Displaying publications 141 - 160 of 426 in total

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  1. Wang H, Liu K, He Z, Chen Y, Hu Z, Chen W, et al.
    Mar Pollut Bull, 2024 Apr;201:116198.
    PMID: 38428045 DOI: 10.1016/j.marpolbul.2024.116198
    Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.
  2. Hemagirri M, Chen Y, Gopinath SCB, Adnan M, Patel M, Sasidharan S
    Biogerontology, 2024 Apr 15.
    PMID: 38619670 DOI: 10.1007/s10522-024-10104-y
    Polyalthia longifolia is well-known for its abundance of polyphenol content and traditional medicinal uses. Previous research has demonstrated that the methanolic extract of P. longifolia leaves (PLME, 1 mg/mL) possesses anti-aging properties in Saccharomyces cerevisiae BY611 yeast cells. Building on these findings, this study delves deeper into the potential antiaging mechanism of PLME, by analyzing the transcriptional responses of BY611 cells treated with PLME using RNA-sequencing (RNA-seq) technology. The RNA-seq analysis results identified 1691 significantly (padj 
  3. Zhang S, Liu Q, Yang C, Li X, Chen Y, Wu J, et al.
    Reprod Biomed Online, 2024 Apr;48(4):103727.
    PMID: 38402677 DOI: 10.1016/j.rbmo.2023.103727
    RESEARCH QUESTION: Does type 1 diabetes mellitus (T1DM) affect reproductive health of female patients? What is the potential mechanism of reproductive dysfunction in female patients caused by T1DM?

    DESIGN: Preliminary assessment of serum levels of female hormones in women with or without T1DM. Then histological and immunological examinations were carried out on the pancreas, ovaries and uteri at different stages in non-obese diabetic (NOD) and Institute of Cancer Research (ICR) mice, as well as assessment of their fertility. A protein array was carried out to detect the changes in serum inflammatory cytokines. Furthermore, RNA-sequencing was used to identify the key abnormal genes/pathways in ovarian and uterine tissues of female NOD mice, which were further verified at the protein level.

    RESULTS: Testosterone levels were significantly increased (P = 0.0036) in female mice with T1DM. Increasing age in female NOD mice was accompanied by obvious lymphocyte infiltration in the pancreatic islets. Moreover, the levels of serum inflammatory factors in NOD mice were sharply increased with increasing age. The fertility of female NOD mice declined markedly, and most were capable of conceiving only once. Furthermore, ovarian and uterine morphology and function were severely impaired in NOD female mice. Additionally, ovarian and uterine tissues revealed that the differentially expressed genes were primarily enriched in metabolism, cytokine-receptor interactions and chemokine signalling pathways.

    CONCLUSION: T1DM exerts a substantial impairment on female reproductive health, leading to diminished fertility, potentially associated with immune disorders and alterations in energy metabolism.

  4. Kerishnan JP, Gopinath SC, Kai SB, Tang TH, Ng HL, Rahman ZA, et al.
    Int J Med Sci, 2016;13(6):424-31.
    PMID: 27279791 DOI: 10.7150/ijms.14475
    The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5(+)/6(+)) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients.
  5. Chang CY, Krishnan T, Wang H, Chen Y, Yin WF, Chong YM, et al.
    Sci Rep, 2014;4:7245.
    PMID: 25430794 DOI: 10.1038/srep07245
    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.
  6. Syahmi AR, Vijayarathna S, Sasidharan S, Latha LY, Kwan YP, Lau YL, et al.
    Molecules, 2010 Nov;15(11):8111-21.
    PMID: 21072022 DOI: 10.3390/molecules15118111
    Elaeis guineensis (Arecaceae) is widely used in West African traditional medicine for treating various ailments. An evaluation on the toxicity of extracts of this plant is crucial to support the therapeutic claims. The acute oral toxicity and brine shrimp lethality of a methanolic extract of this plant was tested. Oral administration of crude extract at the highest dose of 5,000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that E. guineensis is nontoxic. Normal behavioral pattern, clinical signs and histology of vital organs confirm this evidence. The E. guineensis extracts screened for toxicity against brine shrimp had 50% lethal concentration (LC₅₀) values of more than 1.0 mg/mL (9.00 and 3.87 mg/mL, at 6 and 24 h, respectively), confirming that the extract was not toxic. Maximum mortalities occurred at 100 mg/mL concentration while the least mortalities happened to be at 0.195 mg/mL concentration. The results of both tests confirm that E. guineensis is nontoxic and hence safe for commercial utilization.
