ACS Omega

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.

Multiple-drug resistant (MDR) Staphylococcus aureus strains (like methicillin-resistant S. aureus or MRSA) uses an arsenal of antioxidant enzymes to mitigate host-induced oxidative stress. Among them the non-canonical Staphylococcal glutathione peroxidase (SaGpx) plays a crucial role in bacterial redox homeostasis by reducing peroxides via thioredoxin-dependent pathways. Thus, enabling oxidative stress mitigation during host infection. Despite its importance in S. aureus, its role in bacterial pathogenesis remains unexplored. This study aimed to elucidate the possible role of SaGpx in Staphylococcal virulence. First, we determined the high-resolution crystal structure of SaGpx (at 1.65 [A] resolution) using X-ray crystallography. Guided by the catalytic cleft architecture of SaGpx, small-molecule based inhibitors were then rationally designed and synthesized. These inhibitors exhibited good binding affinity to SaGpx and complete enzymatic blockade. These inhibitors exhibited potent anti-S. aureus activity (MICs 6.25-31.25 M) along with no cytotoxicity in L929 fibroblast wound-healing assays. Furthermore, the in vivo antibacterial ability of these inhibitors was evaluated using S. aureus-infected skin wound mouse model, where these compounds show potent antibacterial and wound healing ability supported by subsequent histological as well as immunohistochemical analysis. These findings suggest SaGpx as a possible virulence determinant in S. aureus and position these synthesized inhibitors as promising antivirulence therapeutics. HighlightsO_LIThe high-resolution crystal structure of Staphylococcal glutathione peroxidase is solved. C_LIO_LIBased on the SaGpx catalytic site, ,{beta}-unsaturated ketoesters derivatives are synthesized. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives inhibit SaGpx activity and binds the protein at M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vitro antibacterial activity against S. aureus at low M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vivo antibacterial and wound healing ability S. aureus-infected skin wound mouse model. C_LI

Members of the transforming growth factor-{beta} (TGF-{beta}) superfamily, including inhibins and activins, are structurally related glycoprotein dimers that regulate reproductive and endocrine signaling. Their high degree of molecular similarity presents challenges for label-free analytical discrimination. To evaluate the ability of Raman spectroscopy to distinguish closely related TGF-{beta} superfamily proteins based on intrinsic vibrational fingerprints. Raman spectra of recombinant human Inhibin -subunit, Inhibin B ({beta}B homodimer), and Activin A ({beta}A--{beta}A) were acquired using confocal Raman microscopy with 532 nm excitation. Spectra were baseline-corrected, area-normalized, and analysed using principal component analysis (PCA). Distinct spectral signatures were observed across the 500--1800 cm-1 region. Differences within the S--S stretching region (500--550 cm-1) were consistent with variations in disulfide-bond environments, with the Inhibin -subunit exhibiting the highest relative intensity in this region. Variations in the amide I band (1600--1700 cm-1) suggested differences in protein secondary structure, while aromatic amino acid vibrations provided additional discriminatory features. PCA revealed clear clustering and separation of all three protein classes based on their Raman fingerprints. Raman spectroscopy enables label-free differentiation of structurally related endocrine glycoproteins and demonstrates potential for the structural characterization and classification of inhibin and activin proteins within the TGF-{beta} superfamily.

Heat shock protein 90 (HSP90) assists protein folding and maturation of many important signaling proteins. In various diseases, HSP90 clients contribute to aberrant signaling, and HSP90 inhibition is being explored as a potential therapeutic approach. Commonly researched HSP90 inhibitors target the ATP-binding pocket, thereby disrupting the ATP-induced closure of HSP90. Drugs disrupting the HSP90 ATPase cycle by targeting the closed state of the chaperone have not been developed. Here, we present de novo design and selection of protein binders interacting exclusively with the closed state HSP90. Two such binders, H2 and H4, were identified that feature a similar fold and comparable affinities for HSP90 but display different binding kinetics. The structures of the HSP90 complexes with H2 and H4 were determined by cryo-EM single particle analysis, and they revealed high accuracy of the BindCraft predictions. H2 and H4 compete with p23 at one but not both symmetrical p23 binding sites on HSP90. H2 expressed in HEK293T cells moderately elevated expression of HSP70 and had no effect on the HSP90 level, suggesting muted heat shock response. Overall, this study demonstrates that the de novo binders represent novel and promising tools to probe the potential utility of HSP90 inhibition by targeting its closed state.

