ACS Omega

The accumulation of amyloid-beta (A{beta}) plaques is a hallmark of Alzheimers disease (AD), with A{beta}42 representing the predominant and most aggregation-prone isoform. Reliable preparation of monomeric A{beta}42 is essential for investigating the kinetics and mechanisms of its aggregation into oligomers and fibrils. This study provides a direct comparison of two monomerization protocols for recombinantly expressed A{beta}42: one incorporating size-exclusion chromatography (SEC) and the other relying solely on chemical denaturation, using agents such as NaOH and NH4OH. A{beta}42 was produced in E. coli, purified through urea solubilization followed by HPLC, and subjected to monomerization via the respective methods. Monomeric preparations were evaluated using Thioflavin T (ThT) fluorescence to assess aggregation kinetics, TEM to detect fibrils and preformed aggregates, and NMR spectroscopy. SEC-isolated monomers displayed sigmoidal aggregation profiles in ThT assays, featuring distinct lag, growth, and plateau phases consistent with secondary nucleation-dominated models as determined by AmyloFit analysis. Increasing the initial peptide concentration resulted in higher fibril yields, which was further supported by TEM images showing extensive fibrillization following incubation. In contrast, non-SEC preparations containing pre-existing aggregates detectable by TEM and showed attenuated NMR signals, leading to impaired aggregation behavior. NaOH-denatured samples predominantly exhibited flat ThT curves, whereas NH4OH-denatured samples displayed extended lag phases. NH4OH performance better than NaOH, likely because its gradual pH neutralization reduced peptide structural perturbation. Overall, these findings demonstrate that SEC is critical for obtaining highly pure monomeric A{beta}42 and improving the reproducibility of aggregation assays, highlighting the importance of standardized monomer preparation protocols in AD research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=49 SRC="FIGDIR/small/724608v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a3b9caorg.highwire.dtl.DTLVardef@1fa85d2org.highwire.dtl.DTLVardef@67a83dorg.highwire.dtl.DTLVardef@1564f77_HPS_FORMAT_FIGEXP M_FIG C_FIG

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

BackgroundRising antimicrobial resistance rates, require new therapeutic approaches such as antimicrobial peptides (AMPs), which are part of the innate immune defense, as alternatives to antibiotics. In this study, we aim to unravel the antibacterial activity of human histone H1.2 peptide against Pseudomonas aeruginosa and its potential immune modulatory role. MethodsWe used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen sequences of histone H1 were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. SYTOX green assay, electrophoretic mobility shift assay, and differential proteomics assays were conducted to determine the mode of action of H1.2 peptide fragment. A crystal violet assay was performed to evaluate the inhibition of biofilm formation. The cytotoxicity of the peptide was tested in LDH and Alamar assays. Finally, to visualize the contributions of H1.2 in NETs formation, scanning electron microscopy was performed. ResultsThe H1.2 peptide inhibited the growth of P. aeruginosa in a dose and pH-dependent manner without cytotoxicity towards mammalian THP-1 cells. It acts on intracellular targets to inhibit the growth of P. aeruginosa. STRING analysis from the differential proteomics assay showed that H1.2 targets the downregulation of proteins involved in the biogenesis of outer membrane proteins, including the folding and trafficking of outer membrane proteins across the cytoplasmic membrane. Scanning electron microscopy images showed that H1.2 forms NET-like structures capable of trapping and immobilizing P. aeruginosa. ConclusionThe characterized antimicrobial activity of H1.2 points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/724237v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1778ddborg.highwire.dtl.DTLVardef@26430org.highwire.dtl.DTLVardef@ffbfa2org.highwire.dtl.DTLVardef@7e38ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Sec transport and BAM complex system including chaperone proteins and quality control proteases are inhibited by H1.2 in Pseudomonas aeruginosa.Outer membrane proteins (OMPs) are synthesized in the cytoplasm and transported across the inner membrane via the Sec translocase, assisted by SecA/SecB or ribosomes. In the periplasm, they are escorted by chaperones such as SurA to the BAM complex for insertion into the outer membrane. Here, we show that H1.2, an antimicrobial peptide, targets membrane biogenesis in P. aeruginosa through downregulating Sec translocase (SecA/SecB and SecYEG), SurA, and BAM complex. Therefore, leading to improper transfer, folding and insertion of OMPs into the outer membrane. Normally, misfolded proteins are degraded by the protease MucD to prevent toxic aggregation in the bacteria. However, with H1.2 inhibiting MucD the proteotoxic stress is exacerbated, ultimately compromising bacterial homeostasis and viability. Figure created using BioRender.com.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

