Mechanistic Insights Research Articles

Mechanistic insights into the foaming properties of potato proteins based on protein composition, colloidal state, and air-water interfacial properties

Potato proteins have been shown to be suitable alternatives for animal proteins in food foam formulations but their ability to form and stabilize foam is mechanistically ill-understood. The present study aimed to better understand the foaming properties of a protein fraction isolated from potato trimmings, a by-product from the potato industry. This was achieved by comparing the properties of these in-house isolated proteins to those of commercial and highly purified potato proteins. Protein isolates (53.8 ± 4.7 g/100 g dry weight) were produced from trimmings by alkaline solubilization (pH 9.0) and isoelectric precipitation (pH 4.0). The foaming properties of the in-house isolate at pH 3.0 and 7.0 were better than those of a blend of commercial potato proteins with similar relative amounts of patatin and protease inhibitors as the in-house isolate. Notably, this was the case despite the fact that more proteins were insoluble in the in-house isolate than in the commercial potato protein suspensions. Patatin and protease inhibitors were further shown to both contribute considerably to the formation and stabilization of foams. Interestingly, a beneficial effect on foam stability was found when patatin and protease inhibitors of various colloidal sizes were present. Additionally, impurities in the in-house isolates likely contributed to their superior foaming properties. In conclusion, a protein-rich fraction with good foaming properties can be recovered from potato trimmings. Mechanistic insights were gained into the foaming properties of potato protein (mixtures). This research also highlighted the importance of assessing protein colloidal state when investigating plant protein structure-function relationships.

In Vitro Assessment of Inhibitory Effects of Kinase Inhibitors on CYP2C9, 3A and 1A2: Prediction of Drug-Drug Interaction Risk with Warfarin and Direct Oral Anticoagulants

ObjectiveThis study aimed to evaluate the cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) potential of kinase inhibitors with warfarin and direct oral anticoagulants (DOACs). MethodsAn in vitro CYP probe substrate cocktail assay was used to study the inhibitory effects of fifteen kinase inhibitors on CYP2C9, 3A, and 1A2. Then, DDI predictions were performed using both mechanistic static and physiologically-based pharmacokinetic (PBPK) models. ResultsLinsitinib, masitinib, regorafenib, tozasertib, trametinib, and vatalanib were identified as competitive CYP2C9 inhibitors (Ki = 1.4, 1.0, 1.1, 3.8, 0.5, and 0.1 μM, respectively). Masitinib and vatalanib were competitive CYP3A inhibitors (Ki = 1.3 and 0.2 μM), and vatalanib noncompetitively inhibited CYP1A2 (Ki = 2.0 μM). Moreover, linsitinib and tozasertib were CYP3A time-dependent inhibitors (KI = 26.5 and 400.3 μM, kinact = 0.060 and 0.026 min−1, respectively). Only linsitinib showed time-dependent inhibition of CYP1A2 (KI = 13.9 μM, kinact = 0.018 min−1). Mechanistic static models identified possible DDI risks for linsitinib and vatalanib with (S)-/(R)-warfarin, and for masitinib with (S)-warfarin. PBPK simulations further confirmed that vatalanib may increase (S)- and (R)-warfarin exposure by 4.37- and 1.80-fold, respectively, and that linsitinib may increase (R)-warfarin exposure by 3.10-fold. Mechanistic static models predicted a smaller risk of DDIs between kinase inhibitors and apixaban or rivaroxaban. The greatest AUC increases (1.50-1.74) were predicted for erlotinib in combination with apixaban and rivaroxaban. Linsitinib, masitinib, and vatalanib were predicted to have a smaller effect on apixaban and rivaroxaban AUCs (AUCR 1.22-1.53). No kinase inhibitor was predicted to increase edoxaban exposure. ConclusionsOur results suggest that several kinase inhibitors, including vatalanib and linsitinib, can cause CYP-mediated drug-drug interactions with warfarin and, to a lesser extent, with apixaban and rivaroxaban. The work provides mechanistic insights into the risk of DDIs between kinase inhibitors and anticoagulants, which can be used to avoid preventable DDIs in the clinic.

