Lactoperoxidase (LPO) is a heme-containing enzyme widely distributed in mammalian secretions, contributing to host defense through the generation of antimicrobial oxidants. Unintended inhibition of LPO by therapeutic agents may impair mucosal immunity, making the investigation of potential inhibitors clinically relevant. In this study, the inhibitory effects of 11 acetohydrazide-based sulfonamide derivatives on bovine milk LPO were investigated using combined biochemical and computational approaches. The compounds exhibited diverse inhibition profiles, with IC50 values ranging from 2.32 ± 0.14 to 13.4 ± 2.05 μM. The corresponding Kᵢ values were determined in the low micromolar range, varying from 1.25 ± 0.05 to 7.53 ± 0.03 μM. Among them, 4b-2 (3-pyridyl) and 4a-5 (2-naphthyl) derivatives were identified as the most potent inhibitors, acting through a competitive inhibition mechanism with low micromolar Kᵢ values. The binding mechanisms of relatively active compounds in the enzymatic assay were explored through molecular modeling. The docking study disclosed that compounds 4a-5 and 4b-2 would show higher binding potential to the LPO structure. The two compounds formed stable complexes with the enzyme structure according to the molecular dynamics (MD) simulation study. These interactions were mainly governed by hydrogen bonding and π-π stacking interactions involving key residues in the heme-containing active site, supporting a competitive binding mode consistent with the kinetic data. Overall, the combined biochemical and computational findings suggest that acetohydrazide-based sulfonamides represent promising scaffolds for the development of potent and selective LPO inhibitors and provide new insights into the molecular recognition patterns governing their interaction with the LPO active site.

We present a high-speed force microscopy platform mounted on a confocal microscope with a z sample stage enabling long-range, high-speed force spectroscopy measurements on biological samples. The control software is built on a field-programmable gate array (FPGA)-based data acquisition and processing system, complemented with a custom graphical user insterface (GUI). We introduce smart algorithms based on probe-engagement prediction algorithms that leverage previously measured probe-sample contact to accelerate the probe engagement to mm/s and decelerate it in proximity of contact to user-defined μm/s velocities. This significantly reduces long-range force curve acquisition time and data density. Using this system, we provide proof-of-concept mechanical maps of cell clusters to extract topography and viscoelastic parameters. To further explore the versatility of our system, we probed the forces required to extract membrane tethers from monocytic cells using ultrashort cantilevers functionalised with adhesion molecules. Our system allowed us to cover retract velocities from ~1 μm/s up to ~6000 μm/s, and to explore the dynamics of tether formation at physiologically relevant velocities. Our results show that coupling extended z displacement with the prediction-based engagement algorithms enables rapid, quantitative mechanical mapping of heterogeneous biological samples with large topography variation and supports measurements requiring long distances at high velocities.

Immunoglobulin M (IgM) is the first class of antibody produced in response to pathogens. It also plays an important role in regulating the immune response, including in autoimmunity. IgM pentamers consist of an Fc core composed of Cμ2, Cμ3, and Cμ4 domains, as well as 10 Fab domains composed of Cμ1 and a variable domain. In this study, we use high-resolution atomic force microscopy (AFM) topography to constrain the reconstruction of pentameric IgM Fc core pseudo-atomic model (without Fabs). Reconstruction is performed in two stages using the AFM-Assembly pipeline. First, we fit the known cryo-EM structure of the inner core of the IgM Fc (Cμ3-Cμ4) and the known x-ray structure of Cμ2 beneath the AFM topographs using the DockAFM tool. Second, the two structural units are assembled to generate a complete pentameric IgM Fc core structure. The experimentally constrained reconstruction of the IgM Fc core reveals a divergence in the assembly of the five Cμ2 monomers: the first and last monomers (M1 and M5) are tightly attached to the inner core, while the remaining monomers (M2-M4) are more loosely bound. This reconstruction describes multiple alternative conformations for the attachment of the Cμ2 domains, suggesting that the reconstruction protocol integrates molecular flexibility present in molecules individually deposited on the AFM substrate.

