Competitive Displacement Research Articles

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered “Crab”-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. “Crab”-like configuration of two catalytic peptides (“pincers”) flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for “single-pincer” conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled “Crabs” to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5′-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Antiparasitic Activity of Enterocin M and Durancin-like from Beneficial Enterococci in Mice Experimentally Infected with Trichinella spiralis.

Beneficial/probiotic strains protect the host from pathogens by competitive displacement and production of antibacterial substances, i.e., bacteriocins. The antiparasitic potential of bacteriocins/enterocins and their producing strains in experimental murine trichinellosis were tested as a new therapeutic strategy. Enterocin M and Durancin-like and their producers Enterococcus faecium CCM8558 and Enterococcus durans ED26E/7 were administered daily to mice that were challenged with Trichinella spiralis. Our study confirmed the antiparasitic effect of enterocins/enterococci, which reduced the number of adults in the intestine (Enterocin M-43.8%, E. faecium CCM8558-54.5%, Durancin-like-16.4%, E. durans ED26E/7-35.7%), suppressed the Trichinella reproductive capacity ex vivo (Enterocin M-61%, E. faecium CCM8558-74%, Durancin-like-38%, E. durans ED26E/7-66%), and reduced the number of muscle larvae (Enterocin M-39.6%, E. faecium CCM8558-55.7%, Durancin-like-15%, E. durans ED26E/7-36.3%). The direct effect of enterocins on Trichinella fecundity was documented by an in vitro test in which Durancin-like showed a comparable reducing effect to Enterocin M (40-60%) in contrast to the ex vivo test. The reducing activity of T.spiralis infection induced by Enterocin M was comparable to its strain E. faecium CCM8558; Durancin-like showed lower antiparasitic activity than its producer E. durans ED26E/7.

Multifunctional Self-Signaling nanoMIP and Its Application for a Washing-Free Assay of Human Angiotensin-Converting Enzyme 2.

Molecular imprinting techniques have attracted a lot of attention as a potential biomimetic technology, but there are still challenges in protein imprinting. Herein, multifunctional nanosized molecularly imprinted polymers (nanoMIPs) for human angiotensin-converting enzyme 2 (ACE2) were prepared by epitope imprinting of magnetic nanoparticles-anchored peptide (magNP-P) templates, which were further applied to construct a competitive displacement fluorescence assay toward ACE2. A cysteine-flanked dodecapeptide sequence was elaborately selected as an epitope for ACE2, which was immobilized onto the surface of magnetic nanoparticles and served as a magNP-P template for imprinting. During polymerization, fluorescent monomers were introduced to endow fluorescence responsiveness to the prepared self-signaling nanoMIPs. A competitive displacement fluorescence assay based on the nanoMIPs was established and operated in a washing-free manner, yielding a wide range for ACE2 (0.1-6.0 pg/mL) and a low detection limit (0.081 pg/mL). This approach offers a promising avenue in the preparation of nanoMIPs for macromolecule recognition and expands potential application of an MIP in the detection of proteins as well as peptides.

Exploring the molecular basis of tucatinib interaction with human serum albumin: A spectroscopic and computational analysis

This study delves into the kinetics of interaction among anticancer drug tucatinib (TCT) and human serum albumin (HSA), a pivotal protein that transports substances in the bloodstream of humans. Employing biophysical techniques alongside in-silico approaches, the TCT-HSA complex formation is substantiated. This binding interaction is unequivocally confirmed by the inverse relationship between temperature and Stern-Volmer constant (KSV) and the hyperchromicity in UV–visible spectra. The binding constant (Kb) value (5.34 x 104 M−1) indicates a moderate affinity between TCT and HSA, with thermodynamic analysis pointing towards stabilization through hydrogen bonds and hydrophobic and van der Waals interactions. Moreover, despite causing alterations in the vicinity of Tyr and Trp amino acids, the introduction of TCT to HSA confers a substantial protective effect against the thermal denaturation of the protein. Circular dichroism unveils a decrease in the content of alpha helices upon TCT binding. At the same time, CD thermal analysis hints at a marginal increase in the melting temperature of HSA, indicating heightened stability at elevated temperatures. Molecular docking and competitive displacement assays confirm that the preferred binding site for TCT is subdomain IIA (Sudlow's site I) of HSA. Molecular dynamics simulations emphasize the stability of the TCT-HSA complex. This work sheds light on a detailed characterization of the HSA-TCT interaction, elucidating binding mechanisms and their consequential impact on HSA structure and stability.

