Surface Plasmon Resonance Studies Research Articles

Surface plasmon resonance, molecular docking, and molecular dynamics simulation studies of lysozyme interaction with tannic acid

AbstractLysozyme (LZM) is an important enzyme in medicine and industry. Tannic acid (TA) is used in brewing, wine industry, and as a food flavor enhancer. In nutritional and food science, LZM interacts with TA, notably in wine and saliva. This study aimed to investigate the binding interaction between LZM and TA using surface plasmon resonance, molecular docking, and molecular dynamics simulation. Chicken egg white lysozyme (CEWLZM) was applied as a model protein. Tri‐N‐acetylchitotriose (NAG3), the known inhibitor of CEWLZM, was used in the redocking experiments to determine the precise binding location within the complex. The average binding energies obtained from docking NAG3 and tannic acid to the target structure of CEWLZM were found to be −6.46 ± 0.05 kcal/mol and −7.52 ± 0.39 kcal/mol, respectively. The binding free energy of the CEWLZM‐TA complex was then calculated as −27.61 kcal/mol by MMPBSA based on MD simulation trajectories. The observed interactions between the ligands and the lysozyme structure were mainly driven by hydrophobic, van der Waals, and H‐bond interactions formed by the active site residues. MD simulations showed consistent and satisfactory binding distances between CEWLZM and TA throughout the analysis. SPR analysis was performed using 1X PBS buffer (pH 7.4) as coupling and running buffers, 30 μL/min as flow rate, and 2.5 mg/mL CEWLZM. Serial concentrations of TA (20–150 μM) were injected through immobilized CEWLZM, and the KD value of CEWLZM‐TA binding was obtained as 4.17 × 10−5 M. This study could enhance existing literature and pave the way for future research in food science and oral biology.

Design and investigation of celastrol‐peptide nanoassemblies and their binding interactions with superoxide dismutase 1 and its mutants

AbstractThe misfolding and aggregation of superoxide dismutase 1 (SOD1) and its mutants has been implicated in amyotrophic lateral sclerosis (ALS). In this study, we have created three peptide conjugates with the antioxidant pentacyclic terpene celastrol and examined their interactions with SOD1 and its mutants A4V and G93A. The peptides YYIVS, MPDAHL, and GSGGL are derived from natural sources and are known for their inherent antioxidant properties. Docking studies revealed that most conjugates showed strong binding with the metal binding and electrostatic loops as well as the β1, β5, and β6 hydrophobic core of SOD1. The conjugates were synthesized and self‐assembled into nanoassemblies. Surface plasmon resonance studies further confirmed the binding interactions of the nanoassemblies with the SOD1 proteins. The nanoassemblies were found to internalize into HEK293T cells. The HEK 293T cells were then transfected with GFP fused WT (Wild Type), A4V and G93A SOD1 mutants. Flow cytometry revealed that treatment with celastrol‐peptide nanoassemblies, affected the fluorescence of the SOD1 protein, implying their role in modulating SOD1, particularly for the mutants. N–Acetyl–Leu–Leu–Norleucinal (ALLN) induced SOD1 aggregation was also affected upon treatment with the nanoassemblies. These results suggest that the nanoassemblies may potentially modulate the activity and structure of SOD1.

Localized surface plasmon resonance and atomic force microscopy study of model lipid membranes and their interactions with amyloid and melatonin

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. The toxicity of amyloid to neuronal cell surfaces arises from interactions between small intermediate aggregates, namely amyloid oligomers, and the cell membrane. The nature of these interactions changes with age and disease progression. In our previous work, we demonstrated that both membrane composition and nanoscale structure play crucial roles in amyloid toxicity, and that membrane models mimicking healthy neuron were less affected by amyloid than model membranes mimicking AD neuronal membranes. This understanding introduces the possibility of modifying membrane properties with membrane-active molecules, such as melatonin, to protect them from amyloid-induced damage. In this study, we employed atomic force microscopy and localized surface plasmon resonance to investigate the protective effects of melatonin. We utilized synthetic lipid membranes that mimic the neuronal cellular membrane at various stages of AD and explored their interactions with amyloid-β (1–42) in the presence of melatonin. Our findings reveal that the early diseased membrane model is particularly vulnerable to amyloid binding and subsequent damage. However, melatonin exerts its most potent protective effect on this early-stage membrane. These results suggest that melatonin could act at the membrane level to alleviate amyloid toxicity, offering the most protection during the initial stages of AD.

