Mechanistic Insights Research Articles

Imperatorin attenuates CCl4-induced cirrhosis and portal hypertension by improving vascular remodeling and profibrogenic pathways

BackgroundCirrhosis leads to portal hypertension (PHT), affecting survival with limited treatment options. This study investigated Imperatorin (IMP), a furanocoumarin with anti-inflammatory and hypotensive properties, for its therapeutic role and mechanisms in cirrhotic PHT. MethodsHepatic stellate cells (HSCs) inhibition by IMP was evaluated using LX-2 cell line. Rat cirrhosis was induced via CCl4 for 16 weeks. Experimental group were orally administered IMP (15/25 mg/kg/day) for 4 weeks. We subsequently examined portal pressure (PP), cirrhosis, inflammation, angiogenesis, and vascular remodeling. Network pharmacology was employed for mechanistic insights. ResultsIMP significantly inhibited the fibrogenesis in HSCs and suppressed cell viability. CCl4 exposure induced cirrhosis, inflammation, angiogenesis, vascular remodeling and PHT. IMP significantly reduced PP from 22.85 ± 3.88 mmHg to 6.67 ± 0.6 mmHg, diminished collagen deposition and pro-fibrotic factor expression, alleviated inflammation, and improved liver function. Vessel wall thickness in superior mesenteric arteries was restored, and intra-/extrahepatic angiogenesis was inhibited via VEGF and vWF. Furthermore, IMP induced sinusoidal vasodilation by upregulating eNOS and GCH1. Enrichment analysis indicated that IMP was involved in various biological processes associated with cirrhosis, such as the regulation of blood pressure, tissue remodeling, response to inflammation, and regulation of angiogenesis, etc. Additionally, IMP suppressed hepatic expression of TGF-β both in vitro and in vivo, which was further supported by KEGG analysis. ConclusionOur research demonstrated that IMP significantly mitigated cirrhosis PHT by reducing hepatic fibrosis and inflammation, curbing angiogenesis and vascular remodeling, and promoting vasodilation. This protective mechanism appears to be facilitated through the downregulation of TGF-β.

Unusual Cascade Reactions of 8-Acetoxy-6-hydroxymethyllimonene with Salicylic Aldehydes: Diverse Oxygen Heterocycles from Common Precursors.

Chiral oxygen-containing heterocyclic compounds are of great interest for the development of pharmaceuticals. Monoterpenes and their derivatives are naturally abundant precursors of novel synthetic chiral oxygen-containing heterocyclic compounds. In this study, acid catalyzed reactions of salicylic aldehydes with (-)-8-acetoxy-6-hydroxymethyllimonene, readily accessible from α-pinene, leads to the formation of chiral polycyclic products of various structural types. Three of the six isolated chiral heterocyclic products obtained from salicylic aldehyde contain previously unknown polycyclic ring types. Having carried out the reaction in the presence of Brønsted or Lewis acids (Amberlyst 15, trifluoromethanesulfonic acid, trifluoroacetic acid and boron trifluoride etherate) or aluminosilicates (montmorillonite K10, halloysite nanotubes), we found that the nature of products depends on the catalyst as well as the reaction conditions (reaction time, reactant ratio, presence or absence of solvent). Detailed mechanistic insight on the complex cascade reactions for product formation is provided with extensive experimental and quantum mechanical computational studies.

Propagation of transient explosive synchronization in a mesoscale mouse brain network model of epilepsy

Abstract Generalized epileptic attacks, which exhibit widespread disruption of brain activity, are characterized by recurrent, spontaneous, and synchronized bursts of neural activity that self-initiate and self-terminate through critical transitions. Here we utilize the general framework of explosive synchronization (ES) from complex systems science to study the role of network structure and resource dynamics in the generation and propagation of seizures. We show that a combination of resource constraint and adaptive coupling in a Kuramoto network oscillator model can reliably generate seizure-like synchronization activity across different network topologies, including a biologically derived mesoscale mouse brain network. The model, coupled with a novel algorithm for tracking seizure propagation, provides mechanistic insight into the dynamics of transition to the synchronized state and its dependence on resources; and identifies key brain areas that may be involved in the initiation and spatial propagation of the seizure. The model, though minimal, efficiently recapitulates several experimental and theoretical predictions from more complex models and makes novel experimentally testable predictions.

Dioxygen Activation by Gold(I)-Distorted Porphyrin Dinuclear Complexes.