Structure of Aspergillus flavus populations associated with maize in Greece, Spain, and Serbia: Implications for aflatoxin biocontrol on a regional scale.

Aspergillus flavus is the most frequently identified producer of aflatoxins. Non-aflatoxigenic members of the A. flavus L strains are used in various continents as active ingredients of bioprotectants directed at preventing aflatoxin contamination by competitive displacement of aflatoxin producers. The current research examined the genetic diversity of A. flavus L strain across southern Europe to gain insights into the population structure and evolution of this species and to evaluate the prevalence of genotypes closely related to MUCL54911, the active ingredient of AF-X1. A total of 2173L strain isolates recovered from maize collected across Greece, Spain, and Serbia in 2020 and 2021 were subjected to simple sequence repeat (SSR) genotyping. The analysis revealed high diversity within and among countries and dozens of haplotypes shared. Linkage disequilibrium analysis indicated asexual reproduction and clonal evolution of A. flavus L strain resident in Europe. Moreover, haplotypes closely related to MUCL54911 were found to belong to the same vegetative compatibility group (VCG) IT006 and were relatively common in all three countries. The results indicate that IT006 is endemic to southern Europe and may be utilized as an aflatoxin mitigation tool for maize across the region without concern for potential adverse impacts associated with the introduction of an exotic microorganism.

Macrophage mannose receptor CD206 targeting of fluoride-18 labeled mannosylated dextran: A validation study in mice

PurposeAluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated mannosylated dextran derivative (Al[18F]F-NOTA-D10CM) is a new tracer for PET imaging. We report here on in vitro and in vivo validation of the tracer’s ability to target the macrophage mannose receptor CD206.MethodsFirst, the uptake of intravenously (i.v.) administered Al[18F]F-NOTA-D10CM was compared between wild-type (WT) and CD206−/− knockout (KO) mice. C57BL/6N mice were injected with complete Freund’s adjuvant (CFA) in the left hind leg and the uptake of Al[18F]F-NOTA-D10CM after i.v. or intradermal (i.d.) injection was studied at 5 and 14 days after CFA induction of inflammation. Healthy C57BL/6N mice were studied as controls. Mice underwent PET/CT on consecutive days with [18F]FDG, i.v. Al[18F]F-NOTA-D10CM, and i.d. Al[18F]F-NOTA-D10CM. After the last imaging, Al[18F]F-NOTA-D10CM was i.v. injected for an ex vivo biodistribution study and autoradiography of inflamed tissues. Blood plasma samples were analyzed using high-performance liquid chromatography. To evaluate the specificity of Al[18F]F-NOTA-D10CM binding, an in vitro competitive displacement study was performed on inflamed tissue sections using autoradiography. CD206 expression was assessed by immunohistochemical staining.ResultsCompared with WT mice, the uptake of Al[18F]F-NOTA-D10CM was significantly lower in several CD206−/− KO mice tissues, including liver (SUV 8.21 ± 2.51 vs. 1.06 ± 0.16, P < 0.001) and bone marrow (SUV 1.63 ± 0.37 vs. 0.22 ± 0.05, P < 0.0001). The uptake of i.v. injected Al[18F]F-NOTA-D10CM was significantly higher in inflamed ankle joint (SUV 0.48 ± 0.13 vs. 0.18 ± 0.05, P < 0.0001) and inflamed foot pad skin (SUV 0.41 ± 0.10 vs. 0.04 ± 0.01, P < 0.0001) than in the corresponding tissues in healthy mice. The i.d.-injected Al[18F]F-NOTA-D10CM revealed differences between CFA-induced lymph node activation and lymph nodes in healthy mice. Ex vivo γ-counting, autoradiography, and immunohistochemistry supported the results, and a decrease of ~ 80% in the binding of Al[18F]F-NOTA-D10CM in the displacement study with excess NOTA-D10CM confirmed that tracer binding was specific. At 60 min after i.v. injection, an average 96.70% of plasma radioactivity was derived from intact Al[18F]F-NOTA-D10CM, indicating good in vivo stability. The uptake of Al[18F]F-NOTA-D10CM into inflamed tissues was positively associated with the area percentage of CD206-positive staining.ConclusionThe uptake of mannosylated dextran derivative Al[18F]F-NOTA-D10CM correlated with CD206 expression and the tracer appears promising for inflammation imaging.Graphical abstract

