Biochimica et Biophysica Acta (BBA) - Biomembranes

Significant cellular processes, including protein sorting, signal transduction, and pathogen entry, amongst others, are associated with membrane microdomains, also known as lipid rafts. Lipid rafts, due to their unique biophysical properties compared to their surrounding environment, which stem from their distinct lipid and protein profiles, have garnered interest in methods and techniques that tune their coexisting liquid-ordered/liquid-disordered state, aiming to disrupt or destabilize them. Since cholesterol stabilizes the membrane domain, cholesterol-depleting compounds like cyclodextrin can be used to destabilize and disrupt the membrane rafts. Overall, given the membrane rafts importance in biological processes, it is crucial to understand the biophysical factors that influence its stability. In this study, we present a new method for disrupting and dissolving lipid rafts in a model system of phase-separated supported lipid bilayer (SLB) patches composed of DOPC, DPPC, and cholesterol. Using fluorescence microscopy to monitor the liquid ordered (Lo) and liquid disordered (Ld) phases of the SLB patches, we observed that adding DOPC liposomes causes a transformation of the co-existing Ld and Lo phases into a single-phase bilayer. On the other hand, adding liposomes that match the lipid content of the phase-separated SLB patch increase the areas of the existing Ld and Lo phases. This work also offers a new method for redistributing raft-localized molecules, confirmed by tracking the redistribution of cholera toxin bound to GM1 after domain dissolution with DOPC liposomes. The work describes an alternative method for dynamically altering membrane composition and dissolving domains via liposome addition, rather than lipid depletion or exchange.

The plasma membrane is a laterally heterogeneous environment in which lipid organization plays a central role in regulating protein function. In model systems, this heterogeneity is often described in terms of coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases, commonly associated with the lipid raft concept. Despite extensive experimental and computational efforts, the molecular determinants governing protein partitioning between these domains remain poorly understood, largely due to the limited number of systems studied. Here, we address this challenge using a high-throughput computational approach, systematically analyzing the partitioning behavior of almost 5,000 helical transmembrane peptides in phase-separating lipid membranes. Across all simulations, we find that none of the peptides exhibit a clear preference for the Lo phase, while the vast majority partition into the Ld phase. This observation is consistent with experimental results in simplified membrane systems and suggests that commonly used ternary lipid mixtures may not fully capture the physicochemical environment governing protein sorting in biological membranes. In addition, we identify a subset of peptides that preferentially localize at the Lo/Ld interface. These interfacial peptides display distinct sequence characteristics, indicating that boundary localization is governed by specific combinations of residue composition and spatial arrangement rather than a single dominant feature. Overall, our results reveal that transmembrane helix partitioning in model membranes is dominated by a preference for disordered environments, with interfacial localization emerging as a distinct and potentially functional behavior.

Membrane association is governed by the thermodynamics of amino acid partitioning between water and the lipid bilayer. Here, we quantified amino acid side-chain insertion energetics in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer using unbiased molecular dynamics simulations. Equilibrium depth distributions of 28 analogs, including multiple protonation states, were converted into potentials of mean force (PMFs) by Boltzmann inversion. The resulting PMFs reproduced the main features of bilayer partitioning. Hydrophobic analogs favored the bilayer core, aromatic analogs were stabilized in interfacial regions, and polar or charged analogs remained unfavorable in the hydrophobic interior. A diglycine analog representing the peptide backbone behaved similarly to uncharged polar residues. Depth-dependent pKa profiles and orientational analyses further showed how protonation equilibria and aromatic-ring alignment influence insertion energetics. Agreement with experimental hydrophobicity scales supports the robustness of the approach. These results provide an efficient and internally consistent framework for characterizing bilayer insertion energetics and establish a reference for future studies in more complex lipid environments. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/723583v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@127b12org.highwire.dtl.DTLVardef@14de924org.highwire.dtl.DTLVardef@53b27org.highwire.dtl.DTLVardef@16e8ee4_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCEMembrane-associated proteins represent a large fraction of the proteome and include many major drug targets, yet quantitative understanding of their interactions with lipid bilayers remains limited. Here, we present an unbiased molecular dynamics framework for systematically determining amino acid side-chain insertion free energies in a model bilayer. By deriving potentials of mean force directly from equilibrium depth distributions, this approach enables internally consistent comparisons across residue classes and protonation states without biasing restraints. The resulting free-energy profiles reproduce established hydrophobicity trends and show how protonation equilibria and aromatic-ring orientation modulate bilayer partitioning. This scalable strategy provides a quantitative reference for residue-level membrane thermodynamics and establishes a foundation for extending insertion energetics to more diverse lipid compositions and more complex membrane-associated systems.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