Mechanistic insights into stepwise activation of malachite for enhancing surface reactivity and flotation performance

Mechanistic insights into stepwise activation of malachite for enhancing surface reactivity and flotation performance

Mechanistic Insights on Metformin and Arginine Implementation as Repurposed Drugs in Glioblastoma Treatment.

As the most common and aggressive primary malignant brain tumor, glioblastoma is still lacking a satisfactory curative approach. The standard management consisting of gross total resection followed by radiotherapy and chemotherapy with temozolomide only prolongs patients' life moderately. In recent years, many therapeutics have failed to give a breakthrough in GBM treatment. In the search for new treatment solutions, we became interested in the repurposing of existing medicines, which have established safety profiles. We focused on the possible implementation of well-known drugs, metformin, and arginine. Metformin is widely used in diabetes treatment, but arginine is mainly a cardiovascular protective drug. We evaluated the effects of metformin and arginine on total DNA methylation, as well as the oxidative stress evoked by treatment with those agents. In glioblastoma cell lines, a decrease in 5-methylcytosine contents was observed with increasing drug concentration. When combined with temozolomide, both guanidines parallelly increased DNA methylation and decreased 8-oxo-deoxyguanosine contents. These effects can be explained by specific interactions of the guanidine group with m5CpG dinucleotide. We showed that metformin and arginine act on the epigenetic level, influencing the foreground and potent DNA regulatory mechanisms. Therefore, they can be used separately or in combination with temozolomide, in various stages of disease, depending on desired treatment effects.

EIF4G2 Promotes Hepatocellular Carcinoma Progression via IRES-dependent PLEKHA1 Translation Regulation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

Insight of the molecular mechanism of inhibitors located at different allosteric sites regulating the activity of wild type and mutant KRAS (G12)

As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor as AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ . Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors.

Occurrence, Toxicodynamics, and Mechanistic Insights for Atrazine Degradation in the Environment

Occurrence, Toxicodynamics, and Mechanistic Insights for Atrazine Degradation in the Environment

Mechanistic Insight of High-Valent First-Row Transition Metal Complexes for Dehydrogenation of Ammonia Borane.

Designing an efficient and cost-effective catalyst for ammonia borane (AB) dehydrogenation remains a persistent challenge in advancing a hydrogen-based economy. Transition metal complexes, known for their C-H bond activation capabilities, have emerged as promising candidates for AB dehydrogenation. In this study, we investigated two recently synthesized C-H activation catalysts, 1 (CoIV-dinitrate complex) and 2 (NiIV-nitrate complex), and demonstrated their efficacy for AB dehydrogenation. Using density functional theory calculations and a detailed analysis, we elucidated the AB dehydrogenation mechanism of these complexes. Our results revealed that both complexes 1 and 2 can efficiently dehydrogenate AB at room temperature, although the abstraction of molecular H2 from these complexes requires slightly elevated temperatures. We utilized H2 binding free energy calculations to identify potentially active sites and observed that complex 2 can release two equivalents of H2 at a temperature slightly higher than room temperature. Furthermore, we investigated AB dehydrogenation kinetics and thermodynamics in iron (Fe)-substituted systems, complexes 3 and 4. Our results showed that the strategic alteration of the central metal atom, replacing Ni in complex 2 with Fe in complex 4, resulted in enhanced kinetics and thermodynamics for AB dehydrogenation in the initial cycle. These results underscore the potential of high-valent first-row transition metal complexes for facilitating AB dehydrogenation at room temperature. Additionally, our study highlights the beneficial impact of incorporating iron into such mononuclear systems, enhancing their catalytic activity.

Catalytic pyrolysis mechanism of lignin moieties driven by aldehyde, hydroxyl, methoxy, and allyl functionalization: the role of reactive quinone methide and ketene intermediates.