Interactions between gold-based materials and dioxygen (O2) have motivated researchers to understand reaction mechanisms for O2 activation by homo- and heterogeneous gold catalysts. In this work, gold(I) porphyrin dinuclear complexes were synthesized with a saddle-distorted porphyrin ligand. The gold(I) porphyrin complexes showed unprecedented O2 activation in the presence of protic solvents to form gold(III) tetradentate porphyrin complexes. Mechanistic insights into the O2 activation by the gold(I) center were elucidated by spectroscopic measurements and theoretical calculations, revealing that dissociation of halides on the gold(I) center by alcohol solvents and hydrogen bonding of an N-H proton in the distorted porphyrin with dioxygen played important roles in establishing the unique reactivities of gold(I) complexes.

Exploration of Optimum Configurations of Electro-Assisted Anaerobic Digester for Sulfate-Reducing Conditions: Significance of Membranes and Mechanistic Insights

Exploration of Optimum Configurations of Electro-Assisted Anaerobic Digester for Sulfate-Reducing Conditions: Significance of Membranes and Mechanistic Insights

STAU1-mediated CNBP mRNA degradation by LINC00665 alters stem cell characteristics in ovarian cancer

BackgroundTo investigate the role of lncRNA LINC00665 in modulating ovarian cancer stemness and its influence on treatment resistance and cancer development.MethodsWe isolated ovarian cancer stem cells (OCSCs) from the COC1 cell line using a combination of chemotherapeutic agents and growth factors, and verified their stemness through western blotting and immunofluorescence for stem cell markers. Employing bioinformatics, we identified lncRNAs associated with ovarian cancer, with a focus on LINC00665 and its interaction with the CNBP mRNA. In situ hybridization, immunohistochemistry, and qPCR were utilized to examine their expression and localization, alongside functional assays to determine the effects of LINC00665 on CNBP.ResultsLINC00665 employs its Alu elements to interact with the 3’-UTR of CNBP mRNA, targeting it for degradation. This molecular crosstalk enhances stemness by promoting the STAU1-mediated decay of CNBP mRNA, thereby modulating the Wnt and Notch signaling cascades that are pivotal for maintaining CSC characteristics and driving tumor progression. These mechanistic insights were corroborated by a series of in vitro assays and validated in vivo using tumor xenograft models. Furthermore, we established a positive correlation between elevated CNBP levels and increased disease-free survival in patients with ovarian cancer, underscoring the prognostic value of CNBP in this context.ConclusionslncRNA LINC00665 enhances stemness in ovarian cancer by mediating the degradation of CNBP mRNA, thereby identifying LINC00665 as a potential therapeutic target to counteract drug resistance and tumor recurrence associated with CSCs.

LYSMD proteins promote activation of Rab32-family GTPases for lysosome-related organelle biogenesis.

Lysosome-related organelles (LROs) are specialized lysosomes with cell type-specific roles in organismal homeostasis. Dysregulation of LROs leads to many human disorders, but the mechanisms underlying their biogenesis are not fully understood. Here, we identify a group of LYSMD proteins as evolutionarily conserved regulators of LROs. In Caenorhabditis elegans, mutations of LMD-2, a LysM domain-containing protein, reduce the levels of the Rab32 GTPase ortholog GLO-1 on intestine-specific LROs, the gut granules, leading to their abnormal enlargement and defective biogenesis. LMD-2 interacts with GLO-3, a subunit of GLO-1 guanine nucleotide exchange factor (GEF), thereby promoting GLO-1 activation. Mammalian homologs of LMD-2, LYSMD1, and LYSMD2 can functionally replace LMD-2 in C. elegans. In mammals, LYSMD1/2 physically interact with the HPS1 subunit of BLOC-3, the GEF of Rab32/38, thus promoting Rab32 activation. Inactivation of both LYSMD1 and LYSMD2 reduces Rab32 activation, causing melanosome enlargement and decreased melanin production in mouse melanoma cells. These findings provide important mechanistic insights into LRO biogenesis and functions.