  7. Lin YP, Luo Y, Chen Y, Lamers MM, Zhou Q, Yang XH, et al.
    BMC Infect Dis, 2016;16:102.
    PMID: 26932451 DOI: 10.1186/s12879-016-1379-4
    Dengue virus is transmitted by mosquito around the tropical and sub-tropical regions. There was a large-scale dengue epidemic in Guangdong province, China during 2014 and around fifty thousands dengue fever cases, including six deaths, have been reported. In this study, we aimed to understand the clinical characteristics of hospitalized patients with laboratory-confirmed dengue virus (DENV) infection and determined the origin of the virus from the outbreak.
  8. Torey A, Vijayarathna S, Jothy SL, Gothai S, Chen Y, Latha LY, et al.
    J Tradit Complement Med, 2016 Jan;6(1):97-104.
    PMID: 26870686 DOI: 10.1016/j.jtcme.2014.11.017
    Candida albicans has become resistant to the commercially available, toxic, and expensive anti-Candida agents that are on the market. These factors force the search for new antifungal agents from natural resources. Cassia spectabilis had been traditionally employed by healers for many generations. The possible mechanisms of the C. spectabilis leaf extract were determined by potassium leakage study and the effect of the extract on the constituents of the cell wall and enzymes as well as the morphological changes on C. albicans cells were studied along with cytotoxicity assays. The cytotoxicity result indicated that the extract is nontoxic as was clearly substantiated by a half maximal inhibitory concentration (IC50) value of 59.10 μg/mL. The treated cells (C. spectabilis extract) demonstrated potassium leakage of 1039 parts per million (ppm) compared to Amphotericin B (AmpB)-treated cells with a released potassium value of 1115 ppm. The effects of the extract on the cell wall proteins illustrated that there were three major types of variations in the expression of treated cell wall proteins: the presence of new proteins, the absence of proteins, and the amount of expressed protein. The activities of two enzymes, α-glucosidase and proteinase, were determined to be significantly high, thereby not fully coinciding with the properties of the antifungal reaction triggered by C. spectabilis. The morphology of C. albicans cells treated with the C. spectabilis extract showed that the cells had abnormalities and were damaged or detached within the microcolonies. Our study verifies C. spectabilis leaf extract as an effective anti-C. albicans agent.
  9. Kwan YP, Saito T, Ibrahim D, Al-Hassan FM, Ein Oon C, Chen Y, et al.
    Pharm Biol, 2015 Jul 8.
    PMID: 26154521
    Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments.
  10. Gopinath SC, Anbu P, Lakshmipriya T, Tang TH, Chen Y, Hashim U, et al.
    Biomed Res Int, 2015;2015:140726.
    PMID: 26180780 DOI: 10.1155/2015/140726
    Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed.
  11. Masaoka T, Hiraoka A, Okamoto S, Kodera Y, Cao LX, Lu DP, et al.
    Int J Hematol, 1999 Oct;70(3):190-2.
    PMID: 10561913
    The first cooperative study of the Asian Pacific bone marrow transplantation group included 75 patients with early leukemia who received human leukocyte antigen-matched sibling bone marrow transplants and were randomized into granulocyte colony-stimulating factor and control groups. The selected patients were registered from 10 centers in six countries and areas within Asia (Beijing, Taipei, Hong Kong, Japan, Korea, and Malaysia). The incidence of grades II-IV acute graft-vs.-host disease was 22.2%, and the 2-year survival rate was 62.7%. The period of protective isolation (27.1-66.7 days), period of hospitalization (38.6-130.5 days), and medical costs for 4 months (US $10,300-US $80,803) varied considerably. Good cooperation, i.e., low rate of protocol violation or rapid and precise presentation of case reports, was obtained.