Mononuclear η6-arene ruthenium(II) complexes with pyrazolyl-pyridazine ligands: synthesis, CT-DNA binding, reactivity towards glutathione, and cytotoxicity.

Organometallic η6-arene ruthenium(II) complexes with 3-chloro-6-(1H-pyrazol-1-yl)pyridazine (Ru1, Ru2, and Ru5) and 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (Ru3-4) N,N' heterocyclic and η6-arene (cymene (Ru1-4) or toluene (Ru 5)) have been synthesized. The ruthenium(II) complexes have common "three-legged piano-stool" pseudo-octahedral structures known for half-sandwich complexes. Evolution of their UV-Visible absorption spectra in PBS buffer or DMSO over 24h confirmed their good solvolysis stability. Titrations of the complexes with the calf thymus DNA (CT-DNA) were monitored using UV-Visible absorption and fluorescence spectroscopies. The complexes interact moderately with CT-DNA and their binding constants are in the order of 104M-1. Competitive binding of the complexes to a DNA-Hoechst 33,258 depicted competitive displacement of Hoechst from DNA's minor grooves. These complexes bind to glutathione forming GSH-adducts through S coordination by replacement of a halide, with the iodo-analogues having higher binding constants than the chloro-complexes. Cyclic voltammograms of the complexes exhibited one electron-transfer quasi-reversible process. Trends in the molecular docking data of Ru1-5/DNA were similar to those for DNA binding constants. Of the five, only Ru1, Ru3 and Ru5 showed some activity (moderate) against the MCF-7 breast cancer cells with IC50 values in the range of 59.2-39.9 for which Ru5 was the most active. However, the more difficult-to-treat cell line, MDA-MB 231 cell was recalcitrant to the treatment by these complexes.

Clinical Pharmacokinetics and Pharmacodynamics of Naloxone.

Naloxone is a World Health Organization (WHO)-listed essential medicine and is the first choice for treating the respiratory depression of opioids, also by lay-people witnessing an opioid overdose. Naloxone acts by competitive displacement of opioid agonists at the μ-opioid receptor (MOR). Its effect depends on pharmacological characteristics of the opioid agonist, such as dissociation rate from the MOR receptor and constitution of the victim. Aim of treatment is a balancing act between restoration of respiration (not consciousness) and avoidance of withdrawal, achieved by titration to response after initial doses of 0.4-2 mg. Naloxone is rapidly eliminated [half-life (t1/2) 60-120 min] due to high clearance. Metabolites are inactive. Major routes for administration are intravenous, intramuscular, and intranasal, the latter primarily for take-home naloxone. Nasal bioavailability is about 50%. Nasal uptake [mean time to maximum concentration (Tmax) 15-30 min] is likely slower than intramuscular, as reversal of respiration lag behind intramuscular naloxone in overdose victims. The intraindividual, interindividual and between-study variability in pharmacokinetics in volunteers are large. Variability in the target population is unknown. The duration of action of 1 mg intravenous (IV) is 2 h, possibly longer by intramuscular and intranasal administration. Initial parenteral doses of 0.4-0.8 mg are usually sufficient to restore breathing after heroin overdose. Fentanyl overdoses likely require higher doses of naloxone. Controlled clinical trials are feasible in opioid overdose but are absent in cohorts with synthetic opioids. Modeling studies provide valuable insight in pharmacotherapy but cannot replace clinical trials. Laypeople should always have access to at least two dose kits for their interim intervention.