Harnessing transcriptomic signals for amyotrophic lateral sclerosis to identify novel drugs and enhance risk prediction

IntroductionAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. This study integrates common genetic association results from the latest ALS genome-wide association study (GWAS) summary statistics with functional genomic annotations with the aim of providing mechanistic insights into ALS risk loci, inferring drug repurposing opportunities, and enhancing prediction of ALS risk and clinical characteristics. MethodsGenes associated with ALS were identified using GWAS summary statistic methodology including SuSiE SNP-based fine-mapping, and transcriptome- and proteome-wide association study (TWAS/PWAS) analyses. Using several approaches, gene associations were integrated with the DrugTargetor drug-gene interaction database to identify drugs that could be repurposed for the treatment of ALS. Furthermore, ALS gene associations from TWAS were combined with observed blood expression in two external ALS case-control datasets to calculate polytranscriptomic scores and evaluate their utility for prediction of ALS risk and clinical characteristics, including site of onset, age at onset, and survival. ResultsSNP-based fine-mapping, TWAS and PWAS identified 118 genes associated with ALS, with TWAS and PWAS providing novel mechanistic insights. Drug repurposing analyses identified six drugs significantly enriched for interactions with ALS associated genes, though directionality could not be determined. Additionally, drug class enrichment analysis showed gene signatures linked to calcium channel blockers may reduce ALS risk, whereas antiepileptic drugs may increase ALS risk. Across the two observed expression target samples, ALS polytranscriptomic scores significantly predicted ALS risk (R2 = 5.1 %; p-value = 3.2 × 10−27) and clinical characteristics. ConclusionsFunctionally-informed analyses of ALS GWAS summary statistics identified novel mechanistic insights into ALS aetiology, highlighted several therapeutic research avenues, and enabled statistically significant prediction of ALS risk.

A Kinetic Model for Assessing Potential Nitrosamine Carcinogenicity.

Understanding the potential carcinogenic potency of nitrosamines is necessary to setting acceptable intake limits. Nitrosamines and the components that can form them are commonly present in food, water, cosmetics, and tobacco. The recent observation of nitrosamines in pharmaceuticals highlighted the need for effective methods to determine acceptable intake limits. Herein, we describe two computational models that utilize properties based upon quantum mechanical calculations in conjunction with mechanistic insights and established data to determine the carcinogenic potency of a variety of common nitrosamines. These models can be applied to experimentally untested nitrosamines to aid in the establishment of acceptable intake limits.

Mechanistic Insights into N2O5-Halide Ions Chemistry at the Air-Water Interface.

The activation of halogens (X = Cl, Br, I) by N2O5 is linked to NOx sources, ozone concentrations, NO3 reactivity, and the chemistry of halide-containing aerosol particles. However, a detailed chemical mechanism is still lacking. Herein, we explored the chemistry of the N2O5···X- systems at the air-water interface. Two different reaction pathways were identified for the reaction of N2O5 with X- at the air-water interface: the formation of XNO2 or XONO, along with NO3-. In the case of the Cl- system, the ClNO2 generation pathway is more favorable, while for the Br- and I- systems, the formation of BrONO and IONO is barrierless, making them the predominant products. Furthermore, the mechanisms of formation of X2 from XNO2 and XONO were also investigated. The high energy barriers of reactions and the high free energies of the products compared to those of the reactants indicate that ClNO2 is stable at the air-water interface. Contrary to the widely held belief regarding X2 producing from the reaction of XNO2 with X-, our calculations demonstrate that BrONO and IONO initially form stable BrONO···Br- and IONO···I- complexes, which then subsequently react with Br- and I- to form Br3- and I3-, respectively. Finally, Br3- and I3- decompose to form Br2 and I2. These findings have significant implications for experimental interpretation and offer new insights into halogen cycling in the atmosphere.

PHF12 regulates HDAC1 to promote tumorigenesis via EGFR/AKT signaling pathway in non-small cell lung cancer

BackgroundLung cancer stands as the second most prevalent malignant neoplasm worldwide. Addressing the underlying mechanisms propelling the progression of non-small cell lung cancer is of paramount importance. In this study, we have elucidated the pivotal role of PHF12 in this context.Materials and methodsWe harnessed clinical lung cancer tissue samples and non-small cell lung cancer cell lines to discern the expression pattern of PHF12. In vitro assays probing cell proliferation were conducted to substantiate the functional impact of PHF12. Furthermore, an in vivo Xenograft model was employed to dissect the role of PHF12. Employing ChIP assays and qRT-PCR, we delved into the intricate binding dynamics between PHF12 and HDAC1. Mechanistic insights into the PHF12-HDAC1 axis in lung cancer progression were pursued via RNA-seq and GSEA analyses.ResultsNotably, PHF12 exhibited a substantial upregulation within tumor tissue, concomitant with its correlation to HDAC1. The trilogy of cell proliferation assays, transwell assays, and the Xenograft model collectively underscored the promoting influence of PHF12 on lung cancer proliferation, both in vitro and in vivo. The ChIP assay unveiled the transcriptional regulatory role of PHF12 in governing HDAC1 expression. This correlation extended to both mRNA and protein levels. PHF12 promotes NSCLC progression through regulating HDCA1 expression. Intriguingly, the rescue of function within NSCLC cell lines post PHF12 knockdown was achievable through HDAC1 overexpression. Additionally, our findings unveiled the capacity of the PHF12-HDAC1 axis to activate the EGFR/AKT signaling pathway, thereby further corroborating its significance in lung cancer progression.ConclusionOur study identified PHF12 as an oncogenic role in lung cancer proliferation and migration for the first time. PHF12 transcriptionally regulate HDAC1 and activate EGFR/AKT signaling pathway in NSCLC progression. PHF12 may serve as an important target in lung cancer therapy.