Atomic Force Microscopy (AFM) based single molecule recognition has emerged as a transformative paradigm in nanoscale biology, enabling direct investigation of biological interactions under physiological conditions. This technology bridges critical gaps between structural characterization and functional analysis by providing unparalleled capabilities for quantifying binding forces, mapping molecular distributions, and resolving kinetic parameters at the single molecule level. The core methodology relies on precision probe functionalization strategies, such as oriented immobilization and polyethylene glycol (PEG) linkers, to ensure specific recognition while minimizing nonspecific interactions. Advanced techniques, including single molecule force spectroscopy (SMFS) and topography and recognition imaging (TREC), allow simultaneous acquisition of morphological and binding data with nanometer resolution, revealing insights into molecular organization and dynamics. Applications span diverse domains, from fundamental studies of antigen-antibody interactions and DNA-protein binding to clinical investigations of membrane receptor distributions in cancer cells and pathological diagnosis based on mechanical properties. Despite challenges in throughput, signal resolution, and standardization, integration with super-resolution microscopy and spectroscopic techniques demonstrates significant potential. Future developments emphasize multimodal correlation, artificial intelligence-assisted data analysis, and clinical translation toward nano biopsy, positioning AFM as a highly valuable and complementary platform for advancing molecular biophysics, drug discovery, and personalized medicine, notably due to its unique capability to combine nanoscale imaging with single-molecule force measurement on the same sample under physiological conditions.

Three-dimensional (3D) cell culture systems are increasingly recognised as more physiologically relevant models than traditional two-dimensional cultures, as they better mimic the native tumour microenvironment and enable the study of complex cellular behaviour. However, applying atomic force microscopy (AFM) to these models is challenging due to the inherent instability of multicellular aggregates, which complicates reproducible mechanical property measurements. To address this, we developed and evaluated two distinct strategies for confining multicellular aggregates for AFM analysis. In the first approach, aggregates were encapsulated in porous 3D-printed truncated conical microstructures fabricated by two-photon polymerisation. These structures were designed to allow medium perfusion, which is hypothesised to improve nutrient and metabolite exchange by enhancing fluid accessibility within the confined environment. In the second, cylindrical cavities were microfabricated into the base of PolyHEMA-coated Petri dishes to provide a simple yet robust platform for aggregate retention. Both methods successfully confined aggregates without compromising cellular integrity, enabling reproducible and reliable measurements of stiffness and viscoelasticity. Human pancreatic cancer cells (PANC-1) were used both as single cells and as monotypic aggregates. Aggregates confined in either system were consistently softer than substrate-attached single cells, indicating reduced cytoskeletal tension and altered remodelling in 3D environments. Among the two approaches, cylindrical cavities provided more reliable aggregate retention, whereas the 3D-printed truncated conical structures may possibly facilitate improved medium access through their porous architecture. Together, these microconfinement strategies enable reproducible mechanical characterisation of multicellular aggregates and extend the applicability of AFM to tumour mechanobiology and the assessment of anticancer therapies.

Programmed Cell Death-1 receptors on the surface of monocytes and T and B cells modulate various immune responses and are key immunotherapy targets. Immunotherapeutic monoclonal antibodies, including Nivolumab, block the activity of this receptor and are a mainstay of cancer therapy. Use of these drugs has been associated with side effects, including local inflammation and systemic fever in some cancer patients. To understand the role of physiologic (37°C), pathophysiologic (39°C) and standard (25°C) temperatures on the formation of the immune complexes between PD-1 and Nivolumab, we used Surface Plasmon Resonance and Molecular Dynamics simulations, at appropriate temperatures. The mean lives obtained from the dissociation curves of Nivolumab:PD-1 complexes at 37°C or 25°C are ~16 times and ~125 times longer, respectively, than those at 39°C (~7 s). These discrepancies are due to N-terminal loop from PD-1 moving away from Nivolumab only at the higher temperatures, as well as due to changes in the epitope:paratope interface.