Effect of glycation-induced concentration-dependent change in albumin structure and alteration in its binding capacity

Reducing sugars causes confirmatory alterations in albumin structure by the nonenzymatic glycation of the amino group of serum albumin. In this study, glucose and its hazardous metabolic products like glyoxal and methylglyoxal were incubated with bovine serum albumin (BSA). The confirmational changes in BSA molecule’s structure by glycating substances was investigated using a variety of spectroscopic methods, including deconvolutionFourier Transform Infra-red (FT-IR) spectroscopy, fluorescence spectroscopy, UV spectroscopy and circular dichroism (CD) spectroscopy. Dynamic fluorescence quenching was observed in the case of glucose, while static quenching was observed in the case of methyl glyoxal and glyoxal. Similarly, employing deconvolution FT-IR spectroscopy and CD spectroscopy for determination of change in secondary structures in terms signature of α-helix, β-turn, β-sheet and random coil modifications. Destabilization or unfolding of the albumin structure, due to the disruption of the hydrogen bonding pattern that stabilizes the albumin manifold, causes a 25-50% reduction in α-helix and a 2-fold increase in β-sheet and turns in glycated BSA. The competitive displacement interaction studies with warfarin were performed using the ultrafiltration technique and quantitative determination of free drug in ultrafiltrate using LC-MS/MS. The binding of carbamazepine (CBZ) or its active metabolite to proteins was unaffected by the glycation of BSA with glucose and methyl glyoxal. Nevertheless, with glyoxal-modified BSA, it changed the binding of selected analytes significantly. Based on in vitro observations and results, it could be anticipated that the serum CBZ concentration variation may be worsened in uncontrolled diabetes circumstances, with an overall variance of 30–40% possible. Communicated by Ramaswamy H. Sarma

Possible Involvement of Protein Binding Inhibition in Changes in Dexmedetomidine Concentration in Extracorporeal Circuits during Midazolam Use.

It was recently reported that the dexmedetomidine concentration within the extracorporeal circuit decreases with co-administration of midazolam. In this study, we investigated whether displacement of dexmedetomidine by midazolam from the binding site of major plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AAG), would increase levels of free dexmedetomidine that could be adsorbed to the circuit. Equilibrium dialysis experiments indicated that dexmedetomidine binds to a single site on both HSA and AAG with four times greater affinity than midazolam. Midazolam-mediated inhibition of the binding of dexmedetomidine to HSA and AAG was also examined. The binding of dexmedetomidine to these proteins decreased in the presence of midazolam. Competitive binding experiments suggested that the inhibition of binding by midazolam was due to competitive displacement at site II of HSA and due to non-competitive displacement at the site of AAG. Thus, our present data indicate that free dexmedetomidine displaced by midazolam from site II of HSA or from AAG is adsorbed onto extracorporeal circuits, resulting in a change in the dexmedetomidine concentration within the circuit.

Ibuprofen-Immobilized Thin Films: A Novel Approach to Improve the Clearance of Protein-Bound Uremic Toxins.