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

Click chemistry can access unnatural N-acetylneuraminic acid–anchored β-cyclodextrin conjugates as potential anti-SARS-CoV-2 entry inhibitors

Multivalent N-acetylneuraminic acid (Neu5Ac) derivatives can be used to block virus entry into host cells by mimicking the natural receptors of the virus. However, the antiviral properties of unnatural Neu5Ac derivatives have not been extensively studied. Our study presents a facile and robust strategy for anchoring multiple unnatural Neu5Ac at the primary face of the β-cyclodextrin (β-CD) core using click chemistry. Further, we evaluated the antiviral activity of these conjugates and found that the monovalent and heptavalent unnatural Neu5Ac conjugates exhibited different antiviral activity against SARS-CoV-2 spike pseudovirus in hACE2-overexpressing BHK-21 cells without significant cytotoxicity (CC50 > 100 µM). The most potent inhibition was observed for conjugate 9c, with the half-maximal inhibitory concentration of 24.78 µM. The surface plasmon resonance study was also performed directly to elucidate the binding affinity of conjugate 9c to three CoVs spike proteins. The overall results suggest that the coupling of multiple unnatural Neu5Ac to the C6 carbons of β-CD with suitable linkage can bind the spike protein, thus blocking the entry of CoVs into host cells, implying that multivalent unnatural Neu5Ac derivatives are promising antiviral entry agents.

Properties of APTES-Modified CNC Films.

Sulfated cellulose nanocrystals' (CNCs') facile aqueous dispersibility enables producing films, fibers, and other materials using only water as a solvent but prevents using sulfated CNCs in applications that require water immersion. We report that modifying CNCs with 3-aminopropyl-triethoxysilane (APTES) via a simple, single-pot reaction scheme dramatically improves the hydrolytic stability of CNC films. The effects of APTES modification on CNCs' properties were studied using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force and optical microscopy, thermogravimetric analysis, dynamic light scattering, and ultimate analysis. Substituting a mere 12.6% of the CNCs' available hydroxyl groups with APTES dramatically increased the hydrolytic stability of shear cast films while only having minor impacts on their mechanical properties. In addition, quartz crystal microbalance with dissipation monitoring (QCMD) and multiparametric surface plasmon resonance (MP-SPR) studies showed that the CNC-APTES films also had a greater irreversible binding with carbofuran, a pesticide and emerging contaminant. These results highlight that APTES modification is a promising method for increasing the utility of sulfated CNCs in sensors, adsorbents, and other applications requiring water immersion.

Study of surface plasmon resonance with zirconium oxide multilayer coatings of silver and gold films: reflection polarization modes and sensitivity

We propose a surface plasmon resonance (SPR) sensor based on multilayer films with silver (Ag) and gold (Au) interfaces coated with a thin layer of zirconium oxide (ZrO2) in Kretschmann configuration. A dielectric layer of ZrO2 acts as the prism coating and the second layer is deposited after the metal film in the dielectric/metal/dielectric (DMD) system. In order to achieve the best design for the sensor, the dependence of the sensitivity on the angle of incidence and the thickness of the alternating layer is comprehensively explored. The polarization change results are shown with the angles Ψ and ∆ which are the traditional ellipsometry measurement angles of the optical properties of the DMD system. The advantage of Ag/ZrO2 multilayers over Au and Au/ZrO2 for SPR refractive index detection in terms of sensitivity, as well as the Ag/ZrO2 interface offers the possibility of studies with promising features for SPR users to generalize this type of interface. The proposed DMD sensor is expected to find application in biochemical sensing due to its resolution.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).