The standard set of 20 genetically-encoded amino acids (C20) exhibits a statistically non-random distribution in primarily two structurally-relevant physicochemical properties: hydrophobicity and molecular volume, and to a lesser extent charge. It remains an open question, however, whether evolutionary pressures similarly optimized the same alphabet for the distribution of functionally-relevant properties, such as reactivity. In this study, we used semi-empirical quantum chemistry simulations to calculate the highest occupied molecular orbital and lowest unoccupied molecular orbital (HOMO-LUMO) gaps for 84 xeno amino acids and constructed 10 million random 20-mer amino acid alphabets to determine where C20 fit amongst this background. The HOMO-LUMO gap measurements demonstrated that C20, similar to hydrophobicity and volume, also exhibits a non-random distribution. However, unlike hydrophobicity and volume, this distribution is non-random across an unevenly broad range. The results expand upon previous theory and suggest HOMO-LUMO gap energies as one synthetic biologists may consider when developing novel protein design tools or designing functional xeno amino acid alphabets. HighlightsO_LILifes amino acid alphabet is non-randomly distributed within an expanded computationally-generated chemistry space generated from large-scale quantum chemistry simulations. C_LIO_LIAmino acid alphabet coverage theory applies beyond structurally-relevant physicochemical descriptors to include functionally-relevant properties like reactivity as measured by frontier molecular orbitals C_LIO_LIFindings here provide a theoretical framework to guide the design of novel proteins and development of synthetic amino acid alphabets. C_LI

This paper presents the results of a structural analysis of chlorogenic acid isolated from a 70% ethanol extract of red clover (Trifolium pratense) callus culture. X-ray phase analysis showed that the sample was crystalline and single-phase and crystallized in an orthorhombic unit cell with the following parameters: a = 36.7548(5) [A], b = 11.0770(3) [A], c = 7.7947(2) [A], V = 3173.46(11) [A]3, R-Bragg = 0.347 %, Rexp = 4.75 %, Rwp = 5.83 %, Rp = 4.39 %, GOF = 1.23 %. NMR spectroscopy data (1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC) confirmed that the chemical structure and purity of the sample fully corresponded to chlorogenic acid, with no chemical impurities detected. Complete proton and carbon atom assignments are provided.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Silver has been used as a biocide for centuries, mostly in health-oriented applications. However, as a biocide, silver is toxic not only to its intended targets, mainly bacteria and fungi, but also to all living cells. Because of this toxicity, it is desirable to use forms of silver that maximize the required biocidal activity while minimizing the amount of silver that will be released in the environment at the end of life of the product. Silver nano objects are a good compromise for such requirements. The high surface to volume ratio allows for good reactivity and thus good biocidal activity, while the small amount of silver present in nano objects allows for a limited environmental release at the product end of life. In this work, we tested three types of silver nano objects. The first type, polyvinylpyrrolidone-coated silver nanoparticles (nAg-PVP) were used as a control nanoparticle, as this type of nanoparticle is now widespread. We also manufactured and tested maltodextrin-coated silver nanoparticles (nAg-MD) and micrometric (20 {micro}m in two dimensions and a few nanometers in the third one) silver flakes ({micro}AgSF). For these three silver nano objects, we investigated the biocidal activity by stringent tests using both Staphylococcus aureus and Escherichia coli as target bacteria. In addition, we investigated toxicity on mammalian macrophages or keratinocytes cell lines, as well as on an insect hemocyte cell line. Our results showed that the two innovative silver nano objects (nAg-MD and even more {micro}AgSF), showed both a better bactericidal activity and a lesser toxicity than the reference nAg-PVP nanoparticles. In addition, we also checked that beyond toxicity, the silver nano objects did not induce an inflammatory reaction, making them safer to use.