Chronic kidney disease (CKD), a pressing global health issue, affects millions and leads to end-stage renal disease (ESRD). Hemodialysis (HD) is a crucial treatment for ESRD, yet its limited efficiency in removing protein-bound uremic toxins (PBUTs) results in high morbidity and mortality rates. A high affinity of pharmaceutical drugs for human serum albumin (HSA) can be leveraged to compete effectively with PBUTs for the same HSA binding sites, thereby enabling them to be capable of displacing these toxins. One such drug is ibuprofen (IBF), known for its very high affinity for HSA and sharing the same binding site as indoxyl sulfate (IS). This study explores the development of IBF-immobilized cellulose acetate-based (CA-based) thin films. The films were created by reacting CA with IBF-modified silica precursors at varying concentrations. The presence of IBF in CA/TEOS/APTES-IBF-3 and CA/TEOS-IBF-25 films, containing 3 and 25 wt % IBF, respectively, was confirmed through 1H NMR spectra. Competitive displacement binding assays indicated that while the incorporation of 3 wt % IBF showed no significant enhancement in IS displacement, the 25 wt % IBF film increased the dialyzed IS by 1.3 when normalized to non-IBF films. Furthermore, there was a 1.2-fold decrease in the total percentage of IS, and the free percentage of IS increased 1.3 to 3.0 times. Although direct systemic infusion of IBF in HD patients achieves a 2.4 times higher removal of IS, it is impractical due to the risks it poses to ESRD patients. The IBF-immobilized films offer the advantage of localized binding, thus eliminating the need for systemic exposure. This innovative approach lays a foundation for developing more efficient HD membranes, aiming to address the challenging issue of PBUT elimination and potentially enhance the quality of life and treatment outcomes for ESRD patients.

Minimal impacts of invasive Scaevola taccada on Scaevola plumieri via pollinator competition in Puerto Rico

IntroductionScaevola taccada and Scaevola plumieri co-occur on shorelines of the Caribbean. Scaevola taccada is introduced in this habitat and directly competes with native dune vegetation, including S. plumieri, a species listed as locally endangered and threatened in Caribbean locations. This study addresses whether the invasive S. taccada also impacts the native S. plumieri indirectly by competing for pollinators and represents the first comparative study of insect visitation between these species.MethodsInsect visitation rates were measured at sites where species co-occur and where only the native occurs. Where species cooccur, insect visitors were captured, identified and analyzed for the pollen they carry. Pollen found on open-pollinated flowers was analyzed to assess pollen movement between the two species. We also compared floral nectar from each species by measuring volume, sugar content, and presence and proportions of amine group containing constituents (AGCCs).ResultsOur results demonstrate that both species share insect visitors providing the context for possible pollinator competition, yet significant differences in visitation frequency were not found. We found evidence of asymmetrical heterospecific pollen deposition in the native species, suggesting a possible reproductive impact. Insect visitation rates for the native were not significantly different between invaded and uninvaded sites, suggesting that the invasive S. taccada does not limit pollinator visits to S. plumieri. Comparisons of nectar rewards from the invasive and the native reveal similar volumes and sugar concentrations, but significant differences in some amine group containing constituents that may enhance pollinator attraction.ConclusionOur analysis finds no evidence for pollination competition and therefore S. taccada’s main impacts on S. plumieri are through competitive displacement and possibly through reproductive impacts as a consequence of heterospecific pollen deposition.

Competitive Displacement and Agonistic Character Displacement, or the Ghost of Interference Competition.

AbstractInterference competition can drive species apart in habitat use through competitive displacement in ecological time and agonistic character displacement (ACD) over evolutionary time. As predicted by ACD theory, sympatric species of rubyspot damselflies (Hetaerina spp.) that respond more aggressively to each other in staged encounters differ more in microhabitat use. However, the same pattern could arise from competitive displacement if dominant species actively exclude subordinate species from preferred microhabitats. The degree to which habitat partitioning is caused by competitive displacement can be assessed with removal experiments. We carried out removal experiments with three species pairs of rubyspot damselflies. With competitive displacement, removing dominant species should allow subordinate species to shift into the dominant species' microhabitat. Instead, we found that species-specific microhabitat use persisted after the experimental removals. Thus, the previously documented association between heterospecific aggression and microhabitat partitioning in this genus is most likely a product of divergence in habitat preferences caused by interference competition in the evolutionary past.

