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The nematode Caenorhabditis elegans contains genes for two types of ferritin (ftn-1 and ftn-2) that express FTN-1 and FTN-2. We have expressed and purified both proteins and characterized them by X-ray crystallography, cryo-electron microscopy, transmission electron microscopy, dynamic light scattering, and kinetically by oxygen electrode and UV-vis spectroscopy. Both show ferroxidase activity, but although they have identical ferroxidase active sites, FTN-2 is shown to react approximately 10 times faster than FTN-1, with L-type ferritin character over longer time periods. We hypothesize that the large variation in rate may be due to differences in the three- and four-fold channels into the interior of the protein 24-mer. FTN-2 is shown to have a wider entrance into the three-fold channel than FTN-1. Additionally, the charge gradient through the channel of FTN-2 is more pronounced, with Asn and Gln residues in FTN-1 replaced by Asp and Glu residues in FTN-2. Both FTN-1 and FTN-2 have an Asn residue near the ferroxidase active site that is a Val in most other species, including human H ferritin. This Asn residue has been observed before in ferritin from the marine pennate diatom Pseudo-mitzchia multiseries. By replacing this Asn residue with a Val in FTN-2, we show that the reactivity decreases over long time scales. We therefore propose that Asn106 is involved in iron transport from the ferroxidase active site to the central cavity of the protein.

Ultrasmall gold nanoclusters (AuNC) show great promise for application in theranostics due to their unique optical and physicochemical properties; however, the associated nanotoxicology concerns need to be carefully considered because of their high diffusion across the cellular barrier. Herein, new insights into the role of surface modification of 2 nm AuNC on their toxicity with impact on the metabolism of COS-7 fibroblast-like cells are revealed. AuNCs are chemically modified with either a monodentate-thiolated molecule (AuNC-MHA) or a modified-bidentate sulfobetaine zwitterionic molecule (AuNC-ZwBuEt). Uptake and localization inside fibroblasts and the resultant influence on cell ultrastructure are carefully evaluated using scanning transmission electron microscopy (STEM) and cryo-soft-X-ray tomography (cryo-SXT). At concentrations of >= 25 mu g Au mL(-1), AuNC-ZwBuEt are cytotoxic toward COS-7 cells and are observed to cross the nuclear membrane. Cryo-SXT analysis shows that fibroblasts develop an acute stress response in the form of swollen mitochondria, nuclear membrane blebbing, and large cytoplasmic vacuoles as early as 1 h postexposure. By contrast, AuNC-MHA are not cytotoxic toward COS-7 cells. Endosomal escape and translocation of the AuNC-ZwBuEt into the nucleus and other organelles may be the cause for the observed cytotoxicity and highlight the need for further study of metal nanocluster-cell interactions.

Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs' specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.

The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.

Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.

Advances in electron microscopy (EM) such as electron tomography and focused ion-beam scanning electron microscopy provide unprecedented, three-dimensional views of cardiac ultrastructures within sample volumes ranging from hundreds of nanometres to hundreds of micrometres. The datasets from these samples are typically large, with file sizes ranging from gigabytes to terabytes and the number of image slices within the three-dimensional stack in the hundreds. A significant bottleneck with these large datasets is the time taken to extract and statistically analyse three-dimensional changes in cardiac ultrastructures. This is because of the inherently low contrast and the significant amount of structural detail that is present in EM images. These datasets often require manual annotation, which needs substantial person-hours and may result in only partial segmentation that makes quantitative analysis of the three-dimensional volumes infeasible. We present CardioVinci, a deep learning workflow to automatically segment and statistically quantify the morphologies and spatial assembly of mitochondria, myofibrils and Z-discs with minimal manual annotation. The workflow encodes a probabilistic model of the three-dimensional cardiomyocyte using a generative adversarial network. This generative model can be used to create new models of cardiomyocyte architecture that reflect variations in morphologies and cell architecture found in EM datasets. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Diabetic cardiomyopathy is a leading cause of heart failure in diabetes. At the cellular level, diabetic cardiomyopathy leads to altered mitochondrial energy metabolism and cardiomyocyte ultrastructure. We combined electron microscopy (EM) and computational modelling to understand the impact of diabetes-induced ultrastructural changes on cardiac bioenergetics. We collected transverse micrographs of multiple control and type I diabetic rat cardiomyocytes using EM. Micrographs were converted to finite-element meshes, and bioenergetics was simulated over them using a biophysical model. The simulations also incorporated depressed mitochondrial capacity for oxidative phosphorylation (OXPHOS) and creatine kinase (CK) reactions to simulate diabetes-induced mitochondrial dysfunction. Analysis of micrographs revealed a 14% decline in mitochondrial area fraction in diabetic cardiomyocytes, and an irregular arrangement of mitochondria and myofibrils. Simulations predicted that this irregular arrangement, coupled with the depressed activity of mitochondrial CK enzymes, leads to large spatial variation in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio profile of diabetic cardiomyocytes. However, when spatially averaged, myofibrillar ADP/ATP ratios of a cardiomyocyte do not change with diabetes. Instead, average concentration of inorganic phosphate rises by 40% owing to lower mitochondrial area fraction and dysfunction in OXPHOS. These simulations indicate that a disorganized cellular ultrastructure negatively impacts metabolite transport in diabetic cardiomyopathy. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

