its severe health effects on humans. Among the many metals, lead (Pb), which is a toxic and harmful element, has the most widespread global distribution. Pb pollution is a major problem of water pollution in developing countries and nations. The most common sources of lead in drinking water are lead pipes, faucets, and plumbing fixtures. Adsorption is the most efficient method for metal removal, and activated carbon has been used widely in many applications as an effective adsorbent, but its high production costs have created the necessity for a low-cost alternative adsorbent. Biochar can be a cost-effective substitute for activated carbon in lead adsorption because of its porous structure, irregular surface, high surface-to-volume ratio, and presence of oxygenated functional groups. Extensive research has explored the remarkable potential of biochar in adsorbing Pb from water and wastewater through batch and column studies. Despite its efficacy in Pb removal, several challenges hinder the real application of biochar as an adsorbent. These challenges include variability in the adsorption capacity due to the diverse range of biomass feedstocks, production processes, pH dependence, potential desorption, or a leaching of Pb from the biochar back into the solution; the regeneration and reutilization of spent biochar; and a lack of studies on scalability issues for its application as an adsorbent. This manuscript aims to review the last ten years of research, highlighting the opportunities and engineering challenges associated with using biochar for Pb removal from water. Biochar production and activation methods, kinetics, adsorption isotherms, mechanisms, regeneration, and adsorption capacities with process conditions are discussed. The objective is to provide a comprehensive resource that can guide future researchers and practitioners in addressing engineering challenges.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3–8.1 to values for H44R/H48Q ADH of 8.0–9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds.Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.

Exploring the pH dependence of an improved PETase

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the Leaf-branch Compost Cutinase (LCC) enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0 while the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme towards a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity, and underscores the importance of studying the enzyme at the liquid/solid interface.

Exploring the sustainable synthesis pathway and comprehensive characterization of magnetic hybrid alumina nanoparticles phase (MHAl-NPsP) as highly efficient adsorbents and selective copper ions removal

This study introduces a novel method for producing magnetic hybrid alumina nanoparticles phase (MHAl-NPsP) tailored specifically for efficient copper (II) ion removal from wastewater. The synthesized MHAl-NPsP underwent comprehensive characterization, including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) revealing its rough and porous surface morphology, X-ray diffraction (XRD) analysis, transmission electron microscopy (TEM) analysis, BET analysis for surface area measurements, TGA analysis confirming high thermal stability, vibrating sample magnetometry (VSM) analysis confirming successful synthesis through detection of magnetic properties, and X-ray Photoelectron Spectroscopy (XPS) analysis. Remarkably, MHAl-NPsP demonstrated an exceptional adsorption capacity of 52.5 mg/g under optimized conditions of pH 3.5 and an initial copper concentration of 30 mg/L, surpassing previous results significantly. Detailed investigation into adsorption kinetics revealed a pseudo-second-order model, suggesting a predominant chemisorption mechanism. Moreover, analysis using the Langmuir isotherm model showed excellent fitting with an R² value of 0.994, indicating monolayer coverage as the primary adsorption mode. Notably, pH dependency studies indicated enhanced adsorption efficiency with decreasing pH levels, highlighting the significant role of electrostatic interactions. This study underscores the effectiveness and environmental sustainability of the green synthesis approach employed for MHAl-NPsP. Utilizing their magnetic properties, MHAl-NPsP facilitate easy separation and retrieval of adsorbed copper ions, making them highly promising for practical applications in wastewater treatment. The findings advocate for the development of eco-friendly adsorbents to tackle water pollution challenges, providing promising solutions for sustainable environmental remediation.

On the Substrate Scope of Dimethylsulfonium Propionate Lyases toward Dimethylsulfoxonium Propionate Derivatives

AbstractThe six dimethylsulfonium propionate (DMSP) lyases DddQ, DddW, DddP, DddY, DddK and DddL catalyze the elimination of dimethyl sulfide from DMSP and can also cleave the marine metabolite dimethylsulfoxonium propionate (DMSOP) to DMSO and acrylate. In this study the potency of all six enzymes for the conversion of four DMSOP analogs with longer alkyl chains that were synthesized in three steps from 3‐mercaptopropionic acid was investigated. For this purpose, the pH dependency of the enzyme reactions was determined, showing optimal conditions at pH 8 for all enzymes but DddP, for which an optimum of pH 6 was found. Efficient transformations were observed for DddQ, DddW, DddY and DddK, for which the Michaelis‐Menten kinetics were determined for all four substrate analogs. HPLC analysis of the dialkylsulfoxides obtained with the most efficient enzyme DddW revealed that these compounds were obtained with a low to moderate enantiomeric excess (up to 25 % ee), demonstrating that this enzyme has a preference for one of the enantiomers of the DMSOP analogs.