Exploring the binding mechanism of Pomalidomide drug with CT-DNA: Insights from multi-spectroscopic, molecular docking and simulation studies

Exploration of the interaction of a thalidomide analogue immunomodulatory and anti-angiogenic drug pomalidomide (POD) with calf thymus DNA (CT-DNA) was carried out using multi-spectroscopic (UV–Vis, fluorescence, and circular dichroism), molecular docking and metadynamics simulation studies in Tris–HCl buffer (pH 7.4). The results revealed that POD binds to the nucleic acid via groove binding mode. The proposed binding mode is strongly supported by competitive dye displacement assay using ethidium bromide (EB) and rhodamine B (RB) dyes, KI quenching studies and viscosity measurements. The thermodynamic parameters such as ∆Ho (60 kJ mol−1), ∆So (297 JK−1 mol−1) and ∆Go (-22 kJ mol−1) indicated that the POD/CT-DNA interaction is spontaneous and is stabilized predominantly by hydrophobic forces. The proposed groove binding mode of interaction of POD with CT-DNA was further confirmed by a computer-aided molecular docking studies and analysis of free energy surface contour derived from the molecular simulation study.

Tuning N-heterocyclic carbene wingtips to form electrochemically stable adlayers on metals.

Self-assembled monolayers (SAMs) are employed in electrochemical biosensors to passivate and functionalize electrode surfaces. These monolayers prevent the occurrence of undesired electrochemical reactions and act as scaffolds for coupling bioaffinity reagents. Thiols are the most common adlayer used for this application; however, the thiol-gold bond is susceptible to competitive displacement by naturally occurring solvated thiols in biological fluids, as well as to desorption under continuous voltage interrogation. To overcome these issues, N-heterocyclic carbene (NHC) monolayers have been proposed as an alternative for electrochemical biosensor applications due to the strong carbon-gold bond. To maximize the effectiveness of NHCs for SAMs, a thorough understanding of both the steric effects of wingtip substituents and NHC precursor type to the passivation of electrode surfaces is required. In this study, five different NHC wingtips as well as two kinds of NHC precursors were evaluated. The best performing NHC adlayers can be cycled continuously for four days (over 30 000 voltammetric cycles) without appreciably desorbing from the electrode surface. Benchmark thiol monolayers, in contrast, rapidly desorb after only twelve hours. Investigations also show NHC adlayer formation on other biosensor-relevant electrodes such as platinum and palladium.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that invivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

Design, synthesis and characterization of tetra substituted 2,3-dihydrothiazole derivatives as DNA and BSA targeting agents: advantages of the visible-light-induced multicomponent approach.

This report describes the visible-light-induced one-pot multicomponent regioselective synthesis of a series of 5-aroyl-3-((arylidene)amino)-2-((arylidene)hydrazono)-4-methyl-2,3-dihydrothiazoles as DNA and BSA targeting agents. The multicomponent condensation of thiocarbohydrazide and aldehydes with α-bromo-1,3-diketones, generated in situ by the bromination of unsymmetrical 1,3-diketones with NBS using white LED light as an environmental friendly source in the presence of EtOAc solvent furnished the titled 2,3-dihydrothiazole derivatives in excellent yields. The exact regioisomeric structure was identified unambiguously by employing multinuclear 2D-NMR spectroscopy [1H-13C] HMBC; [1H-13C] HMQC and [1H-15N] HMBC. Furthermore, the binding characteristics of the synthesized 2,3-dihydrothiazole derivatives were assessed with double-stranded calf-thymus DNA duplex (ct-DNA) and bovine serum albumin (BSA). Initial screening of all the synthesized 2,3-dihydrothiazole derivatives using various in silico techniques including molecular reactivity analysis, Lipinski rule and molecular docking, concluded 5-(4'-chlorobenzoyl)-3-((4''-methoxybenzylidene)amino)-2-(4'''-methoxybenzylidene)hydrazono)-4-methyl-2,3-dihydrothiazole derivative 6a as the most suitable compound for studying binding interaction with DNA and BSA. Additionally, to illustrate the ex vivo binding mode of 6a with DNA and BSA, several spectroscopic techniques viz. UV-visible, circular dichroism (CD), steady-state fluorescence and competitive displacement assays were carried out.

