Specific Binding Sites Research Articles

A concept of natural genome reconstruction. Part 2. Effect of extracellular double-stranded DNA fragments on hematopoietic stem cells

In this part of the study, the first component of the concept of “natural genome reconstruction” is being proven. It was shown with mouse and human model organisms that CD34+ hematopoietic bone marrow progenitors take up fragments of extracellular double-stranded DNA through a natural mechanism. It is known that the process of internalization of extracellular DNA fragments involves glycocalyx structures, which include glycoproteins/protein glycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors. The bioinformatic analysis conducted indicates that the main surface marker proteins of hematopoietic stem cells belong to the indicated groups of factors and contain specific DNA binding sites, including a heparin-binding domain and clusters of positively charged amino acid residues. A direct interaction of CD34 and CD84 (SLAMF5) glycoproteins, markers of hematopoietic stem cells, with double-stranded DNA fragments was demonstrated using an electrophoretic mobility shift assay system. In cells negative for CD34, which also internalize fragments, concatemerization of the fragments delivered into the cell occurs. In this case, up to five oligonucleotide monomers containing 9 telomeric TTAGGG repeats are stitched together into one structure. Extracellular fragments delivered to hematopoietic stem cells initiate division of the original hematopoietic stem cell in such a way that one of the daughter cells becomes committed to terminal differentiation, and the second retains its low-differentiated status. After treatment of bone marrow cells with hDNAgr, the number of CD34+ cells in the colonies increases to 3 % (humans as the model organism). At the same time, treatment with hDNAgr induces proliferation of blood stem cells and their immediate descendants and stimulates colony formation (mouse, rat and humans as the model organisms). Most often, the granulocyte-macrophage lineage of hematopoiesis is activated as a result of processing extracellular double-stranded DNA. The commitment process is manifested by the appearance and repair of pangenomic single-strand breaks. The transition time in the direction of differentiation (the time it takes for pangenomic single-strand breaks to appear and to be repaired) is about 7 days. It is assumed that at the moment of initiation of pangenomic single-strand breaks, a “recombinogenic situation” ensues in the cell and molecular repair and recombination mechanisms are activated. In all experiments with individual molecules, recombinant human angiogenin was used as a comparison factor. In all other experiments, one of the experimental groups consisted of hematopoietic stem cells treated with angiogenin.

Photocatalytic Organic Semiconductor-Bacteria Imprinted Polymers for Highly Selective Determination of Staphylococcus aureus at the Single-Cell Level.

This work utilized a combination of photocatalytic organic semiconductors and bacteria to create a photocatalytic organic semiconductor-bacterial biomixture system based on a bacteria imprinted polymers (OBBIPs-PEC) sensor, for the detection of S. aureus with high sensitivity in "turn-on" mode at the single-cell level. This outstanding sensor arises from an integration of two different types of semiconductor materials to form heterojunctions. As well this sensor involves combining a semiconductor material with cationic side chains and an electron transport chain within a natural cellular environment, in which the cationic side chain of poly(fluorene-co-phenylene) organic semiconductor at 2-(4-mesyl-2-nitrobenzoyl)-1,3-cyclohexanedione (PFP-OC@MNC) demonstrated the ability to penetrate the cell membrane of S. aureus and interact with specific binding sites through electrostatic interactions. As the cavities in the BIPs were occupied by S. aureus, during light irradiation, the electrons stimulated by the photoexcitation process in the manufactured PFP-OC@MNC semiconductors were successfully transmitted to S. aureus, where these electrons played a role in the regeneration of NADH and FADH2, and then the presence of S. aureus acted as a proficient electron acceptor for photoexcited electrons; thereby the PEC response of the OBBIPs-PEC sensor was significantly enhanced. Of note, it exhibited high selectivity for S. aureus over other bacteria and maintained excellent performance in complex matrices, distinguishing S. aureus with concentrations as low as 10 CFU/mL. This work dramatically reduces the influence of interference factors in the traditional mode and offers a powerful way for microorganism detection in food and environmental fields.