HYPOTHESIS: The ability exhibited by insect wings to resist microbial infestation is a unique feature developed over 400 million years of evolution in response to lifestyle and environmental pressures. The self-cleaning and antimicrobial properties of insect wings may be attributed to the unique combination of nanoscale structures found on the wing surface. EXPERIMENTS: In this study, we characterised the wetting characteristics of superhydrophobic damselfly Calopteryx haemorrhoidalis wings. We revealed the details of air entrapment at the micro- and nano scales on damselfly wing surfaces using a combination of spectroscopic and electron microscopic techniques. Cryo-focused-ion-beam scanning electron microscopy was used to directly observe fungal spores and conidia that were unable to cross the air-liquid interface. By contrast, bacterial cells were able to cross the air-water interface to be ruptured upon attachment to the nanopillar surface. The robustness of the air entrapment, and thus the wing antifungal behaviour, was demonstrated after 1-week of water immersion. A newly developed wetting model confirmed the strict Cassie-Baxter wetting regime when damselfly wings are immersed in water. FINDINGS: We provide evidence that the surface nanopillar topography serves to resist both fungal and bacterial attachment via a dual action: repulsion of fungal conidia while simultaneously killing bacterial cells upon direct contact. These findings will play an important role in guiding the fabrication of biomimetic, anti-fouling surfaces that exhibit both bactericidal and anti-fungal properties.

It is commonly accepted that nanoparticles (NPs) can kill bacteria; however, the mechanism of antimicrobial action remains obscure for large NPs that cannot translocate the bacterial cell wall. It is demonstrated that the increase in membrane tension caused by the adsorption of NPs is responsible for mechanical deformation, leading to cell rupture and death. A biophysical model of the NP–membrane interactions is presented which suggests that adsorbed NPs cause membrane stretching and squeezing. This general phenomenon is demonstrated experimentally using both model membranes and Pseudomonas aeruginosa and Staphylococcus aureus, representing Gram‐positive and Gram‐negative bacteria. Hydrophilic and hydrophobic quasi‐spherical and star‐shaped gold (Au)NPs are synthesized to explore the antibacterial mechanism of non‐translocating AuNPs. Direct observation of nanoparticle‐induced membrane tension and squeezing is demonstrated using a custom‐designed microfluidic device, which relieves contraction of the model membrane surface area and eventual lipid bilayer collapse. Quasi‐spherical nanoparticles exhibit a greater bactericidal action due to a higher interactive affinity, resulting in greater membrane stretching and rupturing, corroborating the theoretical model. Electron microscopy techniques are used to characterize the NP–bacterial‐membrane interactions. This combination of experimental and theoretical results confirm the proposed mechanism of membrane‐tension‐induced (mechanical) killing of bacterial cells by non‐translocating NPs.

The properties of casein micelles are known to be affected by modifications to the environment, such as variations in pH or the addition of salts, yet the scientific literature typically considers the effects of one factor at a time, while in industrial processes, several modifications are performed simultaneously. The aim of this study was to assess the impact of multifactorial environmental modifications on the colloidal, structural and rennet coagulation properties of casein micelles in a simplified model system. A key finding was that dense regions (~20 nm in size) could be released from the casein micelle. The addition of NaCl and CaCl2 had opposing effects, i.e. enhancing or limiting this micellar disruption, respectively. A decrease in pH had the strongest impact on the mineral balance, causing the colloidal CaP to solubilize and the micelle to swell. The rennet clotting time was impacted by variations in pH and NaCl content. Interestingly, a consideration of all three levels of casein micelle structure and their interactions was needed to explain variations in the firmness of rennet gels. This study illustrates the complex interplay of factors affecting micellar structure and improves our understanding of how micelles can be manipulated to control their properties.