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

Carbon monoxide (CO) poisoning is responsible for around 50,000 emergency department visits per year in the U.S. alone. With the present standard of care, persistent neurological sequelae occur in [~]30-40% of severe CO poisoning cases. Currently, there is no available targeted molecular antidote for CO poisoning. In previous work, we have developed an antidotal therapy for CO poisoning based on an engineered hemeprotein, human neuroglobin (Ngb-H64Q-CCC). Intravenous infusion of Ngb-H64Q-CCC removes CO from the circulating red blood cells and improves survival in a lethal CO-poisoning mouse model. However, the infusion of heme-containing proteins has inherent heme toxicity risks that may limit the dose that can be used safely without liver or kidney toxicity. In order to overcome these problems, we have investigated the development of immobilized Ngb in a solid matrix. This approach allows for the development of a CO removal system using an extracorporeal blood circulating system coupled with a stationary matrix with immobilized Ngb-H64Q-CCC. Such system avoids drug infusion and possible organ injury, allows for antidote recycling, and provides advantages for storage and handling of the antidote. By assessing the efficacy of Ngb-H64Q-CCC immobilized through different linkage strategies, we have identified N-hydroxysuccinimide agarose resin as a viable stationary phase. The immobilized protein shows preserved heme redox activity, can be chemically reduced/oxidized for activation/CO release purposes, and retains its CO removal capacity after successive regeneration cycles. We expect that this novel approach will advance the development of new scavenger-based therapies for CO poisoning.

Introduction: Thromboinflammation, which represents the pathological interplay between inflammation and thrombosis, is a leading cause of global mortality. Current therapies are frequently associated with an increased risk of bleeding and do not adequately address the inflammatory component of the disease. The African tree Guibourtia tessmannii represents a promising source of natural anti-inflammatory compounds. This study aimed to synthesize and characterize silver nanoparticles using an aqueous bark extract of G. tessmannii (GT-AgNPs) and to evaluate their anti-inflammatory and anticoagulant properties. Methods: GT-AgNPs were synthesized by reducing silver nitrate with an aqueous extract of G. tessmannii bark. The nanoparticles were comprehensively characterized using UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, and scanning electron microscopy. In vitro anti-inflammatory activity was evaluated through inhibition of bovine serum albumin denaturation, whereas in vivo anti-inflammatory activity was assessed using the carrageenan-induced rat paw edema model. Anticoagulant activity was investigated by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT), corresponding to the intrinsic and extrinsic coagulation pathways, respectively. Results: The synthesis successfully produced GT-AgNPs with an average particle size of approximately 20 nm. Both the aqueous extract and GT-AgNPs exhibited marked anti-inflammatory activity. The nanoparticles achieved 95% inhibition of protein denaturation in vitro and 95% inhibition of carrageenan-induced paw edema in vivo at a dose of 0.4 mg/kg body weight after 5 h. Furthermore, both the extract and GT-AgNPs demonstrated dose-dependent anticoagulant activity. Conclusion: The study demonstrated that GT-AgNPs, synthesized from the bark of G. tessmannii, possess significant anti-inflammatory and anticoagulant properties. These findings highlight the potential of GT-AgNPs as nanotherapeutic candidates for the management of thrombo-inflammatory disorders.