Membrane blebbing is a dynamic cellular phenomenon that plays a key role in numerous biological processes such as migration, apoptosis, cytokinesis, and extracellular vesicle (EV) formation. This phenomenon is traditionally observed via high-resolution imaging techniques on fixed samples, since it is difficult to quantify and monitor on live cells. This aspect limits our understanding of their temporal evolution and mechanical behavior. In this work, we present and validate a novel approach based on Atomic Force Microscopy (AFM) force spectroscopy combined with a custom data analysis pipeline that is capable to detect and map bleb- and EV-like structures directly on live cells. The method leverages the mechanical signatures embedded in force-distance (FD) curves-specifically, the presence of breakthrough points, which indicate the rupture or collapse of soft, vesicular structures. A custom MATLAB algorithm was developed to automatically identify and spatially map FD curves exhibiting EV-like mechanical behavior, based on signal smoothing, inflection point analysis, and user-defined force thresholds. This algorithm was at first validated against isolated EVs in liquid and then applied to live cultures of MDA-MB-231 breast cancer cells, monitored over 3, 7, 10, and 14 days to follow the temporal changes in membrane activity. This method enables the quantitative mapping of vesicle- and bleb-associated curves on the cell surface, highlighting their spatial distribution. A progressive increase in the number of EV-like events was observed up to Day 10, followed by a decline at Day 14-suggesting a dynamic remodeling of the membrane during cell culture maturation or stress. Moreover, they exhibit a preferential localization at the cell periphery. To validate the correspondence between EV-like force signatures and blebs, standard AFM high-resolution images were acquired on the same cells fixed and then analyzed using a Circular Hough Transform-based algorithm to detect and count blebs based on their circular geometry. The time trends obtained from both techniques showed good agreement, confirming the robustness of the force spectroscopy-based detection on live samples. This approach represents a significant advancement in the use of AFM for live-cell analysis. By extracting meaningful biophysical information from FD curves beyond standard elasticity metrics, we demonstrate that AFM spectroscopy can serve as a high-content, non-invasive tool for the detection of nanoscale membrane phenomena.

Achiral bivalent ligands capable of simultaneous occupation of both hormone binding sites of transthyretin generate a substantial and complex induced near ultraviolet circular dichroism spectrum during protein binding, revealing the dynamics of this process. Reduced temperature and pH slow the interaction and reveal two phases consistent with formation of an encounter complex and progression of the interaction through the core of the transthyretin tetramer. The x-ray structure of the protein-ligand complex confirms the endpoint of the binding trajectory and shows evidence of plasticity in the structure, with substantial disturbances of some mainchain and sidechains within and adjacent to the binding channel. This study highlights the effective complementarity of CD and x-ray investigations.

Canine parvovirus type 2 (CPV-2) is a highly contagious and often fatal canine pathogen, with antigenic variants (CPV-2a, 2b, 2c), maternally derived antibody (MDA) interference, and lack of Differentiating Infected from Vaccinated Animals (DIVA) capability limiting current vaccines. Using an immunoinformatics-based reverse vaccinology approach, we identified conserved B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes from CPV-2 structural (VP1, VP2) and nonstructural (NS1, NS2) proteins. These were screened for antigenicity, immunogenicity, safety, and stability. The screening identified three B-cell, two CTL, and five HTL epitopes. Out of these, two representative epitopes were utilized for in vitro evaluation. The peripheral blood mononuclear cells (PBMCs) were collected from 10 dogs 28 days after vaccination with a live-attenuated whole-virus CPV vaccine and pooled. This pool was expected to contain memory cytotoxic T cells recognizing a broad viral epitope repertoire. On antigenic stimulation, PCPV2 induced significant proliferation of canine PBMCs, with the highest response at 33.33 μg/mL, alongside ~15.67-fold upregulation of IFN-γ mRNA, indicating a memory T-cell response. A multi-epitope vaccine was designed by integrating validated epitopes with the TLR-4 agonist RS09 and a Capripoxvirus-derived DIVA marker, linked by AAY/EAAAK spacers. The vaccine construct can be effectively delivered via a recombinant canine adenovirus-2 vector for efficient in vivo expression and cellular response. Structural modeling, molecular docking, immune simulations, molecular dynamic simulations, and codon optimization predicted strong receptor binding, stability, IgG/T-cell responses, and expression feasibility in Escherichia coli and canine cells. These findings support PCPV2 as a potent immunogen and demonstrate the promise of the designed construct for a potential adenovirus-vectored vaccine; however, in vivo validation will be essential to confirm efficacy, cross-variant protection, and DIVA performance.