Selective Novel Metal‐Coordinated Biomedical Agents Encompassing Tetradentate Salen Ligand: Structural Elucidation, DFT Calculation, Cytotoxic, and Antioxidant Activities Supported by Molecular Docking Approach

ABSTRACTSeveral physicochemical and analytical methods were employed to elucidate the structural analysis of some novel complexes derived from the {3,4‐bis‐[(3‐ethoxy‐2‐hydroxy‐benzylidene)‐amino]‐phenyl}‐phenyl‐methanone (ESAB ligand). Decomposition point determination, elemental analysis (CHN), spectroscopy (IR, NMR, and mass spectrometry), conductivity, magnetic susceptibility, UV–Vis spectrum study, and theoretical investigations were among these methods. With conductance values ranging from 7.5 to 15.12 Ω−1 cm2 mol−1, molar conductance values showed that the Zn (II), Cu (II), and Ru (III) complexes are nonelectrolytes in fresh DMSO solutions, with the exception of the ESABRu complex, which is a monoelectrolyte. According to IR spectra, the ligand uses the (N & O) donor sites from the (C=N & C‐O) groups in the ligand moiety to coordinate through the metal ions in a tetradentate form. A 1:1 (metal:ligand) molar ratio was proposed by Job's approach based on analytical data from solution complexation. According to the stability constant (Kf) values, the complexes' stability order was found to be ESABRu > ESABCu > ESABZn. The pH profile showed that the complexes under study are stable throughout a broad pH range, usually between pH = 4 and pH = 10. The complexes' geometric structures and ligand coordination capabilities were inferred with the use of magnetic and electronic spectrum studies. The DFT method uses quantum chemical simulations to evaluate the electronic structures of the ligand under investigation and its complexes. The unbound ligand's B3LYP level, B3LYP/6/311G* degree, and the complexes' B3LYP/6/311G**/LANL2DZ functional categories were employed in the computations of the density function concept (DFT). The findings revealed the consistency between the experimental and DFT computations. Natural bond orbital (NBO) analysis was used to investigate bond strength between molecules' charge exchange, hyperconjugative connections, and molecular equilibrium. By computing the hyperpolarizability (β) and molecular polarizability (α) parameters, the ensuing nonlinear optical properties were investigated, leading to a number of surprising optical properties for the produced compounds. The antipathogenic activity of the generated materials was experimentally verified against a subset of gram (+) and gram (−) bacteria as well as some fungi using the agar well diffusion method. Additionally, hepatic cellular carcinomas, cells from colon and breast cancer, were utilized to test the cytotoxic activity of the ESAB ligand and its metal chelates. Furthermore, the examined compounds' ability to suppress the DPPH radical was examined. Additionally, simulations of molecular docking were performed to ascertain how the produced compounds attached to the specific protein binding sites.

Specific recognition mechanism of an antibody to sulfated tyrosine and its potential use in biological research.

Post-translational modification of proteins is a crucial biological reaction that regulates protein functions by altering molecular properties. The specific detection of such modifications in proteins has made significant contributions to molecular biology research and holds potential for future drug development applications. In HIV research, for example, tyrosine sulfation at the N-terminus of C-C chemokine receptor type 5 (CCR5) is considered to significantly enhance HIV infection efficiency. However, antibodies specific to sulfated CCR5 still need to be developed. In this study, we successfully generated an antibody that specifically recognized the sulfated N-terminal peptide of CCR5 through rabbit immunization and panning via phage display using a CCR5 N-terminal peptide containing sulfate modification. We used various physicochemical methods in combination with molecular dynamics simulation to screen for residues that could be involved in recognition of the sulfated peptide by this antibody. We also confirmed that this antibody recognized the sulfated full-length CCR5 on the cell surface, which suggested it should be useful as a research tool that could lead to the development of novel therapeutics. Although the antibody binding did not inhibit HIV infection, it could be also described as sulfation site-specific binding, beyond sulfation-specific binding.

High hydrostatic pressure promotes gene transcription via a cystathionine-β-synthase domain-containing protein in the hyperthermophilic archaeon Pyrococcus yayanosii.