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that regulates diverse cytosolic acetylation through its two catalytic deacetylase domains and a C-terminal zinc finger ubiquitin-binding domain (ZnF-UBD). This ZnF-UBD mediates key protein-protein interactions (PPIs) that couple deacetylation and ubiquitin-dependent degradation. While most HDAC6 inhibitors target the catalytic domains, the ZnF-UBD represents an underexplored target. Here, we validate previously reported small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction and describe novel N-alkyl moieties based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone and phthalazinone scaffolds, a literature and modeling-guided scaffold hop revealed potential for an extended phthalazinone series. Results obtained both in fluorescence polarization (FP) and differential scanning fluorimetry (DSF) confirm this hypothesis. Additionally, late-stage diversification yields compounds with improved predicted physicochemical properties. Finally, machine-learning-based co-folding affinity predictions correlate with experimental IC{square}{square} rank order, highlighting their utility in PPI inhibitor design. These studies continue expanding the chemical space of HDAC6 ZnF-UBD inhibitors and build upon existing foundations for future therapeutic and mechanistic exploration of HDAC6- ubiquitin signaling.

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

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

Production of recombinant proteins frequently yields inclusion bodies that must undergo refolding to yield active protein. Here, we optimized the refolding conditions for the recombinant leucyl aminopeptidase (rPepL) from Lactocaseibacillus casei expressed in inclusion bodies from E. coli. Several chemical additives were assessed for how well they facilitated an increase in refolding efficiency. The best, 0.5 M L-arginine, yielded 50.8% refolding. The addition of stabilizers, such as sucrose and glycerol, with L-arginine further increased yields to 85%. Urea at lower concentrations (0.25-0.5 M) also facilitated an increase in the refolding yield when co-added with L-arginine, whereas guanidinium chloride inhibited it. Sugars and polyols exhibited dose-dependent effects, with ranges for optima also defined. Fluorescence spectroscopy verified enhancements in the refolding under the optimized conditions. Molecular dynamics simulation under mixed solvent conditions provided atomic insights about stabilizing interactions that are likely to facilitate increased refolding. The results show that a series of aggregation suppressors and protein stabilizers can, in a collaborative way, increase the refolding efficiency for the recombinant proteins from the inclusion bodies. The protocol with the optimization using the additives L-arginine, sucrose, and glycerol is an efficient method for the production of active rPepL. This article outlines the best refolding method to recover recombinant leucyl aminopeptidase from inclusion bodies of E. coli using L-arginine combined with sucrose and glycerol. The combined experimental observations and computational simulations elucidate the molecular process of additive-induced stabilization, which elucidates how aggregation inhibition and hydrogen-bonded stabilization act synergistically. The results presented herein answer both mechanistic understanding and experimental guidance for improving protein refolding.

Recombinant human lactopontin (rhLPN), an equivalent of human milk lactopontin, is of increasing interest for human nutrition applications due to its roles in mineral binding, gastrointestinal function and immune modulation. These properties depend strongly on post-translational modifications, particularly phosphorylation and glycosylation. Here, we report the production of rhLPN in Kluyveromyces lactis at laboratory and pilot scale and present a comprehensive molecular comparison with native human lactopontin (nhLPN) isolated from human milk. Mass spectrometry-based peptide mapping confirmed the primary structure and identified extensive phosphorylation, consistent with the native protein. Middle-up analyses demonstrated closely matched phosphoform distributions between rhLPN and nhLPN, while glycosylation profiling revealed a defined population of low-complexity O-glycoforms localized to the N-terminus. Functional assessment demonstrated substantially greater iron binding by phosphorylated rhLPN compared with dephosphorylated and non-phosphorylated forms. Similar phosphorylation-dependent behaviour was observed for bovine lactopontin, supporting a conserved role for phosphorylation in mineral interaction. Across five 750 L pilot scale batches, both phosphorylation and glycoform distributions were highly consistent, indicating robust process reproducibility. Together, these results demonstrate that rhLPN produced in K. lactis recapitulates key structural and functional attributes of nhLPN, supporting its suitability as a scalable ingredient for nutrition applications.

