Biochimica et Biophysica Acta (BBA) - Biomembranes

Staphylococcus aureus (S. aureus) is a clinically relevant pathogen capable of adapting its membrane composition in response to environmental stress. In this adaptive process, bacterial carotenoids play a crucial role. Although staphyloxanthin (STX) is the main carotenoid produced by the bacterium, S. aureus also synthesizes other pigmented intermediates that play an unknown role in regulating membrane biophysical properties. In this study, we purified 4,4-diaponeurosporenoic acid (4,4'-DNPA) from S. aureus carotenoid extracts and evaluated its effect on the thermotropic and biophysical properties of representative membrane models. The highly rigid triterpenoid 4,4'-DNPA is one of the last precursors in the biosynthesis of STX and is found in high concentrations in the stationary phase of S. aureus. Phase transition temperatures were determined using infrared spectroscopy, while interfacial hydration and hydrophobic core dynamics were investigated using fluorescence spectroscopy through Laurdan generalized polarization and DPH anisotropy. The results show that 4,4'-DNPA increases the main phase transition temperature of lipid bilayers in a concentration-dependent manner. This is in contrast to STX that decreases the transition temperature. This difference is consistent with the additional fatty acid present in STX that changes its effect on the phase behavior. Furthermore, 4,4'-DNPA reduced the interfacial hydration levels and restricted hydrophobic-core dynamics at higher concentrations, consistent with increased molecular order and stability. 4,4'-DNPA therefore complements STX in increasing membrane order and lipid packing. These findings support the notion that the production of bacterial carotenoids functions as a biophysical regulatory mechanism of lipid packing in S. aureus membranes.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Inflammation is a fundamental immune response but, when dysregulated, contributes to the pathogenesis of numerous inflammatory disorders. Although there are several conventional anti-inflammatory drugs which are effective, their long term use is often associated with adverse side effects, which highlights the need for safer alternative therapeutic drugs. Probiotic derived membrane vesicles (MVs) have recently emerged as biologically active nanostructures capable of modulating host immune responses. In the present study, MVs isolated from Lactobacillus acidophilus MTCC 10307 were evaluated for their anti-inflammatory efficacy and safety profile using in vitro and in vivo models. In RAW 264.7 macrophages, L. acidophilus MVs significantly attenuated lipopolysaccharide induced expression of the pro-inflammatory mediators Il-1{beta}, Il-6, and iNOS, accompanied by reduced nitric oxide and reactive oxygen species production which was abolished in the proteinase K treated MVs. The protein levels of NF{kappa}B and IL1{beta} were also reduced in the treatment groups. Repeated dose oral toxicity studies revealed no adverse effects, as evidenced by body weight and histopathological evaluation of major organs. The anti-inflammatory properties of L. acidophilus MVs were further validated in an in vivo hind paw edema model, which shows inflammation resolution demonstrated by molecular and histological analysis. Proteomic analysis using LC-MS/MS identified the presence of surface-layer protein A (SlpA) which is a potential bioactive component which might contribute to the observed immunomodulatory effects. Collectively, these findings demonstrate that L. acidophilus MVs exert potent anti-inflammatory activity while maintaining an excellent safety profile using integrated in vitro and in vivo models.

The conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f68004org.highwire.dtl.DTLVardef@14e9a98org.highwire.dtl.DTLVardef@18771d3org.highwire.dtl.DTLVardef@141fc6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dengue disease remains a significant global health threat, with current vaccines exhibiting variable efficacy and safety concerns. Virus-like particles (VLPs) offer a promising alternative by mimicking native virus structures without infectious genomes. We engineered a mammalian expression plasmid encoding Dengue-1 prM and E proteins, optimized for secretion using Japanese Encephalitis virus signal sequences, and transiently expressed it in HeLa cells. Purified VLPs exhibited spherical morphology ([~]39 nm diameter) consistent with native virions, as confirmed by transmission electron microscopy. Immunization of mice with these VLPs elicited robust Dengue-1 specific IgG antibody responses. Our study demonstrates production of immunogenic Dengue-1 VLPs in HeLa cells, highlighting their potential as a vaccine candidate and a tool for serodiagnosis. Further characterization of VLP epitopes and protective efficacy is warranted to advance vaccine development. ImportanceDengue remains a significant global health challenge, with serotype 1 being one of the dominant strains causing recurrent outbreaks in Nepal. Existing vaccines demonstrate limited efficacy and pose significant safety concerns, particularly in seronegative populations. To address these limitations, this study explores virus-like particles (VLPs) as a safer alternative vaccine platform. VLPs elicit robust immunogenicity by mimicking the structure of native virus while completely lacking genetic components. This study combines DENV1 structural proteins with optimized expression systems to enhance immunogenicity. This work is particularly significant as the first dengue vaccine research conducted in Nepal, directly addressing antigenic mismatches between existing commercial vaccines and locally circulating viral strains. Furthermore, the study provides scalable platform for developing region-specific dengue vaccines for other serotypes and flaviviruses.