Cystathionine-β-synthase (CBS) domains are ubiquitously prevalent in all kingdoms of life. Remarkably, in archaea, proteins consisting of solely CBS domains are widespread. However, the biological functions of CBS proteins in archaea are still unknown. Here, we identified a high hydrostatic pressure regulator (HhpR) that comprises four CBS domains serving as a transcriptional activator via specifically binding to the UAS (upstream activating sequence) motif situated within the promoter region of an operon in a hyperthermophilic archaeon Pyrococcus yayanosii under high hydrostatic pressure (HHP). By combining molecular dynamics simulations,in vitro and in vivo assays, we revealed the potential binding interfaces between HhpR and its specific DNA binding site. Particularly, one stem-loop region in HhpR (termed as 'Arm') was found to play a critical role in regulating the transcription activity,and the 192 position in the Arm region is an essential site in dictating the conformational changes of HhpR at HHP condition. Our work provides novel insights into the structure-function relationship of CBS-containing proteins that participate in archaeal gene regulation as general transcriptional activators.

SPD_0410 negatively regulates capsule polysaccharide synthesis and virulence in Streptococcus pneumoniae D39.

Streptococcus pneumoniae capsular polysaccharide (CPS) is a crucial virulence factor for this pathogenic bacterium and is partially under transcriptional control. In this study, we used electrophoretic mobility shift assays and DNA enzyme footprinting to identified the hypothetical protein SPD_0410 as a negative regulator of cps locus. Our results showed that the D39Δspd0410 mutant strain exhibited significantly elevated CPS levels compared to the parental strain D39s. SPD_0410 directly binds at two specific sites on the cps promoter. The regulatory effect of SPD_0410 on CPS was weakened after the mutation of specific binding sites in the promoter. RNAseq analysis revealed that the deletion of spd0410 led to alterations in glucose metabolism. However, the altered glucose levels appeared to eliminate the regulation of CPS synthesis by SPD_0410. Deleting the spd0410 gene resulted in higher invasion and phagocytic resistance of bacteria and in vivo mouse experiments confirmed that D39Δspd0410 caused more severe systemic disease than the parental strain D39s. Our results indicated that SPD_0410 negatively regulates the synthesis of S. pneumoniae capsules and can directly alter pneumococcal virulence.

Functional epitope mapping of cell surface glucose-regulated protein 94: A combinatorial approach for therapeutic targeting.

Glucose-regulated protein 94 (GRP94) overexpression plays a critical role in tumor cell survival across various cancers. Previously, we developed K101.1, a fully human antibody targeting cell surface GRP94, which effectively inhibits tumor angiogenesis in colorectal cancer (CRC). This study aims to identify K101.1's interaction site and further elucidate GRP94's role in tumor angiogenesis. In an HT29 CRC xenograft mouse model, K101.1 reduced tumor growth and angiogenesis by 30% and 69%, respectively, without inducing severe toxicity. Using hydrogen‑deuterium exchange mass spectrometry, we identified the binding site of K101.1 on GRP94, analyzing 225 peptides with 94.5% sequence coverage and a redundancy score of 4.20. This revealed a linear epitope (Glu26-Ala34) as the specific binding site. The binding was further validated via enzyme-linked immunosorbent assay, and human umbilical vein endothelial cell tube formation assays using a synthetic epitope peptide corresponding to the identified epitope. Our findings suggest that this epitope is functionally involved in GRP94-mediated angiogenesis and is integral to its proangiogenic activity. By integrating epitope profiling with complementary experimental approaches, this study advances the understanding of GRP94's role in CRC angiogenesis and provides new insights for developing targeted cancer therapies and vaccines, offering promising avenues for CRC prevention and treatment.

Sodium alginate-crosslinked montmorillonite nanosheets hydrogel for efficient gallium recovery.

An efficient adsorbent for Ga(III) recovery was developed by applying the geochemical principles of Ga mineralization, using Al-rich clay minerals with a natural affinity for Ga as the raw material. Sodium alginate (SA) facilitated the cross-linked assembly of montmorillonite nanosheets (MMTNS), forming a three-dimensional structured hydrogel. This was achieved through electrostatic interactions between -OH groups on the edges of MMTNS and -COO- groups in SA, as well as the complexation of Ca2+ and -COO- groups. The resulting hydrogel maintained a porous structure while preserving the layered arrangement of MMTNS, significantly enhancing the adsorption capacity for Ga(III). Thermodynamic analysis revealed that Ga(III) adsorption was both endothermic and spontaneous. The Elovich model indicated that heterogeneous chemisorption dominated the adsorption process. Ga(III) adsorption followed the Langmuir isotherm model, indicating that it was controlled by specific binding sites and occurred via uniform monolayer adsorption, with a maximum capacity of 85.95mg/g. The adsorption mechanism involved ion exchange interactions, chelation of functional groups, and electrostatic interactions. After 4cycles, the hydrogel retained an adsorption capacity of 65.4mg/g. Additionally, the hydrogel demonstrated good selectivity for Ga(III) in a quaternary ion solution system. This hydrogel shows significant potential as a candidate for Ga(III) recovery.