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

Conformational plasticity of RNAs plays important roles in recognizing RNA-binding proteins, and is often modulated by their binding partners. Here, we investigate RNA conformational preferences in a non-redundant dataset of 263 protein-RNA complexes to characterize the structural landscape associated with protein recognition. RNA dinucleotide segments are analyzed using seven backbone torsion angles ({delta}1, {varepsilon}1, {zeta}1, 2, {beta}2, {gamma}2, and {delta}2), two glycosidic torsion angles ({chi}1 and {chi}2) and the pseudo-torsion angle . Focusing on dinucleotide steps present in both interface and non-interface regions, we performed density-based clustering using selected backbone torsion angles to identify recurrent conformational states. We identify 28 distinct RNA dinucleotide conformers containing at least ten members each. Among these, eight conformers represent previously unreported nucleotide conformers (NtCs), including the transitional and the non-canonical states AB06, AB07, BB21, BB22, OP32, OP33, IC08 and IC09. Several of these conformers are preferentially enriched at protein-binding interfaces, suggesting their involvement in local conformational adaptation during protein-RNA recognition. The newly identified conformers span transitional A-B geometries, distorted B-like states, open conformations and compact intercalated structures, highlighting the remarkable structural plasticity of RNA in ribonucleoprotein complexes. Overall, this study expands the current understanding of RNA conformational space and provides a refined RNA dinucleotide conformer library for protein-RNA complexes. These findings will facilitate the identification of novel RNA structural motifs and improved RNA structural modeling, docking protein-RNA complexes and deep learning-based prediction frameworks for describing RNA tertiary structures.

Marine macroalgae are valuable sources of bioactive compounds. In this study, we thus investigated the chemical composition and biological activity of an extract from the green seaweed Ulva lacinulata, composed of small bioactive compounds. Comprehensive compositional analyses and high-resolution mass spectrometry revealed its diverse molecular profile composed in particular of peptides/amino acid derivatives, saccharides, low-chain fatty diacids, oxylipins and minerals. Its anti-inflammatory activity was assessed after 6 h pre-treatment in LPS-stimulated cultured RAW 264.7 macrophages, showing that it significantly and dose-dependently reduced the expression and/or secretion of pro-inflammatory cytokines such as TNF-alpha; and IL-6, and targeted the NF-kB signaling cascade. It modulated the SIRT1-AMPK signaling axis and increased the LC3-II/LC3-I ratio, supporting the activation of a controlled autophagic response. This work highlighted the potential of this marine-derived extract as a safe and effective functional ingredient for the development of functional food and/or dietary supplements targeting chronic low-grade inflammation.

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study reveals the role of the hypothetical protein (HP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural inhibitors that disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, critical for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations validated its stable 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 [A]). Virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (-9.1 and -9.0 kcal/mol, respectively) and forming hydrogen bonds, {pi}-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) confirmed complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 [A] and 8.13 [A]) compared to the control and apoprotein. The ADME/Tox profiles highlighted favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Principal component, probability density function, and binding free energy analyses underscore ligand-induced conformational stability. This study proposes the molecular characterisation of the HP and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.

Lysolipin I (1) is a highly bioactive xanthone with strong antibacterial and cytotoxic properties. Given the limited number of lysolipin analogues, discovery of new natural lysolipin derivatives is important for understanding their structure-activity relationships. A soil-derived Streptomyces sp. P8-2B18 harbors a putative lysolipin biosynthetic gene cluster and LC-MS based metabolomic analysis revealed the production of lysolipin I along with unreported analogues. Large-scale fermentation followed by isolation led to the discovery of four new analogues, lysolipins J-M (2- 5), the structures of which were elucidated by mass spectrometric and NMR spectroscopic data analyses. Lysolipin L features a five-membered lactam F ring, which was unprecedented in reported lysolipins. Lysolipin M has a novel skeleton, with an extra methyl (Me-36) and a glycosyl group replacing a 1,3-oxane ring in lysolipin I. While lysolipins I, J and K displayed strong activity against Staphylococcus aureus and Aspergillus flavus with MIC values ranging from 0.25 to 4 g/mL and lysolipin L showed only moderate activities, lysolipin M was inactive (>50 g/mL). Lysolipins I-K showed potent cytotoxic activity against prostate cancer cell lines LNCaP and C4-2B, with IC50 values in the submicromolar range. In contrast, lysolipin L exhibited no cytotoxicity and lysolipin M exhibited substantially reduced potency. Their broad, non-selective bioactivities restricted their applicability as therapeutic agents.