  12. Chen Y, Ge H, Zheng Y, Zhang H, Li Y, Su X, et al.
    J Agric Food Chem, 2020 Jun 03;68(22):6190-6201.
    PMID: 32379465 DOI: 10.1021/acs.jafc.0c01250
    The present study aims to design a milk fat globule membrane (MFGM)-inspired structured membrane (phospholipid- and protein-rich) for microencapsulation of docosahexaenoic acid (DHA) oil. DHA-enriched oil emulsions were prepared using different ratios of sunflower phospholipid (SPL), proteins [whey protein concentrate (WPC), soy protein isolate (SPI), and sodium caseinate (SC)], and maltodextrin and spray-dried to obtain DHA microcapsules. The prepared DHA oil emulsions have nanosized particles. SPLs were found to affect the secondary structure of WPC, which resulted in increased exposure of the protein hydrophobic site and emulsion stability. SPL also reduced the surface tension and viscosity of the DHA oil emulsions. In vitro digestion of the spray-dried DHA microcapsules showed that they were able to effectively resist gastric proteolysis and protect their bioactivity en route to the intestine. The DHA microcapsules have a high lipid digestibility in the small intestine with a high DHA hydrolysis efficiency (74.3%), which is higher than that of commercial DHA microcapsules.
  13. Gopinath SC, Lakshmipriya T, Chen Y, Arshad MK, Kerishnan JP, Ruslinda AR, et al.
    Appl Microbiol Biotechnol, 2016 Aug;100(16):6955-69.
    PMID: 27350620 DOI: 10.1007/s00253-016-7686-2
    Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens.
  14. Zeng D, Zhou T, Ong WJ, Wu M, Duan X, Xu W, et al.
    ACS Appl Mater Interfaces, 2019 Feb 13;11(6):5651-5660.
    PMID: 30615433 DOI: 10.1021/acsami.8b20958
    Sub-5 nm ultra-fine iron phosphide (FeP) nano-dots-modified porous graphitic carbon nitride (g-C3N4) heterojunction nanostructures are successfully prepared through the gas-phase phosphorization of Fe3O4/g-C3N4 nanocomposites. The incorporation of zero-dimensional (0D) ultra-small FeP nanodots co-catalysts not only effectively facilitate charge separation but also serve as reaction active sites for hydrogen (H2) evolution. Herein, the strongly coupled FeP/g-C3N4 hybrid systems are employed as precious-metal-free photocatalysts for H2 production under visible-light irradiation. The optimized FeP/g-C3N4 sample displays a maximum H2 evolution rate of 177.9 μmol h-1 g-1 with the apparent quantum yield of 1.57% at 420 nm. Furthermore, the mechanism of photocatalytic H2 evolution using 0D/2D FeP/g-C3N4 heterojunction interfaces is systematically corroborated by steady-state photoluminescence (PL), time-resolved PL spectroscopy, and photoelectrochemical results. Additionally, an increased donor density in FeP/g-C3N4 is evidenced from the Mott-Schottky analysis in comparison with that of parent g-C3N4, signifying the enhancement of electrical conductivity and charge transport owing to the emerging role of FeP. The density functional theory calculations reveal that the FeP/g-C3N4 hybrids could act as a promising catalyst for the H2 evolution reaction. Overall, this work not only paves a new path in the engineering of monodispersed FeP-decorated g-C3N4 0D/2D robust nanoarchitectures but also elucidates potential insights for the utilization of noble-metal-free FeP nanodots as remarkable co-catalysts for superior photocatalytic H2 evolution.
  15. Li G, Tang H, Chen Y, Yin Y, Ogawa S, Liu M, et al.
    Mol Cell Endocrinol, 2018 02 05;461:1-11.