Preclinical characterization of CPL304110 as a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3 for gastric, bladder, and squamous cell lung cancer.

Fibroblast Growth Factor Receptors (FGFRs) are a family of receptor tyrosine kinases expressed on a plethora of cell membranes. They play crucial roles in both embryonic development and adult tissue functions. There is an increasing amount of evidence that FGFR-mediated oncogenesis is mainly related to gene amplification, activating mutations, or translocation in tumors of various histological types. Dysregulation of FGFRs has been implicated in a wide variety of neoplasms, such as bladder, gastric, and lung cancers. Given their functional significance, FGFRs emerge as promising targets for cancer therapy. Here, we introduce CPL304100, an innovative and highly potent FGFR1-3 kinase inhibitor demonstrating excellent in vitro biological activity. Comprehensive analyses encompassed kinase assays, cell line evaluations, PK/PD studies surface plasmon resonance studies, molecular docking, and in vivo testing in mouse xenografts. CPL304110 exhibited a distinctive binding profile to FGFR1/2/3 kinase domains, accompanied by a good safety profile and favorable ADMET parameters. Selective inhibition of tumor cell lines featuring active FGFR signaling was observed, distinguishing it from cell lines lacking FGFR aberrations (FGFR1, 2, and 3). CPL304110 demonstrated efficacy in both FGFR-dependent cell lines and patient-derived tumor xenograft (PDTX) in vivo models. Comparative analyses with FDA-approved FGFR inhibitors, erdafitinib and pemigatinib, revealed certain advantages of CPL304110 in both in vitro and in vivo assessments. Encouraging preclinical results led the way for the initiation of a Phase I clinical trial (01FGFR2018; NCT04149691) to further evaluate CPL304110 as a novel anticancer therapy.

Platanosides from Platanus × acerifolia: New molecules, SAR, and target validation of a strong lead for drug-resistant bacterial infections and the associated sepsis

Three undescribed (1–3) and nine known (4–12) platanosides were isolated and characterized from a bioactive extract of the May leaves of Platanus × acerifolia that initially showed inhibition against Staphylococcus aureus. Targeted compound mining was guided by an LC-MS/MS-based molecular ion networking (MoIN) strategy combined with conventional isolation procedures from a unique geographic location. The novel structures were mainly determined by 2D NMR and computational (NMR/ECD calculations) methods. Compound 1 is a rare acylated kaempferol rhamnoside possessing a truxinate unit. 6 (Z,E-platanoside) and 7 (E,E-platanoside) were confirmed to have remarkable inhibitory effects against both methicillin-resistant S. aureus (MIC: ≤ 16 μg/mL) and glycopeptide-resistant Enterococcus faecium (MIC: ≤ 1 μg/mL). These platanosides were subjected to docking analyses against FabI (enoyl-ACP reductase) and PBP1/2 (penicillin binding protein), both of which are pivotal enzymes governing bacterial growth but not found in the human host. The results showed that 6 and 7 displayed superior binding affinities towards FabI and PBP2. Moreover, surface plasmon resonance studies on the interaction of 1/7 and FabI revealed that 7 has a higher affinity (KD = 1.72 μM), which further supports the above in vitro data and is thus expected to be a novel anti-antibacterial drug lead.

Comparative Analysis of Inhibitor Binding to Peroxiredoxins from Candidatus Liberibacter asiaticus and Its Host Citrus sinensis.