Mechanistic Insights into Curvature Formation in Synthetic Vesicles.

The mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to dynamically induce asymmetry in an artificially bilayer membrane. The molecules were designed around an extended π-conjugated hydrophobic core with tertiary amine terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life-like materials.

Epilepsy due to potential loss of ATP6V1B2 function with mechanistic insight by a Drosophila Vha55 model.

ATP6V1B2 encodes the subunit of the vacuolar H+-ATPase, which is an enzyme responsible for the acidification of intracellular organelles and essential for cell signaling and neurotransmitter release. The aim of the study is to identify the correlation between ATP6V1B2 and epilepsy. Trio-exome sequencing was performed. Reverse Transcription-PCR and Quantitative real-time PCR analyses were carried out to determine whether this variant leads to nonsense-mediated mRNA decay (NMD). Drosophila models with knocked-down homologous genes of ATP6V1B2 were generated to study the causal relationship between the ATP6V1B2 and the phenotype of epilepsy. We described a 5-year-old male with a novel variant c.1163delT(p.Tyr389IlefsTer13) in ATP6V1B2, who presented with epilepsy. The expression level of the premature termination codon (PTC) transcript was normal in the patient, which indicated that NMD evasion existed in the PTC transcript. We generated an animal model using Drosophila to study the knock down effects of Vha55, which is the ATP6V1B2 ortholog in fly. The Vha55 knockdown flies show seizure-like behaviors and climbing defects. This study expands the variation spectrum of the ATP6V1B2 gene. Cross-species animal model demonstrates the causal relationship between ATP6V1B2 defect and epilepsy, and shed new insights into the disease mechanism caused by ATP6V1B2 LOF variants.

Biocatalysis enables the scalable conversion of biobased furans into various furfurylamines

Biobased furans have emerged as chemical building blocks for the development of materials because of their diverse scaffolds and as they can be directly prepared from sugars. However, selective, efficient, and cost-effective scalable conversion of biobased furans remains elusive. Here, we report a robust transaminase (TA) from Shimia marina (SMTA) that enables the scalable amination of biobased furanaldehydes with high activity and broad substrate specificity. Crystallographic and mutagenesis analyses provide mechanistic insights and a structural basis for understanding SMTA, which enables a higher substrate conversion. The enzymatic cascade process established in this study allows one-pot synthesis of 2,5-bis(aminomethyl)furan (BAMF) and 5-(aminomethyl)furan-2-carboxylic acid from 5-hydroxymethylfurfural. The biosynthesis of various furfurylamines, including a one-pot cascade reaction for BAMF generation using whole cells, demonstrates their practical application in the pharmaceutical and polymer industries.

Exerting pulling forces in fluids by directional disassembly of microcrystalline fibres.

Biomolecular polymerization motors are biochemical systems that use supramolecular (de-)polymerization to convert chemical potential into useful mechanical work. With the intent to explore new chemomechanical transduction strategies, here we show a synthetic molecular system that can generate forces via the controlled disassembly of self-organized molecules in a crystal lattice, as they are freely suspended in a fluid. An amphiphilic monomer self-assembles into rigid, high-aspect-ratio microcrystalline fibres. The assembly process is regulated by a coumarin-based pH switching motif. The microfibre crystal morphology determines the monomer reactivity at the interface, resulting in anisotropic etching. This effect exerts a directional pulling force on microscopic beads adsorbed on the crystal surface through weak multivalent interactions. We use optical-tweezers-based force spectroscopy to extract mechanistic insights into this process, quantifying a stall force of 2.3 pN (±0.1 pN) exerted by the ratcheting mechanism produced by the disassembly of the microfibres.

New insights into the cellular and molecular mechanisms of skeletal muscle fatigue: The Marion J. Siegman Award Lectureships.