Tetramethylpyrazine attenuates the cancer stem cell like-properties and doxorubicin resistance by targeting HMGCR in breast cancer.

Tetramethylpyrazine (TMP), a key bioactive constituent derived from Ligusticum wallichii Franchat, has demonstrated efficacy in mitigating multidrug resistance (MDR) in human breast cancer (BC) cells. However, the precise mechanisms underlying its action remain poorly understood. Cancer stem cells (CSCs) are widely recognized as the primary contributors to MDR. This investigation seeks to elucidate the role and mechanisms through which TMP counteracts MDR by attenuating CSC-like characteristics. Various assays, including flow cytometry, sphere formation, and Western blotting, were employed to evaluate TMP's effects on breast cancer stem cell (BCSC)-like phenotypes in vitro. In vivo, extreme limiting dilution assays and immunohistochemistry (IHC) were executed to assess the impacts of TMP on BCSC frequency and the levels of stemness markers. Mechanistically, RNA sequencing was performed to uncover the key biological processes involved in TMP's effects on BCSCs. Further experiments, encompassing micro scale thermophoresis (MST), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and amino acid mutation analyses, were utilized to identify the essential targets and corresponding binding sites of TMP. Finally, the effects of TMP on BCSC-like phenotypes were confirmed using cells with mutated amino acid residues, which allowed us to investigate the specificity of TMP's binding sites. To further evaluate the impact of TMP on drug resistance, doxorubicin-resistant MCF7 (MCF-7ADR) cells, along with corresponding cell lines harboring mutated amino acid residues, were employed. TMP was found to inhibit BCSC-like properties both in vitro and in vivo, evidenced by a reduction in the CD44+/CD24- population, sphere formation capability, and expression of stemness markers. Mechanistic studies revealed that TMP targets 3‑hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. TMP binds to Asp-767 of HMGCR, thereby inhibiting its activity and reducing cholesterol synthesis. The influence of TMP on BCSC-like phenotypes was nullified by overexpression of wild-type HMGCR, while mutations in the binding site of HMGCR had no effect on TMP's inhibition of BCSC-like properties. Additionally, TMP mitigated MDR by targeting HMGCR. These findings suggest that TMP alleviates MDR by reducing BCSC-like traits through targeting HMGCR and disruption of cholesterol biosynthesis in BC. This provides new insights into the mechanisms through which TMP alleviates MDR and offers new lead compound for exploring HMCGR antagonists.

Tungsten disulphide nanosheet modulated fluorescent gold nanocluster immunoprobe for the detection of tau peptide: Alzheimer's disease biomarker.

The neuronal tau peptide serves as a key biomarker for neurodegenerative diseases, specifically, Alzheimer's disease, a condition that currently has no cure or definitive diagnosis. The methodology to noninvasively detect tau levels from body fluids remains a major hurdle for a rapid and simple diagnostic approach. Thus, developing new detection methods for sensing tau protein levels is crucial. In this work, we report an immunoprobe based on anti-tau antibody (mAb-tau)-conjugated fluorescent gold nanoclusters (AuNCs) quenched with tungsten disulphide nanosheets (WS2 NS) for the detection of tau protein in human serum samples. The mAb-tau conjugated probe is designed to provide a specific binding site for the tau peptide by strong antigen-antibody interface. The WS2 NS surface quenches the fluorescence of mAb-tau@AuNCs, which is subsequently recovered by the addition of tau peptide in a linear concentration range (63.3-615.38 pg mL-1). The enhancement in fluorescence of WS2 NS@mAb-tau@AuNCs enables the quantification of tau peptide in concentrations pertinent to human serum tau levels in Alzheimer's patients. The developed probe achieves a limit of detection (LOD) and limit of quantification (LOQ) of 6.54 pg mL-1 and 21.8 pg mL-1 for tau peptide in PBS buffer. The study is further extended in spiked human serum samples, with satisfactory recovery percentages in the range of 94.37-117.53%. The technique holds promise as an immunoprobe for tau peptide detection and has potential in developing an economically viable probe for the clinical diagnosis of tau-related neurodegenerative disorders.