Mesoporous metal-organic networks have attracted widespread interest owing to their potential applications in diverse fields including gas storage, separations, catalysis, and drug delivery. Despite recent advances, the synthesis of metal-organic networks with large and ordered mesochannels (>20 nm), which are important for loading, separating, and releasing macromolecules, remains a challenge. Herein, we report a templating strategy using sacrificial double cubic network polymer cubosomes (Im3̅m) to synthesize ordered mesoporous metal-phenolic particles (meso-MPN particles) with a large-pore (∼40 nm) single cubic network (Pm3̅m). We demonstrate that the large-pore network and the phenolic groups in the meso-MPN particles enable high loadings of various proteins (e.g., horseradish peroxidase (HRP), bovine hemoglobin, immunoglobulin G, and glucose oxidase (GOx)), which have different shapes, charges, and sizes (i.e., molecular weights spanning 44-160 kDa). For example, GOx loading in the meso-MPN particles was 362 mg g-1, which is ∼6-fold higher than the amount loaded in commercially available SiO2 particles with an average pore size of 50 nm. Furthermore, we show that HRP, when loaded in the meso-MPN particles (486 mg g-1), retained ∼82% activity of free HRP in solution and can be recycled at least five times with a minimal (∼13%) decrease in HRP activity, which exceeds HRP performance in 50 nm pore SiO2 particles (∼36% retained activity and ∼30% activity loss when recycled five times). Considering the wide selection of naturally abundant polyphenols (>8000 species) and metal ions available, the present cubosome-enabled strategy is expected to provide new avenues for designing a range of meso-MPN particles for various applications.

Exosomes are promising disease diagnostic markers and drug delivery vehicles, although their use in practice is limited by insufficient homogeneous quantities that can be produced. We reveal that exposing cells to high frequency acoustic irradiation stimulates their generation without detriment to cell viability by exploiting their innate membrane repair mechanism, wherein the enhanced recruitment of calcium ions from the extracellular milieu into the cells triggers an ESCRT pathway known to orchestrate exosomal production. Given the high post-irradiation cell viabilities (≈95%), we are able to recycle the cells through iterative irradiation and post-excitation incubation steps, which facilitate high throughput production of a homogeneous population of exosomes-a particular challenge for translating exosome therapy into clinical practice. In particular, we show that approximately eight- to ten-fold enrichment in the number of exosomes produced can be achieved with just 7 cycles over 280 mins, equivalent to a yield of around 1.7-2.1-fold/h.

The mechano-bactericidal activity of nanostructured surfaces has become the focus of intensive research toward the development of a new generation of antibacterial surfaces, particularly in the current era of emerging antibiotic resistance. This work demonstrates the effects of an incremental increase of nanopillar height on nanostructure-induced bacterial cell death. We propose that the mechanical lysis of bacterial cells can be influenced by the degree of elasticity and clustering of highly ordered silicon nanopillar arrays. Herein, silicon nanopillar arrays with diameter 35 nm, periodicity 90 nm and increasing heights of 220, 360, and 420 nm were fabricated using deep UV immersion lithography. Nanoarrays of 360-nm-height pillars exhibited the highest degree of bactericidal activity toward both Gram stain-negative Pseudomonas aeruginosa and Gram stain-positive Staphylococcus aureus bacteria, inducing 95 ± 5% and 83 ± 12% cell death, respectively. At heights of 360 nm, increased nanopillar elasticity contributes to the onset of pillar deformation in response to bacterial adhesion to the surface. Theoretical analyses of pillar elasticity confirm that deflection, deformation force, and mechanical energies are more significant for the substrata possessing more flexible pillars. Increased storage and release of mechanical energy may explain the enhanced bactericidal action of these nanopillar arrays toward bacterial cells contacting the surface; however, with further increase of nanopillar height (420 nm), the forces (and tensions) can be partially compensated by irreversible interpillar adhesion that reduces their bactericidal effect. These findings can be used to inform the design of next-generation mechano-responsive surfaces with tuneable bactericidal characteristics f

The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI–related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.

Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the ‘delayed death’ effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

BACKGROUND: With the advent of new high-throughput electron microscopy techniques such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion-beam scanning electron microscopy (FIB-SEM) biomedical scientists can study sub-cellular structural mechanisms of heart disease at high resolution and high volume. Among several key components that determine healthy contractile function in cardiomyocytes are Z-disks or Z-lines, which are located at the lateral borders of the sarcomere, the fundamental unit of striated muscle. Z-disks play the important role of anchoring contractile proteins within the cell that make the heartbeat. Changes to their organization can affect the force with which the cardiomyocyte contracts and may also affect signaling pathways that regulate cardiomyocyte health and function. Compared to other components in the cell, such as mitochondria, Z-disks appear as very thin linear structures in microscopy data with limited difference in contrast to the remaining components of the cell. METHODS: In this paper, we propose to generate a 3D model of Z-disks within single adult cardiac cells from an automated segmentation of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The proposed fully automated segmentation scheme is comprised of three main modules including "pre-processing", "segmentation" and "refinement". We represent a simple, yet effective model to perform segmentation and refinement steps. Contrast stretching, and Gaussian kernels are used to pre-process the dataset, and well-known "Sobel operators" are used in the segmentation module. RESULTS: We have validated our model by comparing segmentation results with ground-truth annotated Z-disks in terms of pixel-wise accuracy. The results show that our model correctly detects Z-disks with 90.56% accuracy. We also compare and contrast the accuracy of the proposed algorithm in segmenting a FIB-SEM dataset against the accuracy of segmentations from a machine

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.