The catalytic pyrolysis of guaiacol-based lignin monomers, vanillin, syringol, and eugenol over commercial HZSM-5 has been investigated using operando Photoelectron Photoion Coincidence (PEPICO) spectroscopy to unveil the reaction mechanism by detecting reactive intermediates, such as quinone methides and ketenes, and products. Vanillin shares the decomposition mechanism with guaiacol due to prompt and efficient decarbonylation, which allows us to control this reaction leading to a phenol selectivity increase by switching to a faujasite catalyst and decreasing the Si/Al ratio. Syringol first demethylates to 3-methoxycatechol, which mainly dehydroxylates to o- and m-guaiacol. Ketene formation channels over HZSM-5 are less important here than for guaiacol or vanillin, but product distributionremains similar. C3 addition to guaiacol yields eugenol, which shows a more complex product distribution upon catalytic pyrolysis. By analogies to monomers with simplified functionalization, namely allylbenzene, 4-allylcatechol, and 4-methylcatechol, the eugenol chemistry could be fully resolved. Previously postulated reactive semi-quinone intermediates are detected spectroscopically, and their involvement opens alternative pathways to condensation and phenol formation. Allyl groups, produced by dehydroxylation of the β-O-4 bond, may not only decompose via C1/C2/C3 loss, but also cyclize to indene and its derivatives over HZSM-5. This comparably high reactivity leads to an unselective branching of the chemistry and to a complex product distribution, which is difficult to control. Indenes and naphthalenes are also prototypical coke precursors efficiently deactivating the catalyst. We rely on these mechanistic insights to discuss strategies to fine-tune process conditions to increase the selectivities of desired products by enhancing either vanillin and guaiacol or supressing eugenol yields from native lignin.

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

Mechanistic Insights into the Role of Elements in Ni‐Co‐P Catalysts for Electrochemical Conversion of 5‐Hydroxymethylfurfural to 2,5‐Furandicarboxylic Acid

Mechanistic Insights into the Role of Elements in Ni‐Co‐P Catalysts for Electrochemical Conversion of 5‐Hydroxymethylfurfural to 2,5‐Furandicarboxylic Acid

Development and characterization of a preclinical mouse model of alkali-induced limbal stem cell deficiency

PurposeLimbal stem cell deficiency (LSCD) secondary to ocular surface alkali burn is a blinding condition that features corneal conjunctivalization. Mechanistic insights into its pathophysiology are lacking. Here, we developed a mouse model that recapitulates human disease to comprehensively delineate the clinicopathological features of a conjunctivalized cornea. MethodsLSCD was induced in the right eyes of 6-8-week-old C57BL/6 male and female mice (n = 151) by topical administration of 0.25N sodium hydroxide on the cornea. Uninjured left eyes served as controls. Clinical, histological, phenotypic, molecular, and immunological assessments were performed at multiple time-points over 6-months. ResultsClinically, alkali burn caused persistent corneal opacity (p = 0.0014), increased punctate staining (p = 0.0002), and reduced epithelial thickness (p = 0.0082) compared to controls. Total LSCD was confirmed in corneal whole mounts by loss of K12 protein (p < 0.0001) and mRNA expression (p = 0.0090). Instead, K8+, K13+, K15+ and MUC5AC+ conjunctival epithelia prevailed. 20 % of injured corneas developed islands of K12+ epithelia, suggesting epithelial transdifferentiation. Squamous metaplasia was detected in 50 % of injured corneas. Goblet cell density peaked early post-injury but decreased over time (p = 0.0047). Intraepithelial corneal basal nerve density remained reduced even at 6-months post-injury (p = 0.0487). ConclusionsWe developed and comprehensively characterized a preclinical mouse model of alkali-induced LSCD. Understanding the pathophysiological processes that transpire on the ocular surface in LSCD is key to discovering, testing, and advancing biological and pharmacological interventions that can be dispensed prior to or in conjunction with stem cell therapy to rehabilitate the cornea and restore vision.

Novel Chemical Routes for Carbon Dioxide and Methane Production from Lignin Photodegradation: The Role of Environmental Free Radicals.