BackgroundTo control leishmaniasis, chemotherapy drugs are currently under development. However, these drugs often exhibit poor efficacy and are associated with toxicity, adverse effects, and drug resistance. At present, no specific drug is available for the treatment of leishmaniasis. Meanwhile, vaccine research is ongoing. Recent studies have analysed some experimental vaccines using mathematical models. AimIn previous work, drug targeting was focused on the entire human body rather than specifically addressing infected macrophages and parasites. In our current approach, we aim to eliminate infected macrophages and parasites through nano-drug design. Specifically, we utilise two types of nanoparticles: iron oxide and citric acid-coated iron oxide. Moving forward, we plan to advance this strategy using mathematical modelling of macrophage-parasite interactions. MethodsWe design PDE-based models of macrophages and parasites, incorporating cytokine dynamics, to support nano-drug development. Drug efficacy is estimated using posterior distributions to analyse phenotypic fluctuations of macrophages and parasites during the design phase. We investigate implicit and semi-implicit treatment schemes, focusing on energy decay properties. To model drug flow during treatment, we introduce a three-phase moving boundary problem. Comparative analyses are conducted to evaluate macrophage and parasite behaviour with and without treatment. Finally, the entire framework is implemented within a virtual lab environment. ResultsThe results show that the nano-drug exhibits better efficacy compared to combined drug doses. We analysed and compared two types of nano-drug particles: iron oxide and citric acid-coated iron oxide. We discuss how the drug effectively targets and eliminates infected macrophages and parasites. ConclusionOur models results and simulations will support researchers conducting further studies in nano-drug design for leishmaniasis. These simulations are performed within a virtual lab environment.

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

The apparent pKa of ionizable lipids in lipid nanoparticles (LNPs) is a key determinant of RNA encapsulation during formulation and endosomal release after cellular uptake. However, it is difficult to predict the effective pKa of a given ionizable lipid solely from its solution pKa, because it is sensitive to the membranes composition, as well as solution conditions such as the salt concentration. We developed a simple continuum electrostatics model, based on Gouy-Chapman theory, to predict the shift in effective pKa for ionizable lipids in lipid bilayers as a function of salt concentration and membrane composition. We derive equations for the surface potential and fraction of lipids charged, which are solved self-consistently as a function of solution pH to extract the titration curve and effective pKa. The model shows that the shift in effective pKa is largest when the concentration of titratable lipid is high, and the effect is diminished by increasing salt concentration. We provide a python implementation of the model and an interactive notebook that will allow users to further easily explore the predicted pKa shifts as a function of formulation variables.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Calculation of density maps from atomic models is essential for structural studies using crystallography and electron cryo-microscopy (cryoEM). These maps serve various purposes, including atomic model building, refinement, visualization, and validation. However, accurately comparing model-calculated maps to experimental data poses challenges, particularly because the resolution of cryoEM experimental maps varies across the map. Traditional crystallography methods generate finite-resolution maps with uniform resolution throughout the unit cell volume, while most modern software in cryoEM employ Gaussian-like functions to generate these maps, which does not adequately account for atomic model parameters and resolution. Recent work by Urzhumtsev & Lunin (2022, IUCr Journal, 9, 728-734) introduces a novel method for computing atomic model maps that incorporate local resolution and can be expressed as analytically differentiable functions of all atomic parameters. This approach enhances the accuracy of matching atomic models to experimental maps. In this paper, we detail the implementation of this method in CCTBX and Phenix. SynopsisNew tools implemented in CCTBX and Phenix allow the calculation of variable-resolution maps through a sum of atomic images expressed as analytic functions of all atomic parameters, along with their associated local resolution.