In mammalian cells, lipid monolayers support the integrity of lipid droplets (LDs), organelles that function as storage for neutral lipids. Liver-targeting illnesses such as liver cancer interrupt normal LD metabolism and prompt changes in the chemical content of these organelles, which can have effects on structural and organizational behavior of the lipids. In LDs, liver cancer induces concentric crystalline phases of cholesteryl esters (CEs) and triglycerides near the NL-monolayer interface, which become more pronounced as CE concentration increases. Yet, there is little known about how this phenomenon may link to persistence of undigested LDs in liver cancer patients. To shed light on this, all-atom molecular dynamics simulations were used to model LD micropipette aspiration experiments and gain insight into the effect of CE concentration on partitioning, structural, and mechanical properties of LDs. We successfully model micropipette aspiration by application of constant surface tension laterally, which stretches lipid bilayers and monolayers as the magnitude increased. The results show increased phospholipid packing due to insertion of CE fatty tails into the monolayer. Increasing CE concentration induces a non-linear change in surface packing defects on the LDs, notable rigidification, and stiffness. Taken together, these insights improve our understanding of the physical properties at the LD monolayer-core interface during liver cancer progression.

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

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.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

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 molten globule (MG) state is an intermediate in the unfolding pathway of proteins, typically triggered by denaturing agents such as urea, extreme pH, high pressure, or heat. The microscopic details of such states are far from understood. Here we study the MG states in protein Hen Egg-White Lysozyme (PDB ID: 1AKI) using microscopic constant pH molecular dynamics (CpHMD) simulations and experiments across a wide pH range. We observe that the titratable residues act as key drivers of conformational fluctuations, promoting the emergence of MG states at extreme pH. These states display partial unfolding, and small global structural changes (< 7% deviation). Hydration around the fluctuating acidic residues shows reduced water density and weakened hydrogen bonding at low pH. At high pH, hydration around acidic residues increases relative to pH = 7, whereas hydration around basic residues decreases. The translational and rotational dynamics of hydration water also exhibit pronounced pH dependence: the translational diffusion coefficient (Dtrans) increases linearly with decrease in pH in acidic medium and increases linearly with increasing pH in the basic regime. The rotational diffusion (Drot) shows similar dependencies on pH except a break at pH {approx} 4 corresponding to acidic residue pKa values. Our results may be useful to identify ligand binding of lysozyme in extreme pH conditions.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

IntroductionMalaria is still a pressing global health challenge, especially in sub-Saharan Africa, where behavioral factors such as alcohol consumption may exacerbate its impact. The present study is aimed at investigating the pathogenesis of alcohol-exacerbated malaria in Plasmodium berghei-infected an animal model (mice). MethodsMale mice were separated into four treatment groups: control, alcohol control, P. berghei and P. berghei plus acute alcohol treatment groups. Animals were infected with malaria through intraperitoneal injection of P. berghei and an acute dose of ethanol (20% v/v) was introduced 48 hours post-infection. Parasitaemia was monitored using the Giemsa-stained thin blood smears. Haematological parameters were assessed using automated blood analyser. Liver function was evaluated by measuring serum levels of AST and ALT and cytokine profiles (TNF-, INF-{gamma}, IL-6, IL-1{beta}) were quantified using ELISA kits. ResultsResults show that acute alcohol intake led to a significant increase in parasitaemia in the P. berghei group (p<0.01). Haematological analysis revealed a significant (p<0.001) reduction in RBC count, haemoglobin levels, haematocrit percentage, platelet count and others in the P. berghei plus acute alcohol group. Liver enzyme assays revealed an elevated AST and ALT levels (p<0.001) in the P. berghei group. Cytokine analysis revealed a significant (p<0.01) upregulation of pro-inflammatory cytokines (TNF- INF-{gamma}, IL-1{beta} and IL-6), due to acute alcohol. These results suggest that alcohol exacerbates malaria pathogenesis by increasing parasitaemia, promoting immune dysregulation and liver injury, mediated by a shift toward a pro-inflammatory cytokine profile.