Sunlight irradiation significantly mediates plant litter's carbon dynamics and volatile carbon release in semi-arid and arid ecosystems. In this process, carbon loss is controlled by lignin, but the mechanisms of production of CO2 and CH4 during lignin photolysis are ambiguous. In this study, the photomineralization of plant litter and the lignocellulosic component collectively indicate that lignin is a major source of CO2 and CH4 emissions. Characterization and free radical analysis reveal that the production of CO2 is due to the oxidation and ring-opening reaction of the coniferyl alcohol unit, with the subsequent decarboxylation of carboxylic acid as an oxidation product. This reaction involves o-quinone formation by the reactions between O2, superoxide radical (O2·-), and persistent free radicals (PFRs)-bearing lignin. Of this, O2·- contributes to 43.2% of the photogenerated CO2, as a new pathway, derived from the electron transfer from PFRs to O2. Interestingly, photoinduced demethylation of the dimethoxybenzene-type compounds as the photolysis products of lignin results in a never-before-reported CH4 formation chemical route independent of that of O2. This mechanistic insight into the role of lignin in volatile carbon production from the irradiative plant litter will contribute to a deeper understanding of carbon balance in water-limited ecosystems.

Identification of a specific APOE transcript and functional elements associated with Alzheimer’s disease

BackgroundThe APOE gene is the strongest genetic risk factor for late-onset Alzheimer’s Disease (LOAD). However, the gene regulatory mechanisms at this locus remain incompletely characterized.MethodsTo identify novel AD-linked functional elements within the APOE locus, we integrated SNP variants with multi-omics data from human postmortem brains including 2,179 RNA-seq samples from 3 brain regions and two ancestries (European and African), 667 DNA methylation samples, and ChIP-seq samples. Additionally, we plotted the expression trajectory of APOE transcripts in human brains during development.ResultsWe identified an AD-linked APOE transcript (jxn1.2.2) particularly observed in the dorsolateral prefrontal cortex (DLPFC). The APOE jxn1.2.2 transcript is associated with brain neuropathological features, cognitive impairment, and the presence of the APOE4 allele in DLPFC. We prioritized two independent functional SNPs (rs157580 and rs439401) significantly associated with jxn1.2.2 transcript abundance and DNA methylation levels. These SNPs are located within active chromatin regions and affect brain-related transcription factor-binding affinities. The two SNPs shared effects on the jxn1.2.2 transcript between European and African ethnic groups.ConclusionThe novel APOE functional elements provide potential therapeutic targets with mechanistic insight into the disease etiology.

Updated aspects of alpha‐Solanine as a potential anticancer agent: Mechanistic insights and future directions

Updated aspects of alpha‐Solanine as a potential anticancer agent: Mechanistic insights and future directions

Multicomponent double Povarov reaction for julolidine: Synthesis and mechanistic insights

Julolidines constitute a class of N-heterocyclic molecules that behave as molecular rotors. Due to their fluorescent properties, they are applied in a wide range of technological fields, such as the production of solar cells, fluorescent biomarkers or bioimaging, and photoconductive materials. In the present work, we developed a methodology for the synthesis of julolidines through the one-pot tandem reaction-based application of multicomponent double Povarov reaction (MCPR) using BF3.OEt2 as the catalyst (25 mol%), star anise oil rich in trans-anethole as the substrate, and water as the solvent in reactions at room temperature. The julolidines were obtained with total yields ranging from 55 to 95 %. A mechanistic study allowed for the conclusion that these MCPRs take place through an ionic mechanism and also that released protons by the reaction between BF3.Et2O and water are involved in the catalytic process. After extracting the products from the reaction medium, the remaining aqueous phase containing the catalyst can be reused for up to six catalytic cycles with a minimal loss of catalytic efficiency. DFT calculations were conducted to investigate the catalytic cycle using BF3.Et2O and ZnCl2 as catalysts. The results indicate that the formation of iminium ions is a prerequisite for the progress of the reaction (MCPR). When BF3.Et2O and water are involved, the reaction proceeds via Brønsted catalysis, and the formation of iminium ions occurs in an exothermic and spontaneous process. However, when the ZnCl2 is used, the process is Lewis catalyzed to form the first iminium ion and is endergonic, shifting the equilibrium towards the reactants and inhibiting the cyclization.