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase (hPCC) and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryo-EM data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.

Reactive oxygen species generated during inflammation can oxidize viral envelope lipids, with outcomes ranging from modulated infectivity to viral inactivation. For SARS-CoV-2, the molecular mechanisms by which membrane lipid oxidation influences spike protein anchoring remain poorly understood. We use all-atom molecular dynamics (MD) simulations to quantify how graded oxidation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) affects the anchoring of the SARS-CoV-2 spike transmembrane (TM) region in an endoplasmic-reticulum-Golgi intermediate compartment (ERGIC)-like multicomponent membrane. Viral envelopes containing 0, 25, 50, 75, and 100% oxidized POPC (PoxnoPC) corresponding to 0 - 55% oxidation of all PO-type phospholipids were simulated with the spike TM helix and cytoplasmic tail embedded in a POPC/POPE/POPI/POPS/cholesterol mixture. Steered MD and umbrella sampling were used to calculate the potential of mean force (PMF) for extracting the TM+CT region along the membrane normal. Partial oxidation (25 - 75% POPC) produced reductions in the detachment barrier that were not statistically distinguishable from the native system within the sampling uncertainty, whereas full POPC oxidation lowered the anchoring free energy by about 23% (from 606 {+/-} 39 to 464 {+/-} 38 kJ mol-1), indicating that oxidation of roughly half of the glycerophospholipids can measurably weaken spike-membrane coupling. Despite this reduction, the remaining barrier (about 180kBT ) is still large, suggesting that oxidation alone may be insufficient for spontaneous spike detachment and likely acts synergistically with mechanical forces during fusion or immune engagement. Analysis of acyl-chain order parameters, area per lipid, membrane thickness, number-density profiles, and lateral lipid clustering reveals that POPC peroxidation decreases lipid order, thins and softens the bilayer, and disrupts cholesterol-stabilized clusters that refer to large cooperative lipid assemblies (>10 lipids) identified via RDF-based clustering. These oxidation-induced changes reduce hydrophobic matching around the TM helix and facilitate its extraction from the viral envelope. Our results provide a mechanistic link between lipid peroxidation, membrane nanostructure, and spike anchoring, supporting lipid oxidation for example during cold atmospheric plasma or ozone treatment as a physically grounded contributing antiviral mechanism against SARS-CoV-2.

1Long QT syndrome Type 2 (LQT2) is a genetic disorder caused by missense mutations in the KCNH2 gene that encodes the potassium channel KV11.1. Previous studies have shown that most KV11.1 missense mutations with loss-of-function phenotypes result from impaired trafficking from the endoplasmic reticulum to the plasma membrane. To investigate the molecular basis of these defects, we used molecular dynamics simulations to analyze two sets of disease-associated missense mutations: those that suppress and those that maintain normal channel trafficking. We focused initially on the conformational and dynamics differences between wild-type and several mutants of KV11.1 via molecular dynamics simulations when two K+ were placed in the selectivity filter (SF). Our study reveals that missense mutations in the S4 helix allosterically disrupt the selectivity filter, a critical determinant for proper channel trafficking. Trafficking-competent variants largely retained a wild-type selectivity filter structure, whereas trafficking-deficient mutants exhibited pronounced structural perturbations in this region. These findings suggest that certain LQT2-associated missense mutations in KCNH2 impair channel trafficking by compromising the structural integrity of the selectivity filter. We additionally found that second-site variants Y652C in the drug binding vestibule can correct structural defects associated with some mistrafficking variants.

-Synuclein (Syn) remodels cellular membranes through interactions that involve both its structured, membrane-binding N-terminal domain (NTD) and intrinsically disordered C-terminal domain (CTD). While the amphipathic NTD helix is known to insert into lipid bilayers and generate curvature, the contribution of the acidic CTD remains unclear. Here, we dissect the individual and cooperative roles of these domains using Supported Bilayers with Excess Membrane Reservoir (SUPER) templates to quantify membrane remodeling via membrane fission and membrane morphological deformations (i.e., membrane budding and tubulation). We show that both the NTD and CTD independently remodel membranes, while full-length Syn exhibits greater remodeling ability than either the NTD or CTD in isolation. This result demonstrates a synergistic amplification between helix insertion of the NTD and the tethered, disordered CTD. To further probe the mechanism of membrane remodeling by the CTD, we modulated the chain length of the protein, the bulk ionic strength of the solution (i.e., charge screening), and applied relevant polymer scaling laws for disordered proteins. Our results suggest that the membrane remodeling mechanism for the disordered CTD is electrostatic in nature, stemming from protein-protein repulsion at elevated binding densities. Together, our findings reveal a cooperative energetic mechanism in which N-terminal helix insertion biases membrane curvature and the disordered, C-terminal domain adds an additional electrostatic component that helps to overcome the free energy barrier for membrane bending.