Experimental and theoretical unraveling of the electrocatalytic hydrodechlorination of chlorinated phenol using Pd promoted by oxygen vacancies in TiO2 array

The electrocatalytic dichlorination (EHDC) shows a promising potential to degrade chlorinated phenols (CPs). It is also a green technology in reducing the corresponding hazardous impact on the environment. In this study, we constructed a catalytic structure which was Pd on a TiO2 array with oxygen vacancies on carbon fibre paper substrate (Pd/TiO2/CFP) by a relatively mild route. Due to the presence of the oxygen vacancies, the resulting strong metal-support interaction (SMSI) effect has been identified which featured a redistribution of spatial charge in Pd/TiO2. The electron flow from the oxygen vacancies in the TiO2 to Pd facilitated the adsorption of Pd with 4-chlorophenol (4-CP) and endowed the Pd metal with an improved chemical reaction kinetics even at a low Pd loading. Experimentally, we studied the dependence of pH, concentration of 4-CP, and working potential on the reaction conditions of EHDC. The results showed that the conversion of 4-CP reached 100 % within 180 min with an apparent rate constant of 2.9 × 10-2 min−1 and the dominant product was phenol (P). The multi-cycle test illustrated that the Pd/TiO2/CFP electrode showed superior stability. The results also revealed that the comprehensive catalytic behavior was much better than those of the Pd/CFP and the TiO2/CFP electrodes. The density function theory (DFT) calculations indicated that 4-CP was more preferably absorbed on Pd/TiO2/CFP than Pd/CFP due to the SMSI assistance. This study provides a valuable guide in the preparation of precious metal-based electrodes for EHDC with a reduced loading and cost but without performance compromise.

PH Dependence of HSF1 trimerization is shaped by intramolecular interactions

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Abstract 2720: Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines

Abstract Soluble, tumor-derived, pro-tumorigenic (STP) cytokines have been shown to contribute to cancer cell proliferation and drug resistance. But therapeutic targeting of STP cytokines by monoclonal antibodies is limited by high concentrations and persistent production of antigen. We hypothesize that the ability of sweeping antibodies (swAbs) to actively clear soluble antigens will overcome these limitations. SwAbs differ from conventional antibodies via (1) pH-dependent binding of antigen, and (2) increased binding to the neonatal Fc receptor (FcRn). SwAbs facilitate clearance of soluble antigens by releasing antigen in the low pH environment of FcRn-expressing cells. SwAbs are then recycled via FcRn for repeated clearance of antigen.We are developing swAbs against four STP cytokines contributing to cancer cell proliferation, survival, migration or intra-tumoral angiogenesis: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and interleukin 16 (IL-16). We selected four existing neutralizing antibodies against these cytokines with available crystal structures and generated a second antibody targeting interleukin 16 with high affinity (clone 3B6; KD = 13 nM) de novo using phage display. We are using bio-layer interferometry and custom recycling/clearance assays for antibody characterization and pre-clinical validation. To introduce pH dependency into the four antibodies with crystal structures, we performed targeted histidine mutagenesis of residues at the interface with the respective target antigens. For the de novo-generated interleukin-16 antibody, we performed an unbiased histidine scanning approach by mutating residues across all six complementarity-determining regions (CDRs), which resulted in slight increases in pH sensitivity. We then performed a second round of mutagenesis of the most pH-sensitive 3B6 variants by replacing additional residues considered developability liabilities with histidine residues. We found that this approach significantly increased expression and affinity at pH 7 of the resulting 3B6 variants but only modestly increased pH sensitivity. In contrast, replacing a single residue in the heavy CDR1 in clone 14.1, a pre-clinical anti-IL-16 antibody, resulted in substantially increased pH sensitivity (KD(pH=7) = 2 nM; KD(pH<6) = n.b.). We are currently performing pH-sensitivity screenings for the three additional STP-specific antibodies. Our findings indicate that the ability to introduce pH sensitivity is largely dependent on the parental antibody and that some antibodies may not be amenable to this process. In addition, we show that histidine mutagenesis can be an effective approach to enhance expressibility and binding, while also increasing pH sensitivity. This project will answer the question whether anti-STP cytokine swAbs are an effective approach to treat patients with cancer. Citation Format: Jillian M. Baker, Nevil J. Singh, Tim Luetkens. Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2720.

Resveratrol-Loaded Polydimethylsiloxane-Silica Hybrid Materials: Synthesis, Characterization, and Antitumoral Activity.