    PMID: 28801227 DOI: 10.1016/j.mce.2017.08.003
    The LHb expression is up-regulated during puberty in female zebrafish. However, the molecular mechanism underlying how LHb expression is regulated during puberty remains largely unknown. In this study, we found that the mRNA expression levels of lhb, fshb and cyp19a1b were up-regulated along with the puberty onset in zebrafish. Among the three nuclear estrogen receptors (nERs), the esr2b is the only type whose expression is significantly up-regulated during puberty onset in the pituitary. However, in situ hybridization results revealed that lhb mRNA was colocalized with esr1 and esr2a but not esr2b. Exposure to estradiol (E2) significantly stimulates LHb expression in both wild-type and kiss1-/-;kiss2-/-;gnrh3-/- triple knockout pubertal zebrafish. Moreover, exposure of cultured pituitary cells to E2 increased the LHb expression, indicating that the estrogenic effect on LHb expression could be acted at the pituitary level. Finally, we cloned and analyzed the promoter of lhb by luciferase assay. Our results indicated that the E2 responsive regions of lhb promoter for ERα and ERβ2 are identical, suggesting that ERα and ERβ2 could bind to the same half ERE region of the promoter of lhb, exhibiting a classical ERE-dependent pathway. In summary, we demonstrate that E2 could directly act on the pituitary level to stimulate LHb transcription during puberty in zebrafish.
  16. Zhao J, Chang W, Liu L, Xing X, Zhang C, Meng H, et al.
    J Immunol Methods, 2021 02;489:112942.
    PMID: 33333060 DOI: 10.1016/j.jim.2020.112942
    Highly sensitive and easy detection method for Alzheimer's disease (AD) with a suitable biomarker is mandatory for preventing the factors resulting from AD. This research reports a modified ELISA with graphene for the detection of AD biomarker amyloid beta (Aβ) oligomer. Gold nanoparticle (AuNP) conjugated aptamer was used as the capture probe and attached on ELISA-graphene oxide surface through the amine linker. Antibody was used as the detection molecule to reach the maximum detection of Aβ oligomer. Suitable level of APTMS (2%), size of AuNP (30 nm) and aptamer concentration (2 μM) were optimized. This sandwich pattern of aptamer-Aβ oligomer-antibody helps to reach the detection at 50 pM on the optimized ELISA surface and the control experiments in the absence of Aβ oligomer or anti-Aβ oligomer antibody did not show the significant optical detection at 492 nm, indicting the specific detection. Further, Aβ oligomer spiked artificial cerebrospinal fluid did not interfere the detection of Aβ oligomer, confirming the selective detection. This new and modified ELISA surface helps to reach the lower detection of Aβ oligomer and diagnose AD.
  17. Low LE, Wang Q, Chen Y, Lin P, Yang S, Gong L, et al.
    Nanoscale, 2021 Jun 17;13(23):10197-10238.
    PMID: 34027535 DOI: 10.1039/d1nr02127c
    Neurodegenerative disorder is an illness involving neural dysfunction/death attributed to complex pathological processes, which eventually lead to the mortality of the host. It is generally recognized through features such as mitochondrial dysfunction, protein aggregation, oxidative stress, metal ions dyshomeostasis, membrane potential change, neuroinflammation and neurotransmitter impairment. The aforementioned neuronal dysregulations result in the formation of a complex neurodegenerative microenvironment (NME), and may interact with each other, hindering the performance of therapeutics for neurodegenerative disease (ND). Recently, smart nanoassemblies prepared from functional nanoparticles, which possess the ability to interfere with different NME factors, have shown great promise to enhance the diagnostic and therapeutic efficacy of NDs. Herein, this review highlights the recent advances of stimuli-responsive nanoassemblies that can effectively combat the NME for the management of ND. The first section outlined the NME properties and their interrelations that are exploitable for nanoscale targeting. The discussion is then extended to the controlled assembly of functional nanoparticles for the construction of stimuli-responsive nanoassemblies. Further, the applications of stimuli-responsive nanoassemblies for the enhanced diagnosis and therapy of ND are introduced. Finally, perspectives on the future development of NME-tailored nanomedicines are given.
  18. Wong YL, Ramanathan A, Yuen KM, Mustafa WMW, Abraham MT, Tay KK, et al.
    PeerJ, 2021;9:e11548.