The peroxiredoxins (Prxs), potential drug targets, constitute an important class of antioxidant enzymes present in both pathogen and their host. The comparative binding potential of inhibitors to Prxs from pathogen and host could be an important step in drug development against pathogens. Huanglongbing (HLB) is a most devastating disease of citrus caused by Candidatus Liberibacter asiaticus (CLa). In this study, the binding of conoidin-A (conoidin) and celastrol inhibitor molecules to peroxiredoxin of bacterioferritin comigratory protein family from CLa (CLaBCP) and its host plant peroxiredoxin from Citrus sinensis (CsPrx) was assessed. The CLaBCP has a lower specific activity than CsPrx and is efficiently inhibited by conoidin and celastrol molecules. The biophysical studies showed conformational changes and significant thermal stability of CLaBCP in the presence of inhibitor molecules as compared to CsPrx. The surface plasmon resonance (SPR) studies revealed that the conoidin and celastrol inhibitor molecules have a strong binding affinity (KD) with CLaBCP at 33.0µM, and 18.5µM as compared to CsPrx at 52.0µM and 61.6µM, respectively. The docked complexes of inhibitor molecules showed more structural stability of CLaBCP as compared to CsPrx during the run of molecular dynamics-based simulations for 100ns. The present study suggests that the conoidin and celastrol molecules can be exploited as potential inhibitor molecules against the CLa to manage the HLB disease.

Design and sustainable synthesis of small mannose-based glycodendrons as ligands for HIV-1 envelope protein gp120: Toward an explanation for their binding

On the basis of the interesting properties that glucuronic acid-based glycodendrimers and glycodendrons show against Dengue virus through their interaction with its envelope protein, herein we describe the design, sustainable synthesis and anti HIV-1 evaluation of a series of mannose-based glycodendrimers bearing different scaffolds, valency and functional groups at the focal position. Their sustainable chemical synthesis was performed using microwave-assisted copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction to yield glycoconjugates with full conversions (99%), allowing the reduction of the reaction time from 16 h to 60–120 min. Surface plasmon resonance studies have demonstrated that the small mannose-based glycodendrons (divalent derivatives) give the best binding interactions with the HIV-1 gp120 envelope protein, being the most active those that have a methoxymethyl group or an azidomethyl group at the focal position. Molecular modeling studies were carried out to simulate and explain the binding observed by surface plasmon resonance. This work reports a sustainable synthesis of small mannose-based glycodendrons as novel and potent anti HIV lead compounds.

Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

UV–Vis detection of E. coli 0157:H7 using Vitis vinifera and Musa paradaisica modified Au-NPs

Herein, we demonstrate the simple one-pot novel green synthesis of gold nanoparticles (Au-NPs) functionalised with a combination of banana peel (Musa paradaisica) and grape (Vitis vinifera) fruit extracts. The reaction mixture of aqueous gold chloride, banana peel and grape extracts revealed a purple colour after a reaction time of one hour, an indication of the presence and the successful synthesis of gold nanoparticles. The optical and structural properties of the green synthesized nanoparticles were analysed using Ultraviolet-Visible spectroscopy (UV–Vis) and Fourier Transform Infrared Spectroscopy (FTIR) while their surface morphology was determined using X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Small Angle X-Ray (SAX). Furthermore, a quick and simple surface plasmon resonance (SPR) study in the form of an optical sensor for the detection of Escherichia coli 0157:H7 strain was also achieved using UV–Vis. The obtained limit of detection (LOD) value for SPR for the GBPE|Au-NPs|GCE-based system was found to be 1 × 102 CFU/mL, a value well in the range for detection in seawater.•Green synthesis of gold nanoparticles (Au-NPs) was functionalised using banana peel (Musa paradaisica) and grape (Vitis vinifera) fruit extracts as capping and stabilizing agents.•Structural characterization of the Au-NPs was achieved using Ultraviolet-Visible spectroscopy (UV–Vis) and Fourier Transform Infrared Spectroscopy (FTIR) while their surface morphology was determined using X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Small Angle X-Ray (SAX).•The green synthesized Au-NPs were used to detect Escherichia coli 0157:H7 (E. coli 0157:H7) strain using Ultraviolet-Visible spectroscopy (UV–Vis) where the surface plasmon resonance (SPR) was studied.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Pomab a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.