Quantifying Siglec-sialylated ligand interactions: a versatile 19F-T2 CPMG filtered competitive NMR displacement assay.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are integral cell surface proteins crucial for the regulation of immune responses and the maintenance of immune tolerance through interactions with sialic acids. Siglecs recognize sialic acid moieties, usually found at the end of N-glycan and O-glycan chains. However, the different Siglecs prefer diverse presentations of the recognized sialic acid, depending on the type of glycosidic linkage used to link to the contiguous Gal/GalNAc or sialic acid moieties. This fact, together with possible O- or N-substitutions at the recognized glycan epitope significantly influences their roles in various immune-related processes. Understanding the molecular details of Siglec-sialoglycan interactions is essential for unraveling their specificities and for the development of new molecules targeting these receptors. While traditional biophysical methods like isothermal titration calorimetry (ITC) have been utilized to measure binding between lectins and glycans, contemporary techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and biolayer interferometry (BLI) offer improved throughput. However, these methodologies require chemical modification and immobilization of at least one binding partner, which can interfere the recognition between the lectin and the ligand. Since Siglecs display a large range of dissociation constants, depending on the (bio)chemical nature of the interacting partner, a general and robust method that could monitor and quantify binding would be highly welcomed. Herein, we propose the application of an NMR-based a competitive displacement assay, grounded on 19F T2-relaxation NMR and on the design, synthesis, and use of a strategic spy molecule, to assess and quantify sialoside ligand binding to Siglecs. We show that the use of this specific approach allows the quantification of Siglec binding for natural and modified sialosides, multivalent sialosides, and sialylated glycoproteins in solution, which differ in binding affinities in more than two orders of magnitude, thus providing invaluable insights into sialoglycan-mediated interactions.

Spatial and temporal distribution of domestic and wild bee species in mustard with special concern of competitive displacement

Spatial and temporal distribution of domestic and wild bee species in mustard with special concern of competitive displacement

Interactions of Turmeric- and Curcumin-Functionalized Gold Nanoparticles with Human Serum Albumin: Exploration of Protein Corona Formation, Binding, Thermodynamics, and Antifibrillation Studies.

In order to better understand the bioavailability and biocompatibility of polyphenol-assisted surface-modified bioengineered nanoparticles in nanomedicine applications, here, we address a series of photophysical experiments to quantify the binding affinity of serum albumin toward polyphenol-capped gold nanoparticles. For this, two different gold nanoparticles (AuNPs) were synthesized via the green synthesis approach, where curcumin and turmeric extract act as reducing as well as capping agents. The size, surface charge, and surface plasmon bands of the AuNPs were highly affected by the adsorption of human serum albumin (HSA) during protein corona formation, which was investigated using dynamic light scattering (DLS), ξ-potential, ultraviolet-visible (UV-vis) spectroscopy, and transmission electron microscopy (TEM) measurements. Fluorescence-based methods, absorbance, and SERS experiments were carried out to evaluate the binding aspects of AuNPs with HSA. We found that the AuNPs show moderate binding affinity toward HSA (Kb ∼ 104 M-1), irrespective of the capping agents on the surface. Hydrophobic association, along with some contribution of electrostatic interaction, played a key role in the binding process. The binding interaction was more toward the subdomain IIA region of HSA, as indicated by the competitive displacement studies using site-specific binders (warfarin and flufenamic acid). Because of the large surface curvature of small-sized AuNPs, the secondary structural conformations of HSA were slightly altered, as revealed by circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, and surface-enhanced Raman scattering (SERS) measurements. Additionally, the findings of the binding interactions were re-evaluated using molecular dynamics (MD) simulation studies by determining the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and changes in the binding energy of HSA upon complexation with AuNPs. To determine the tentative evidence for pharmacokinetic administration, these biocompatible AuNPs were applied to inhibit the amyloid fibril formation of HSA and monitored by using the thioflavin T (ThT) assay, ANS fluorescence assay, fluorescence microscopic imaging, and FESEM. AuNPs were found to show better resistance toward fibrillation of the adsorbed protein.