Biocompatibility of a biosensor can be achieved by grafting polymer brushes onto a solid surface. These brushes must be able to attract specific analytes or repel unwanted entities. This is obtained with weak polyelectrolyte polymer brushes that shrink or swell depending on external stimuli. In this study, the conformation of polyacrylic acid (PAA) and polyethylene oxide (PEO) polymer brushes was characterized as a function of pH and ionic strength using Atomic Force Microscopy (AFM) in spectroscopic mode. Instead of colloidal tips classically used to measure the mechanical behavior of the brush, force curves were performed with conventional tips for better sensitivity to the interaction between ions and polymer, which is responsible for their conformation. Since force mapping experiments generate thousands of curves, a statistical representation was employed to define the general trend of the curves and facilitate their interpretation. As expected, the neutral PEO is not affected by changes in solution pH and salinity. In contrast, PAA exhibits behaviors depending on the ions present in the solution and increasing salinity; the brush shrinks at low pH with H3O[+] ions and swells with the addition of Na[+] and K[+] ions. The originality of the study also lies in the implementation of an Artificial Intelligence (AI) clustering model applied to force curves to specifically study a 50% PAA/50% PEO mixed polymer brush. This AI model makes it possible to distinguish areas of the surface where only one type of polymer has been grafted and to identify its nature according to its force curve.

The increasing issue of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) necessitates rapid and reliable diagnostic methods. While existing RPA-CRISPR/Cas12a platforms have demonstrated potential for MRSA detection, most rely on single-gene targets or require multiple Cas enzymes. Here, we have developed a novel dual gene detection strategy that simultaneously detects the S. aureus specific femA gene and the methicillin-resistant mecA gene in a single RPA-CRISPR/Cas12a reaction. This integrated approach enables clear discrimination between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) in just 30 min, with results visualized via both fluorescence and lateral flow strips. The assay exhibited high specificity (no cross-reactivity with common pathogens) and a sensitivity of 10 copies/μL, comparable to qPCR. Validation with 39 clinical samples showed 100% concordance with antimicrobial susceptibility testing. Our dual-gene RPA-CRISPR/Cas12a platform represents a significant advancement in point-of-care MRSA diagnostics, offering enhanced accuracy and operational simplicity.

Coronaviruses, among other membrane-enveloped viruses, can remain infectious on surfaces in ambient air for days. Many bacteria and fungi also expose their plasma membranes to air environments. Key survival strategies in air include hydrophobic coatings to prevent water loss and waxy or silica-based resistant cell walls. Yet, many aspects remain largely unexplored due to the lack of suitable nanoscale imaging. While Atomic Force Microscopy (AFM) imaging in the liquid native environment of the biological samples gives access to many details of the membranes, operation in air damages these soft samples, being unable to resolve these fragile structures. Here, we demonstrate that ultra-stiff microelectromechanical (MEMS)-based AFM probes, with an ultra-high stiffness of 200 kN m[-1] and a resonance frequency of 15 MHz, can image soft cell membranes of Halobacterium Sallinarium in ambient air without structural disruption. Although meniscus-mediated tip stabilization has been previously observed for MEMS and optomechanical probes, this study provides the first demonstration of its application to biological imaging. By operating in a capillary-coupled regime with sub-ångström oscillation amplitude, the probe stabilizes at approximately 40% of the meniscus rupture distance, dissipating only ~35% of the maximum interaction energy. This enables noncontact imaging at tip-sample separations of ~5 nm and faithful recovery of the membrane topography (~5 nm thickness). The results establish a pathway for ultra-stiff AFM probe to perform noninvasive imaging of soft biological structures in air.

Retraction: P. Bhattacharjee, T. Ghosh, S. Sarkar, P. Pandya, and K. Bhadra, "Binding Affinity and In Vitro Cytotoxicity of Harmaline Targeting Different Motifs of Nucleic Acids: An Ultimate Drug Designing Approach," Journal of Molecular Recognition 31, no. 4 (2018): e2687. https://doi.org/10.1002/jmr.2687. The above article, published online on 15 December 2017 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Rebecca Wade; and John Wiley & Sons Ltd. A third party reported on PubPeer [1] that images in Figures 9B contained evidence of manipulation; that Figure 9A included an image which had been duplicated from an earlier article by some of the same authors and presented at a different magnification [Sarkar et al. 2016 (https://doi.org/10.1016/j.cbi.2016.08.024)]; and that manipulated versions of an image in Figure 8A had been duplicated in later articles by some of the same authors [Bhattacharjee et al. 2018 (https://doi.org/10.7324/JABB.2018.60401) and Chattopadhyay et al. 2019 (https://doi.org/10.3762/bjoc.15.245)]. The authors responded to an inquiry by the publisher. However, they were unable to provide a sufficient explanation for the evidence of image manipulation and data reuse between this article and other articles by the same author group. Following receipt of the authors' response, the publisher detected additional evidence of image manipulation in Figures 8B and 9A. The retraction has been agreed to because evidence of image duplication and manipulation fundamentally compromises the editors' confidence in the reported conclusions. The authors confirmed that the complete archival of raw data could not be retrieved due to the age of the experiments and stated that they disagree with the wording of the retraction. REFERENCES: [1] Sundasciurus fraterculus. Comments on "Binding affinity and in vitro cytotoxicity of harmaline targeting different motifs of nucleic acids: An ultimate drug designing approach," PubPeer, August-September 2025. https://pubpeer.com/publications/AE3A60EA7C6E78A714B472E1CB953C.