Deciphering regulatory architectures of bacterial promoters from synthetic expression patterns.

For the vast majority of genes in sequenced genomes, there is limited understanding of how they are regulated. Without such knowledge, it is not possible to perform a quantitative theory-experiment dialogue on how such genes give rise to physiological and evolutionary adaptation. One category of high-throughput experiments used to understand the sequence-phenotype relationship of the transcriptome is massively parallel reporter assays (MPRAs). However, to improve the versatility and scalability of MPRAs, we need a "theory of the experiment" to help us better understand the impact of various biological and experimental parameters on the interpretation of experimental data. These parameters include binding site copy number, where a large number of specific binding sites may titrate away transcription factors, as well as the presence of overlapping binding sites, which may affect analysis of the degree of mutual dependence between mutations in the regulatory region and expression levels. To that end, in this paper we create tens of thousands of synthetic gene expression outputs for bacterial promoters using both equilibrium and out-of-equilibrium models. These models make it possible to imitate the summary statistics (information footprints and expression shift matrices) used to characterize the output of MPRAs and thus to infer the underlying regulatory architecture. Specifically, we use a more refined implementation of the so-called thermodynamic models in which the binding energies of each sequence variant are derived from energy matrices. Our simulations reveal important effects of the parameters on MPRA data and we demonstrate our ability to optimize MPRA experimental designs with the goal of generating thermodynamic models of the transcriptome with base-pair specificity. Further, this approach makes it possible to carefully examine the mapping between mutations in binding sites and their corresponding expression profiles, a tool useful not only for developing a theory of transcription, but also for exploring regulatory evolution.

Metal-Independent Correlations for Site-Specific Binding Energies of Relevant Catalytic Intermediates.

Establishing energy correlations among different metals can accelerate the discovery of efficient and cost-effective catalysts for complex reactions. Using a recently introduced coordination-based model, we can predict site-specific metal binding energies (ΔE M) that can be used as a descriptor for chemical reactions. In this study, we have examined a range of metals including Ag, Au, Co, Cu, Ir, Ni, Os, Pd, Pt, Rh, and Ru and found linear correlations between predicted ΔE M and adsorption energies of CH and O (ΔE CH and ΔE O) at various coordination environments for all the considered metals. Interestingly, all the metals correlate with one another under specific surface site coordination, indicating that different metals are interrelated in a particular coordination environment. Furthermore, we have tested and verified for PtPd- and PtIr-based alloys that they follow a similar behavior. Moreover, we have expanded the metal space by taking some early transition metals along with a few s-block metals and shown a cyclic behavior of the adsorbate binding energy (ΔE A) versus ΔE M. Therefore, ΔE CH and ΔE O can be efficiently interpolated between metals, alloys, and intermetallics based on information related to one metal only. This simplifies the process of screening new metal catalyst formulations and their reaction energies.

In Silico evaluation of hyaluronic acid nanoparticle stability and drug encapsulation using molecular dynamics simulations

ABSTRACT Hyaluronic acid-based nanoparticles (HA-NPs) hold promise for drug delivery due to their biocompatibility and biodegradability. However, limited molecular simulations exist to understand their behaviour. This study utilises molecular dynamics to investigate the potential of two HA-NP models: acetylated hyaluronic acid nanoparticles (AcHA-NPs) and hyaluronic acid-ceramide nanoparticles (HACe-NPs), as drug delivery vehicles. We compared the stability and drug loading characteristics of HACe-NPs with different compositions to AcHA-NPs. AcHA-NPs displayed a random, amorphous structure, offering no specific binding sites. The simulations revealed that HACe-NPs exhibit superior stability in aqueous solution compared to AcHA-NPs, which require a high content of organic solvents for stability. Additionally, HACe-NPs self-assembled into micelle-like structures with a well-defined hydrophobic core, potentially providing more efficient drug encapsulation sites compared to the seemingly amorphous structure of AcHA-NPs. Furthermore, the study suggests the presence of specific binding sites within the HACe-NP core for drug molecules, potentially leading to more controlled drug loading. While the simulated nanoparticle size was smaller than experimentally observed sizes, this in silico study provides valuable insights into the fundamental behaviour of HACe-NPs. These findings can guide future research efforts towards optimising HACe-NP design for larger, yet stable, nanoparticles with desired drug loading properties.