Optimizing the Production of Silver Nanoparticles Using Mimosa Diplotricha Leaf Extract

Plant extracts are applicable over the other biological sources due to the availability and ability to act as stabilizing agent during the green nanoparticle synthesise. The present study evaluates the possibility of synthesising silver nanoparticles (SNPs) using plant leaf extracts of invasive weed Mimosa diplotricha. Biological and physical properties of synthesising Silver NPs were also evaluated using the UV-Vis spectroscopy (UV-Vis) and Scanning Electron Microscopy (SEM). To determine the best volume ratio of leaf extract, salt and temperature condition, an experiment was carried out 1:1 and 1:9 (V/V) ratios reacted with (2 mM) of Silver Nitrate under room temperature and 60-80 0C temperature condition. The colour-change conformed formation of Silver NPs. The broad peak obtained at 420-460 nm with UV–Vis surface plasmon resonance studies confirmed that the synthesised nanoparticles were Silver NPs. SEM microscopic studies revealed that spherical shape nanoparticles were formed with the average sizes 28.20 nm. This study shows that M. diplotricha mediated SNPs production is dependent on the concentration, incubation time and temperature of the reaction mixture. The best ratio of plant extract and AgNO3 is 1:1 to optimize the production of SNPs. The temperature range 60-80oC promotes higher SNPs production comparative to the room temperature. Green synthesised SNPs are poly-dispersed without forming agglomeration. Based on the observations, it is possible to conclude that M. diplotricha leaf extract is an effective source for SNPs production. This study is useful to optimize protocols for biological synthesis of SNPs using other plants.

Near- and far-field study of polarization-dependent surface plasmon resonance in bowtie nano-aperture arrays.

Bowtie nano-apertures can confine light into deep subwavelength volumes with extreme field enhancement, making them a useful tool for various applications such as optical trapping, deep subwavelength imaging, nanolithography, and sensors. However, the correlation between the near- and far-field properties of bowtie nano-aperture arrays has yet to be fully explored. In this study, we experimentally investigated the polarization-dependent surface plasmon resonance in bowtie nano-aperture arrays using both optical transmission spectroscopy and photoemission electron microscopy. The experimental results reveal a nonlinear redshift in the transmission spectra as the gap size of the bowtie nanoaperture decreases for vertically polarized light, while the transmission spectra remain unchanged with different gap sizes for horizontally polarized light. To elucidate the underlying mechanisms, we present simulated charge and current distributions, revealing how the electrons respond to light and generate the plasmonic fields. These near-field distributions were verified by photoemission electron microscopy. This study provides a comprehensive understanding of the plasmonic properties of bowtie nano-aperture, enabling their further applications, one of which is the optical switching of the resonance wavelength in the widely used visible spectral region without changing the geometry of the nanostructure.

Preparation of a composite sensor based on a fluorescent and magnetic molecular imprint polymer for metronidazole extraction–detection

We developed a metronidazole (MNZ)-selective sensor, combining magnetic and fluorescent molecularly imprinted polymers (MFMIP) with a non-imprinted reference composite (MFNIP). Using isothermal titration calorimetry (ITC), we optimized the MNZ-to-APTES ratio during polymerization, obtaining a specific MNZ-APTES bonded complex (Kb= 2.575 × 103 L/mol, ΔH° = −1.079 kJ mol−1, TΔS° = 18.389 kJ mol−1, and ΔG° = −19.468 kJ mol−1). The MFMIP composite, incorporating Fe3O4@TEOS magnetite nanoparticles and CdTe@TGA quantum dots, exhibited cavity-specific interactions with MNZ, as confirmed by surface plasmon resonance (SPR) studies. Compared to MFNIP, the MFMIP sensor demonstrated superior selectivity in water samples (5–60 μM, with LOD and LOQ values of 1.28 and 5.00 μM), and even in the presence of interferents. In addition, the use of CdTe quantum dots enabled real-time analysis via Stern–Volmer quenching mechanisms without complex sample preparation. In conclusion, our MFMIP sensor offers sensitive and selective MNZ detection. It presents practical advantages, including real-time analysis and ease of handling, making it a promising extraction–determination system for trace concentrations of MNZ.

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10-30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3-10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable -80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion-ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.