    PMID: 34178453 DOI: 10.7717/peerj.11548
    Background: Oral squamous cell carcinoma (OSCC) has increased in incidence from 1990 to 2017, especially in South and Southeast Asia. It is often diagnosed at an advanced stage with a poor prognosis. Therefore, early detection of OSCC is essential to improve the prognosis of OSCC. This study aims to identify the differentially expressed serum proteins as potential biomarkers for oral squamous cell carcinoma (OSCC).

    Methods: Comparative proteomics profiling of serum samples from OSCC patients, oral potentially malignant disorder (OPMD) patients, and healthy individuals were performed using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) (n = 60) and bioinformatics analysis. The enzyme-linked immunosorbent assay (ELISA) (n = 120) and immunohistochemistry (IHC) (n = 70) were used to confirm our findings.

    Results: The 2-DE analysis revealed that 20 differentially expressed proteins were detected in OPMD and OSCC (p 

  19. Gao M, Sun Y, Wang Q, Ma S, Guo X, Zhou L, et al.
    PMID: 34523748 DOI: 10.1002/bab.2254
    Nanomaterial on the sensing area elevates the biomolecular immobilization by its right orientation with a proper alignment, and zeolite is one of the suitable materials. In this research, the zeolite nanoparticles were synthesized using rice hush ash as the basic source and the prepared zeolite by the addition of sodium silicate was utilized to attach antibody as a probe on a gap-fingered dielectrode surface to identify the colon cancer biomarker, "colon cancer-secreted protein-2" (CCSP-2). Field Emission Scanning Electron Microscopy and Field Emission Transmission Electron Microscopy images confirmed the size of the nanoparticle to be ∼15 nm and the occurrence of silica and alumina. Zeolite was modified on the electrode surface through the amine linker, and then anti-CCSP-2 was attached by an aldehyde linker. On this surface, CCSP-2 was detected and attained the detection limit to be 3 nM on the linear regression curve with 3-5 nM of CCSP-2. Estimated by the determination coefficient of y = 2.3952x - 4.4869 and R2 = 9041 with 3δ (n = 3). In addition, control proteins did not produce the notable current response representing the specific sensing of CCSP-2. This research is suitable to identify CCSP-2 at a lower level in the bloodstream under the physiological condition of a colon cancer patient.
  20. Wang Z, Li P, Ma K, Chen Y, Campana M, Penfold J, et al.
    J Colloid Interface Sci, 2019 May 15;544:293-302.
    PMID: 30861434 DOI: 10.1016/j.jcis.2019.03.011
    The transition from monolayer to multilayer adsorption at the air-water interface in the presence of multivalent counterions has been demonstrated for a limited range of anionic surfactants which exhibit increased tolerance to precipitation in the presence of multivalent counterions. Understanding the role of molecular structure in determining the transition to surface ordering is an important aspect of the phenomenon. The focus of the paper is on the alkyl ester sulfonate, AES, surfactants; a promising group of anionic surfactants, with the potential for improved performance and biocompatibility. Neutron reflectivity measurements were made in aqueous solution and in the presence of NaCl, CaCl2, MgCl2 and AlCl3, for a range of alkyl ester sulfonate surfactants, in which the headgroup and alkyl chain geometries were manipulated. In the regions of monolayer adsorption changing the AES headgroup and alkyl chain geometries results in an increased saturation adsorption and in a more gradual decrease in the adsorption at low concentrations, consistent with a greater adsorption efficiency. Changing the AES headgroup and alkyl chain geometries also results in changes in the transition from monolayer adsorption to more ordered surface structures with the addition of AlCl3 and mixed multivalent electrolytes. A more limited surface layering is observed for the ethyl ester sulfonate, EES, with a C14 alkyl chain. Replacing the C14 alkyl chain with a C18 isostearic chain results in only monolayer adsorption. The results demonstrate the role and importance of the surfactant molecular structure in determining the nature of the surface adsorption in the presence of different electrolytes, and in the tendency to form extended surface multilayer structures.
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