The rapid emergence of antimicrobial resistance, particularly among multidrug-resistant (MDR) and extended-spectrum {beta}-lactamase (ESBL)-producing Escherichia coli, necessitates the development of novel therapeutic strategies. In this study, we report the green synthesis and functionalization of silver nanoparticles (AgNPs) using Azadirachta indica leaf extract conjugated with amoxicillin (Amoxicillin-AI-AgNPs) to enhance antibacterial efficacy. The synthesized nanoparticles were characterized using UV-Vis spectroscopy, FTIR, XRD, DLS, SEM, EDAX, and TEM analyses, confirming the formation of stable, spherical, crystalline nanoparticles with an average size of [~]87 nm and a zeta potential of -28.73 mV. High conjugation efficiency ([~]94%) of amoxicillin with AgNPs was achieved after 96 hours of incubation. Antimicrobial activity assessed against 88 clinical MDR and ESBL-producing E. coli isolates demonstrated significantly enhanced efficacy of Amoxicillin-AI-AgNPs compared to amoxicillin alone, with minimum inhibitory concentrations (MIC) ranging from 1.56 to 6.25 {micro}g/mL and minimum bactericidal concentrations (MBC) between 25-100 {micro}g/mL. Cytotoxicity evaluation on HEK-293 cells revealed a relatively high IC50 value (382.14 {+/-} 6.59 {micro}g/mL), indicating low toxicity at antibacterial doses. The synergistic interaction between AgNPs and amoxicillin likely contributes to improved bacterial inhibition and overcoming resistance mechanisms. Overall, this study highlights the potential of plant-mediated antibiotic- nanoparticle conjugates as an effective and biocompatible approach to combat antibiotic-resistant bacterial infections.

Sickle cell disease (SCD) is caused by a single amino acid substitution in the {beta}S globin chain at 6th position (6E[->]V). This results in polymerization of deoxy state of sickle hemoglobin (HbS), followed by its precipitation and subsequent sickling of erythrocytes. These deformed cells can block small capillaries (vaso-occlusion), causing cardiovascular complications, ultimately leading to ischemia-reperfusion injury, severe oxygen deficiency, and progressive systemic damage. Occasionally, patients with SCD have been observed to produce exorbitantly high levels of fetal hemoglobin (HbF), which has been linked with the inhibition of HbS polymerization. One of the effects of hydroxyurea, the most commonly used therapeutic for SCD, is to elevate HbF levels. However, the mechanism of inhibitory role of HbF on HbS polymerization is largely unknown. This study attempts to gain insights into the mechanisms involved in this process by means of native mass spectrometry, ion mobility mass spectrometry, and hydrogen deuterium exchange-based mass spectrometry (H/DX-MS). The conformational flexibility of asymmetric hemoglobin, HbFS (2{gamma}{beta}S), for the observed regions in the tetrameric molecule appears to be more in the deoxy state as compared to the oxy state, eventually leading to reduced polymerization of sickle hemoglobin in patients with SCD that express elevated HbF levels.

Dendrimers are nanosized molecules with potential antimicrobial applications. This study evaluates the antibacterial and anti-biofilm properties of two cationic dendrimers, NVX-G6 (G6) and NVX-G9 (G9), against clinically relevant bacterial pathogens. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC99) were determined for Escherichia coli, Pseudomonas aeruginosa, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA). The synergy of dendrimers with ceftazidime and vancomycin was evaluated using checkerboard assays. Furthermore, biofilm formation inhibition assays and fluorescent microscopy were performed to assess dendrimer interactions with bacterial biofilms. The results indicate that G6 and G9 exhibit limited direct antibacterial activity at high concentrations (MIC > 1024 {micro}g/mL) but demonstrate synergistic effects when combined with ceftazidime against E. coli and P. aeruginosa (FIC < 0.5). Notably, both dendrimers penetrated and colocalized within established biofilms, with time-dependent reductions in biomass observed after extended incubation, suggesting a role in progressive biofilm disruption rather than acute inhibition of formation, although significant biomass reduction was not observed under standard assay conditions. These findings contribute to the understanding of dendrimer-antibiotic interactions and their implications in antimicrobial and nanomedicine therapy.