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross-bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers, and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum (SR) Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g. accumulation of inorganic phosphate ions and H+) act to directly and indirectly (i.e. via decreased Ca2+ activation) inhibit myosin's force and motion generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases.

Mining electronics waste: Experimental and computational mechanistic insights into organic aqua regia as an extraction method

In the previous decade, organic aqua regia (OAR) was discovered as a mixture of pyridine and thionyl chloride that has been observed to surprisingly yet notably selectively dissolve precious metals, dissolution‐resistant gold. Given the novel combination is two common organic solvents, it has immense potential to contribute to a circular economy of metal recycling, economically and environmentally. The underlying mechanisms behind its metal dissolution and interesting properties are not well‐understood. Using both the original mixture and derivatives, the role of each OAR reagent was analyzed in the dissolution of gold to begin probing and illuminating the novel mechanism. Three mechanisms were proposed: radical based, oxidant and ligand, and Lewis acid and base. Computational means were used to propose possible mechanisms. Experiments were used to explicate the possible mechanisms. Discoveries from the original publication were used to verify assessments and suggest future areas of study for further confirmation. Based on the finding that 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO) does not significantly affect the amount of gold dissolved, the radical mechanism was dismissed. Findings from experiments replacing pyridine with its derivatives disproved the likelihood of the oxidant and ligand mechanism. Finally, with the support of proton nuclear magnetic resonance (1H NMR) and other characterization techniques, the Lewis acid and base mechanism was determined to be the most probable mechanism of OAR. The mechanistic findings reported herein will pave the way for continued understanding of the mechanism to be exploited and optimized for other metals and organometallic chemistry in the recycling of metals.

Investigating the Effects of Transcranial Alternating Current Stimulation on Cortical Oscillations and Network Dynamics

Transcranial electrical brain stimulation techniques like transcranial direct current (tDCS) and transcranial alternating current (tACS) have emerged as potential tools for treating neurological diseases by modulating cortical excitability. These techniques deliver small electric currents to the brain non-invasively through electrodes on the scalp. tDCS uses constant direct current which weakly alters the membrane voltage of cortical neurons, while tACS utilizes alternating current to target and enhance cortical oscillations, though the underlying mechanisms are not fully understood more specifically. To elucidate how tACS perturbs endogenous network dynamics, we simulated spiking neuron network models. We identified distinct roles of the depolarizing and hyperpolarizing phases in driving network activity towards and away from the strong nonlinearity provided by pyramidal neurons. Exploring resonance effects, we found matching tACS frequency to the network’s endogenous resonance frequency creates greater entrainment. Based on this, we developed an algorithm to determine the network’s endogenous frequency, phase, and amplitude, then deliver optimized tACS to entrain network oscillations. Together, these computational results provide mechanistic insight into the effects of tACS on network dynamics and could inform future closed-loop tACS systems that dynamically tune stimulation parameters to ongoing brain activity.

Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

Effect of surfactant and PAM on the settlement of kaolinite particles and its mechanism analysis

High concentrations of fine-grained clay minerals in tailings water are highly detrimental to environmental protection and water recycling. Using kaolinite as the study subject, this research investigates the effects of various cationic surfactants (DDA, DTAB, TTAB, CTAB) and flocculants (APAM, NPAM, CPAM) on the sedimentation of kaolinite particles. The study explores the impact of single agents, combined agents, and the sequence of their addition on kaolinite particle sedimentation. The results indicate that when using individual agents at low concentrations, CTAB outperforms TTAB, DTAB, and DDA, while APAM is more effective than NPAM and CPAM. The optimal performance is achieved with a CTAB concentration of 2×10<sup>-4</sup> mol/L and an APAM dosage of 20 mg/L. When combining agents, the best results are observed when CTAB is added before APAM. By fixing the APAM dosage at 20 mg/L and varying the CTAB concentration, the highest sedimentation rate and lowest turbidity are obtained at a CTAB concentration of 1.5×10<sup>-4</sup> mol/L. Mechanistic insights were obtained through aggregate imaging, area measurement, zeta potential testing, and contact angle testing. Cationic surfactants alter the surface properties of particles, reducing surface electronegativity and increasing hydrophobicity, which diminishes inter-particle repulsion and promotes aggregation, thereby reducing turbidity. Flocculants form larger flocs through adsorption and bridging, accelerating the sedimentation process. When flocculants and cationic surfactants are used together, the resulting flocs are more stable and larger, with an average floc area reaching 5017.6079 µm<sup>2</sup>, indicating a significant reduction in fine particles within the solution.