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis

Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation–immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

Copper-Catalysed Synthesis of (E)-Allylic Organophosphorus Derivatives: A Low Toxic, Mild, Economical, and Ligand-Free Method.

Organophosphorus compounds are fundamental for the chemical industry due to their broad applications across multiple sectors, including pharmaceuticals, agrochemicals, and materials science. Despite their high importance, the sustainable and cost-effective synthesis of organophoshoryl derivatives remains very challenging. Here, we report the first successful regio- and stereoselective hydrophosphorylation of terminal allenamides using an affordable copper catalyst system. This reaction offers an efficient protocol for the synthesis of (E)-allylic organophosphorus derivatives from various types of P-nucleophiles, such as H-phosphonates, H-phosphinates, and secondary phosphine oxides. Key advantages of this ligand-free and atom-economic strategy include low toxicity of the Cu-based catalyst, cost effectiveness, mild reaction conditions, and experimental simplicity, making it competitive with methods that use toxic and expensive Pd-based catalysts. In an effort to comprehend this process, we conducted extensive DFT calculations on this system to uncover the mechanistic insights of this process.

Eggshell-enhanced biochar with in-situ formed CaO/Ca(OH)2 for efficient removal of Pb2+ and Cd2+ from wastewater: Performance and mechanistic insights

This study proposes a green and simple strategy for efficiently removing heavy metals from wastewater by activating biochar with eggshell and in-situ generating CaO/Ca(OH)2. Characterization results revealed that the composite carbon material (BCEg15) successfully incorporated CaO/Ca(OH)2 active sites compared to the raw biochar (BC). Additionally, eggshell activation increased the specific surface area and pore volume of BCEg15 by 4.36 and 6.80 times, respectively, compared to BC. These structural enhancements increased the maximum adsorption capacity of BCEg15 for Pb2+ and Cd2+ by 20.68 and 42 times, respectively, compared to BC. Importantly, the loading of CaO and Ca(OH)2 significantly enhanced the ion exchange and mineral precipitation capacities of the biochar. For instance, compared to BC, the adsorption capacities of BCEg15 attributed to ion exchange (Qe) for Pb2+ and Cd2+ rose by 403.12 mg/g and 197.65 mg/g, respectively, while the adsorption capacities attributed to mineral precipitation (Qp) increased by 19.60 times and 45.20 times, respectively. BCEg15 demonstrated excellent reusability, maintaining adsorption capacities of 461.18 mg/g for Pb2+ and 265.26 mg/g for Cd2+ after five cycles. Additionally, BCEg15 outperformed commercial activated carbon in treating heavy metals from desulfurization wastewater from coal-fired power plants, achieving a removal efficiency of 99.98 % compared to 48.22 %. The primary mechanisms by which BCEg15 removes Pb2+ and Cd2+ were identified as ion exchange, mineral precipitation, complexation with oxygen-containing functional groups, and interactions with π electrons. Density functional theory (DFT) was utilized to delve deeper into the microscopic mechanisms of adsorption. The findings showed that the O-top site of CaO exhibited the greatest stability for heavy metal adsorption. Electron density difference maps and partial density of states (PDOS) results revealed a denser electron cloud and greater orbital overlap between CaO and Pb2+ compared to Cd2+, suggesting a higher affinity and adsorption capacity for Pb2+, consistent with the experimental findings. This study presents an effective method for preparing adsorbents capable of removing Pb2+ and Cd2+ from wastewater and offers theoretical insights into the adsorption mechanisms of metal oxide-incorporated biochar.

Correction: Lopez-Gordo et al. Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights. Viruses 2024, 16, 442

There was an update regarding the affiliation for Kyle Chamberlain [...]