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.

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

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

Liposomes are self-assembled lipid vesicles capable of encapsulating both hydrophilic and hydrophobic therapeutics, making them versatile platforms in drug delivery and biomedical technology. In this study, the limitations of the classical thin-film hydration method were critically evaluated, and a sustainable, systematically optimized strategy was established for generating defined liposomal lamellar phases. Hydration conditions were optimized, and 4 mL of buffer per 10 mg of lipid was determined to be optimal for effective rehydration and improved statistical reliability of vesicle measurements. A refined probe-sonication protocol (20% amplitude, 5 s ON/55 s OFF pulse) enabled controlled transformation of multivesicular vesicles into stable multilamellar and unilamellar vesicles at net ON-times of 90 s and 185 s, respectively, without overheating or contamination. In addition, a Python-based machine-learning tool was developed for vesicle size characterization. Collectively, these optimizations provided a reproducible and sustainable framework for preparing liposomes across different lamellar phases.

Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of [~]9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2{degrees}C proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to optimize PISA then apply the optimized conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, remains a significant public health and veterinary problem in endemic regions. Although chemotherapy and control programs exist, the development of complementary immunotherapeutic tools is increasingly needed. This study evaluated the generation and functional activity of hyperimmune serum (HIS) produced in three adult male castrated llamas (Lama glama) immunized with antigenic material derived from protoscoleces (PSCs) of the parasite. Sera collected after each of the first six immunizations were assessed by ELISA to quantify antigen-specific IgG responses, and their biological effects were tested in vitro using viable PSCs. Motility was measured using video-assisted paired-image scoring across serial serum dilutions (1:2-1:2048), and the methylene blue exclusion assay was used to assess viability. Hyperimmune serum produced a clear, reproducible, dose-dependent inhibition of PSC motility and viability. Higher titers of early inoculations reduced motility by 70-85%, while sera from the fifth and sixth inoculations achieved complete suppression. Naive serum and PBS controls showed no inhibitory effect. ELISA titers strongly correlated with biological activity, indicating that higher humoral responses predicted functional inhibition. These findings demonstrate the feasibility of generating potent anti-Echinococcus granulosus polyclonal antibodies in camelids and support their potential application in passive immunization strategies. The study establishes a foundation for future development of llama-derived immunobiological reagents, including nanobody-based tools, for the control of cystic echinococcosis.

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

Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 {micro}L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples from neonatal intensive care units. For CS quantification, the CS is first enzymatically depolymerised using chondroitinase ABC to release repeating disaccharide units. Matrix complexity is reduced via two rounds of acetonitrile-based protein and lipid precipitation. Disaccharides are separated by hydrophilic interaction liquid chromatography and detected using a Triple Quadrupole Mass Spectrometer, providing robust sensitivity for all CS disaccharides. Method development and validation were performed using pooled mature human milk from term infants. This workflow facilitates detection of all CS disaccharides, with low but reproducible recoveries for total CS. Low- and high-level spike recoveries were 41.3% (RSDr 7.5%, RSDiR 15.9%) and 43.7% (RSDr 24.4%, RSDiR 27.9%), respectively. Despite modest absolute accuracy, precision remained sufficient to make relative comparison of CS concentrations between samples. This method expands the analytical toolkit for human milk glycomics, enabling same day preparation and CS profiling from sample volumes that are 200 times smaller than prior work, supporting future investigations into GAG-mediated functions in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723732v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@176dffborg.highwire.dtl.DTLVardef@16ae4ccorg.highwire.dtl.DTLVardef@d333c2org.highwire.dtl.DTLVardef@1eb3216_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Schematic of sample preparation protocol 25 L of human milk is combined with lyase enzymes and TRIS buffer containing the internal standard prior to incubation. Samples then undergo multiple rounds of centrifugation and refrigeration before analysis via LC-MS/MS. Made using BioRender.com. Glycan nomenclature following Varki et al., 2015. C_FIG