Directed Cell Growth of C2C12 Cells on ECM Free Bioprinted Nano/Micro Scaffolds.

Skeletal muscle cell growth impairment can result in severe health issues, such as reduced mobility, metabolic problems, and cardiovascular issues, which can significantly impact an individual's overall health and lifestyle. To address this issue, it is essential to adopt a multi-faceted approach. Conventional 2D cell culture methods fail to replicate the critical features of in vivo micro/nanoarchitecture, which is crucial for the growth of skeletal muscle cells. In this study, the directed growth of mouse skeletal myoblasts (C2C12) cells on ECM-free biocompatible scaffolds is demonstrated and fabricated using two-photon lithography (TPL). These scaffolds are 2D and 3D and have nano/micro-features derived from chitosan-based carbon quantum dots (Ch-CQDs). Ch-CQDs act as two-photon initiators for TPL and also provide the scaffolds with adequate mechanical strength and specific binding sites. These scaffolds are biocompatible and can support cellular adhesion and growth without the need for ECM coating. The nano/micro scaffolds mimic the in vivo cellular microenvironment, enabling directed cell growth on ECM-free surfaces. The fabricated scaffolds have tunable mechanical strength ranging from 0.09 to 0.75GPa. By using Ch-CQDs, scaffolds are created that promote cell growth and alignment, which is crucial for skeletal muscle cell growth.

Rational design yields RNA-binding zinc finger domains with altered sequence specificity.

Targeting and manipulating endogenous RNAs in a sequence-specific manner is essential for both understanding RNA biology and developing RNA-targeting therapeutics. RNA-binding zinc fingers (ZnFs) are excellent candidates as designer proteins to expand the RNA-targeting toolbox, due to their compact size and modular sequence recognition. Currently, little is known about how the sequence of RNA-binding ZnF domains governs their binding site specificity. Here, we systematically introduced mutations at the RNA-contacting residues of a well-characterized RNA-binding ZnF protein, ZRANB2, and measured RNA binding of mutant ZnFs using a modified RNA-bind-n-seq (RBNS) assay. We identified mutant ZnFs with an altered sequence specificity, preferring to bind a GGG motif instead of the GGU preferred by wild-type ZRANB2. Further, through a series of all-atom molecular dynamics (MD) simulations with ZRANB2 and RNA, we characterized changes in the hydrogen-bond network between the protein and RNA that underlie the observed sequence specificity changes. Our analysis of ZRANB2-RNA interactions both in vitro and in silico expands the understanding of ZnF-RNA recognition rules and serves as a foundation for eventual use of RNA-binding ZnFs for programmable RNA targeting.

Exposed Hsp70-binding site impacts yeast Sup35 prion disaggregation and propagation

The dynamic balance between formation and disaggregation of amyloid fibrils is associated with many neurodegenerative diseases. Multiple chaperones interact with and disaggregate amyloid fibrils, which impacts amyloid propagation and cellular phenotypes. However, it remains poorly understood whether and how site-specific binding of chaperones to amyloids facilitates the concerted disaggregation process and modulates physiological consequences in vivo. Here, we identified binding sites of Ssa1, Sis1, and Hsp104 chaperones for Sup35, the protein determinant of yeast prion [PSI+] yeast. Our biophysical and genetic analyses with various Sup35 deletion mutants and amyloid conformations revealed that the Ssa1-binding to the region outside amyloid core plays a key role in facilitating disaggregation and propagation of yeast prions both in vitro and in vivo. Furthermore, we developed a reconstitution system, including the Ssa1-binding tag and the HAP/Caseinolytic protease P (ClpP) hybrid chaperones, and found that this reconstitution system successfully degraded distinct prion strain conformations. Together, these results show that the properly positioned, exposed Ssa1-binding region in amyloid fibrils influences the efficiency of amyloid disaggregation and propagation, and eventually prion strain phenotypes. More broadly, our findings provide molecular foundations for previous, puzzling observations of prion propagation in vivo, and offer insights into elimination of amyloid deposits in cells.