The proto-oncogene Mas receptor (MasR) is a class-A (orphan-type) G protein-coupled receptor (GPCR) that mediates the cardioprotective effects of Angiotensin 1-7 (Ang 1-7) within the renin-angiotensin system (RAS). Despite its therapeutic relevance, the molecular mechanisms underlying MasR activation remain elusive due to the lack of experimental structural data. In this study, we combined 1-μs all-atom molecular dynamics (MD) simulations of AlphaFold-modeled MasR with energetic network analyses to elucidate ligand-induced activation dynamics. Our results challenge the conventional electrostatic clamp model, revealing a distinct active anchor mechanism. We demonstrate that Ang 1-7 releases the inactive core lock (ARG245-TYR248) to engage a critical anchor cluster (SER109/PHE112). This interaction stabilizes a unique decoupled conformational state: while the NPxxY motif is fully activated and the intracellular vestibule expands via noncanonical communication hubs that substitute for the absent conserved microswitches, TM6 remains in a restricted, compact conformation (~10 Å). Thermodynamically, this partial activation is supported by favorable computed binding energetics and enhanced conformational plasticity. These findings provide a structural rationale for the partial agonist profile of Ang 1-7 and offer a transformative mechanistic framework for therapeutic targeting of the ACE2/Ang 1-7/MasR axis. We propose that rational drug design need not aim for maximal receptor opening; instead, strategies focusing on peptidomimetics or positive allosteric modulators (PAMs) that stabilize this specific compact active state could selectively potentiate protective RAS signaling.

Unexplained recurrent spontaneous abortion (URSA) remains a prevalent obstetric issue, the underlying causes of which are still not fully understood. MicroRNAs (miRNAs) have the potential to influence URSA through their regulation of gene expression. This study aimed to screen key miRNAs and to verify their regulatory roles and potential molecular mechanisms in URSA. Differentially expressed miRNAs and mRNAs in URSA were identified using GEO datasets (GSE178619 and GSE113790). Weighted gene co-expression network analysis (WGCNA) was performed to identify hub miRNAs and mRNAs, followed by construction of a miRNA-mRNA interaction network. Functional enrichment was analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. An oxidized low-density lipoprotein (OX-LDL)-induced trophoblast cell model was established after concentration screening, and gain- and loss-of-function experiments involving miR-146a/b-5p and IRAK1 were conducted to evaluate cellular functions. The targeting relationship between miR-146a/b-5p and IRAK1 was examined using dual-luciferase reporter assays. In vivo, a URSA mouse model was constructed, and lentiviral overexpression of miR-146a/b-5p was applied to assess pregnancy outcomes and NF-κB pathway-related molecular changes. Results showed that GSE178619 identified 34 differentially expressed miRNAs, while GSE113790 revealed 1056 differentially expressed mRNAs. WGCNA highlighted the MEbrown module (112 hub miRNAs) and the MEdarkorange module (673 hub mRNAs) as closely associated with URSA. Eighteen core miRNAs, including miR-146a/b-5p, and 21 core genes, including IRAK1, were identified, with IRAK1 upregulated in URSA and linked to the NF-κB signaling pathway. In vitro, OX-LDL treatment impaired trophoblast cell proliferation and invasion, promoted apoptosis and oxidative stress, reduced miR-146a/b-5p expression, and activated NF-κB signaling through IRAK1. Overexpression of miR-146a/b-5p alleviated these alterations, whereas inhibition of miR-146a/b-5p produced opposite effects that were partially reversed by IRAK1 knockdown. In URSA mice, miR-146a/b-5p overexpression improved pregnancy outcomes, alleviated placental pathological changes, reduced IRAK1 expression, and attenuated NF-κB pathway activation. Together, miR-146a/b-5p regulates URSA progression by targeting IRAK1 and modulating the NF-κB signaling pathway, suggesting its potential as a therapeutic target for URSA.