Prodigiosin is a red pigment produced by various bacteria, including Serratia marcescens. Despite its wide and promising range of biological activities, the large-scale production of prodigiosin is currently limited by its high cost and low yields. Here we propose and optimize an innovative, low-cost, peanut-based solid culture medium that enhances the yield of prodigiosin produced by Serratia marcescens. Colorimetric assays revealed that peanut significantly stimulates prodigiosin synthesis. Further HPLC-MS analysis allowed us to unambiguously identify prodigiosin and shows that our medium specifically improves the yield of prodigiosin. Overall, our innovative culture medium could help lower prodigiosin production costs and, ultimately, open new industrial applications.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18084f7org.highwire.dtl.DTLVardef@99a102org.highwire.dtl.DTLVardef@19a64forg.highwire.dtl.DTLVardef@1f59cff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a88cb5org.highwire.dtl.DTLVardef@adda98org.highwire.dtl.DTLVardef@1ea587corg.highwire.dtl.DTLVardef@574a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

The patch-clamp experimental technique is widely used to study the electrical properties of ion channels in biological and artificial lipid membranes. The key to the high quality of the experiments is the manufacturing of glass pipettes that provide highly electrically resistant contact between the edge of the pipette tip and the lipid bilayer. Preparation of the pipettes is particularly challenging for studies of the mitochondrial membranes due to the need for very small pipette tip sizes. Here, we present a robust procedure for producing pipettes suitable for experiments with native mitochondrial membranes. This procedure involves a two-step approach: initial fabrication of relatively large glass micropipettes using a standard micropipette puller, followed by tip refinement using a microforger to achieve smooth glass surface and reduced opening size. Pipette tip diameters and surface structure were examined using field emission - scanning electron microscopy (FE-SEM) imaging to assess the effects of variable parameters on pipette geometry and size. The resulting pipettes were validated in patch-clamp recording of the mitochondrial inner membranes. This approach enables the reproducible production of optimized pipettes for mitochondrial patch-clamp experiments, improving the quality and throughput of electrophysiological recordings of the mitochondrial ion channels.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is well known for life-threatening acute infections among the human population. The bacterium can withstand most antibiotics by using their high levels of inherent and acquired resistance mechanisms such as Biofilm-EPS, Persistence, and Quorum sensing (QS). Owing to the importance of adaptive antibiotic multi-drug resistance of P. aeruginosa, the current investigation is aimed to explore the phytochemicals derived from mangrove plants as potential agents to control biofilm and drug resistance mechanisms through a multi-mechanistic computational approach. For identifying potential compounds and target, In-silico drug repurposing technique is implemented by docking/virtual screening of 49 phytochemical compounds against 18 proteins involved in the Persister Cell formation, QS, and EPS synthesis in P. aeruginosa which resulted the proteins RelA and SpoT (persistence), PqsA, and PqSR (QS), and PelA and PelB (EPS synthesis) and compounds Taraxerone and Taraxerol to be potential. The results of docking were well corroborated with MD simulations. These targets and compounds explored through in-silico approach, are found to target potential antimicrobial pathways involving EPS synthesis, persistence genes, and QS, aiming to enhance antibiotic efficacy. Further, this study could be reference for in-vivo and in-vitro investigations to evaluate the further effectiveness of the compounds and potentiality of the proteins for MDR therapeutics of P. aeruginosa.

Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.

This study presents the structural verification of baicalin isolated from a hydroethanolic extract of an in vitro Scutellaria baicalensis root culture using X-ray diffraction analysis and a set of NMR spectroscopy techniques. The crystalline molecular structure of the sample was found to correspond to baicalin. The 1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC spectra confirmed that the chemical shifts, signal multiplicities, integral intensities, and spin-spin coupling constants were fully consistent with the structure of the target compound. Minor impurity signals were detected in the aliphatic region of the spectra, with a total content not exceeding 5 mol%. These results confirm the high purity and structural individuality of baicalin, a biologically active flavonoid glycoside of considerable interest.