Immunocastration has emerged as an alternative to surgical and chemical castration for managing reproductive function in animals, yet the development of safe and effective vaccines remains challenging. This study aimed to develop a gonadotropin-releasing hormone (GnRH)-based messenger RNA (mRNA) vaccine and systematically evaluate its immunogenicity, reproductive suppression efficacy, long-term durability, and biosafety in mice and cats. GnRH epitopes were fused to three carrier proteins, Fc, Foldon, and lumazine synthase nanoparticles (pLS) via a flexible linker. After identifying pLS as the optimal scaffold, three mRNA vaccine candidates (GnRH-3, GnRH-4, and GnRH-5) were generated with one, five, or ten tandem GnRH repeats, encapsulated in lipid nanoparticles (LNPs), and assessed in rodent and feline models. Immunogenicity was determined by enzyme-linked immunosorbent assay, gonadal histopathology, hormone measurements, transcriptomic analysis, and mating trials. Among the fusion partners, the pLS-based vaccine (GnRH-3) induced the strongest antibody responses and most pronounced reproductive suppression. Further optimization showed that GnRH-4, containing five tandem GnRH repeats, elicited the highest antibody titers, induced severe gonadal atrophy, and reduced litter size by 93.8% in mice. Transcriptomic analysis revealed that differentially expressed genes in males were enriched in spermatogenesis and motility pathways, whereas those in females were associated with RNA splicing and immune responses. In cats, the optimal regimen was a twoLdose schedule with 50Lg per dose and a 21Lday interval, which induced robust antibody responses lasting at least 12 Lmonths and sustained reproductive suppression. HighLdose (500Lg) administration showed no clinical toxicity or histopathological abnormalities, confirming favorable biosafety. This study successfully developed a pLSLbased GnRH mRNA vaccine (GnRH-4) with five tandem GnRH epitopes that demonstrates strong immunogenicity, longLlasting contraceptive effects, and excellent safety in both rodent and feline models, supporting its potential for clinical application in immunocastration.

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

BackgroundMultidrug-resistant (MDR) Gram-negative bacteria have triggered a critical global health crisis. Polymyxin lipopeptide antibiotics are used as a last-line therapy against these problematic pathogens, but their clinical use is largely limited by severe nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal proximal tubular cells, substantially contributing to their nephrotoxicity. However, it remains unclear how polymyxins interact with hPepT2. MethodsIn this study, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical and cell biology approaches. Bioinformatic modelling predicted the residues essential for the binding of polymyxins with hPepT2. Transporter mutagenesis and molecular analysis were employed to explore the role of each residue in the interaction of hPepT2 and polymyxins. Moreover, we synthesised a series of polymyxin-like analogues with altering the moieties that are critical for binding with hPepT2. The antibacterial activity and nephrotoxicity of these analogues were subsequently assessed. ResultsOur bioinformatic modelling proposed an outward-facing structure of hPepT2 with a possible transport pathway that polymyxins bind to the lateral opening site of hPepT2 (e.g. E214, D215, D317, D342, E622). Molecular assays for transporter function and expression confirmed that D215 residue of hPepT2 is critical for polymyxin binding, while several other residues significantly impact on transporter turnover rate and/or protein expression. Our experimental validations showed that the lipopeptide analogues with altering the Dab1, Dab3, Dab5 and Dab9 moieties of polymyxins demonstrated decreased interactions with hPepT2. Among these synthetic analogues, alanine substitution at Dab3 showed reduced nephrotoxicity in mice while reserved antibacterial activity against a range of bacterial strains. ConclusionsOverall, this proof-of-concept study demonstrated that the computationally predicted and experimentally validated polymyxin-hPepT2 SIR model provides a viable approach for the discovery of novel, safer lipopeptide antibiotics.