Effect of Gold Nanoparticles on the Conformation of Bovine Serum Albumin: Insights from CD Spectroscopic Analysis and Molecular Dynamics Simulations.

With the development of nanotechnology, there is growing interest in using nanoparticles (NPs) for biomedical applications, such as diagnostics, drug delivery, imaging, and nanomedicine. The protein's structural stability plays a pivotal role in its functionality, and any alteration in this structure can have significant implications, including disease progression. Herein, we performed a combined experimental and computational study of the effect of gold NPs with a diameter of 5 nm (5 nm Au-NPs) on the structural stability of bovine serum albumin (BSA) protein in the absence and presence of NaCl salt. Circular dichroism spectroscopy showed a loss in the secondary structure of BSA due to the synergistic effect of Au-NPs and NaCl, and Thioflavin T fluorescence assays showed suppressed β-sheet formation in the presence of Au-NPs in PBS, emphasizing the intricate interplay between NPs and physiological conditions. Additionally, molecular dynamics (MD) simulations revealed that 5 nm Au-NP induced changes in the secondary structure of the BSA monomer in the presence of NaCl, highlighting the initial binding mechanism between BSA and Au-NP. Furthermore, MD simulations explored the effect of smaller Au-NP (3 nm) and nanocluster (Au-NC with the size of 1 nm) on the binding sites of the BSA monomer. Although the formation of stable BSA-Au conjugates was revealed in the presence of NPs of different sizes, no specific protein binding sites were observed. Moreover, due to its small size, 1 nm Au-NC decreased helical content and hydrogen bonds in the BSA monomer, promoting protein unfolding more significantly. In summary, this combined experimental and computational study provides comprehensive insights into the interactions among Au nanosized substances, BSA, and physiological conditions that are essential for developing tailored nanomaterials with enhanced biocompatibility and efficacy.

Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

Role of 4-Phenylbutyric Acid in DNA and Protein Binding and its Functional Analysis

4-Phenylbutyric acid (4-PBA) is a small molecule known for its protein folding capacity to reduce endoplasmic reticulum (ER)-stress. This study aimed to explore the potential of 4-PBA by studying its interactions with DNA and protein and examining its effects on cellular toxicity and antibacterial activity. UV-VIS absorption spectroscopy demonstrated that 4-PBA effectively binds to calf thymus DNA (CT-DNA), as indicated by an evident hyperchromic shift, suggesting stable intercalating interactions. Similarly, the fluorescence quenching assay demonstrated that 4-PBA also interacts with bovine serum albumin (BSA), reducing fluorescence intensity by occupying specific binding sites on the protein. The cytotoxicity analysis using cell counting kit-8 further showed no significant reduction in cell viability of normal human lung epithelial cell line (L132). Subsequently, 4-PBA also exhibited minimal growth inhibition of Escherichia coli and Staphylococcus aureus bacterial strains, indicating limited antibacterial activity under the tested conditions. Additionally, this study provides a basis for future research towards the molecular mechanisms and therapeutic applications of 4-PBA.

Combining synergistic interaction and ion imprinting to improve adsorption capacity for selective uranium extraction from seawater

Abstract To ensuring the demand for uranium by utilizing unconventional uranium resources, the development of materials for selective capturing uranyl ions is increasingly important. Hence, the ion-imprinted polymer (IIP) based on specific binding sites was designed and prepared for selective enrichment of uranium from seawater. The existence of specific adsorption sites and the corresponding adsorption mechanism were confirmed by a series of experimental analyses and supported by density functional theory (DFT) calculations. Under the influence of seawater environment, the maximal uranium uptake of IIP reached 58.31 mg g−1. Significantly, the mass ratio of U and V (Sr or Ni) adsorbed by IIP was greater than 15, and the adsorption capacity did not change obviously after five cycles of use. The strategy combining ion imprinting and synergistic interaction is expected to improve uranium extraction performance.