In this work, hybrid materials within the polydimethylsiloxane-silica (PDMS-SiO2) system, synthesized via the sol-gel method, were developed and characterized for their potential to incorporate and release the bioactive compound resveratrol (RES). RES was incorporated into the materials with a high loading efficiency (>75%) using the rotary evaporator technique. This incorporation induced the amorphization of RES, resulting in enhanced solubility and in vitro release when compared to the free polyphenolic compound. The release profiles displayed pH dependence, exhibiting notably faster release at pH 5.2 compared to pH 7.4. The gradual release of RES over time demonstrated an initial time lag of approximately 4 h, being well described by the Weibull model. In vitro cytotoxicity studies were conducted on human osteosarcoma cells (MG-63), revealing a concentration-dependent decrease in cell viability for RES-loaded samples (for concentrations >50 µg mL-1).

Observations of cyanogen bromide (BrCN) in the global troposphere and their relation to polar surface O3 destruction

Abstract. Bromine activation (the production of Br in an elevated oxidation state) promotes ozone destruction and mercury removal in the global troposphere and commonly occurs in both springtime polar boundary layers, often accompanied by nearly complete ozone destruction. The chemistry and budget of active bromine compounds (e.g., Br2, BrCl, BrO, HOBr) reflect the cycling of Br and affect its environmental impact. Cyanogen bromide (BrCN) has recently been measured by iodide ion high-resolution time-of-flight mass spectrometry (I− CIMS), and trifluoro methoxide ion time-of-flight mass spectrometry (CF3O− CIMS) during the NASA Atmospheric Tomography Mission second, third, and fourth deployments (NASA ATom), and could be a previously unquantified participant in active Br chemistry. BrCN mixing ratios ranged from below the detection limit (1.5 pptv) up to as high as 36 pptv (10 s average) and enhancements were almost exclusively confined to the polar boundary layers in the Arctic winter and in both polar regions during spring and fall. The coincidence of BrCN with active Br chemistry (often observable BrO, BrCl and O3 loss) and high CHBr3/CH2Br2 ratios imply that much of the observed BrCN is from atmospheric Br chemistry rather than a biogenic source. Likely BrCN formation pathways involve the heterogeneous reactions of active Br (Br2, HOBr) with reduced nitrogen compounds, for example hydrogen cyanide (HCN/CN−), on snow, ice, or particle surfaces. Competitive reaction calculations of HOBr reactions with Cl−/Br− and HCN/CN− in solution, as well as box model calculations with bromine chemistry, confirm the viability of this formation channel and show a distinct pH dependence, with BrCN formation favored at higher pH values. Gas-phase loss processes of BrCN due to reaction with radical species are likely quite slow and photolysis is known to be relatively slow (BrCN lifetime of ∼ 4 months in midlatitude summer). These features, and the lack of BrCN enhancements above the polar boundary layer, imply that surface reactions must be the major loss processes. The fate of BrCN determines whether BrCN production fuels or terminates bromine activation. BrCN reactions with other halogens (Br−, HOCl, HOBr) may perpetuate the active Br cycle; however, preliminary laboratory experiments showed that BrCN did not react with aqueous bromide ion (< 0.1 %) to reform Br2. Liquid-phase reactions of BrCN are more likely to convert Br to bromide (Br−) or form a C–Br bonded organic species, as these are the known condensed-phase reactions of BrCN and would therefore constitute a loss of atmospheric active Br. Thus, further study of the chemistry of BrCN will be important for diagnosing polar Br cycling.

Vulnerability of Store-Operated Calcium Entry to Inhibitors and Microenvironment in Cells of Different Breast Cancer Subtypes.

The incidence and development of cancer are highly dependent on pathological disturbances in calcium homeostasis of the cell. One of the major pathways for calcium entry is store-operated calcium entry (SOCE), which functions in virtually all cell types. Changes in the expression level of the main proteins organizing SOCE are observed during the development of various cancer types, particularly breast cancer (BC). This leads to unique SOCE with characteristics individual for each type of BC and requires particular therapeutic approaches. In this study, we tested the sensitivity of SOCE in various BC cells to selective ORAI channel inhibitors and the less selective compounds Leflunomide and Teriflunomide, approved by the FDA for clinical use. We also analyzed the vulnerability of SOCE to the influence of factors typical of the tumor microenvironment: hypoxia and acidification. We have observed that the SOCE inhibitors Leflunomide and Teriflunomide suppress SOCE in the triple-negative BC cell line MDA-MB-231, but not in the luminal A BC cell line MCF-7. MDA-MB-231 cells also demonstrate higher pH dependence of SOCE compared to MCF-7 cells. In addition, the oxygen scavenger sodium dithionide also affects SOCE, stimulating it in MDA-MB-231 cells but inhibiting in MCF-7 cells. Overall, our data highlight the importance of considering the different sensitivities of various BC cell types to inhibitors and to microenvironmental factors such as hypoxia and acidification when developing targeted drugs.