Biology subjects

Price, M.

Publications and source records attributed to Price, M..

18 recordsLinked to original sources

bioRxiv2026

In utero Bisphenol A exposure developmentally reprograms mammary gland fibroblasts

In utero exposure to estrogens is known to increase breast cancer risk in adulthood. The plasticizer bisphenol A (BPA) increases tumor susceptibility in rodent models of in utero exposure, but the mechanism of this increase is unclear. Our lab has previously shown that in utero BPA exposure causes alterations to the tissue microenvironment that are conducive to tumor initiation. We thus sought to understand when these alterations arise and how they are regulated using bulk and single-nuclei epigenomics in adult mammary glands following in utero BPA exposure. Our results indicate that in utero BPA exposure causes developmental reprogramming of mammary fibroblasts that is initiated during puberty and persists into adulthood. Fibroblast-specific changes in chromatin accessibility were identified at enhancers and annotated to genes involved in fibroblast differentiation, suggesting that BPA may exert its effects by reprogramming fibroblast heterogeneity. Single-nuclei multiomics confirmed changes in fibroblast heterogeneity and uncovered estrogen signaling as a putative regulator of the fibroblast cell state that is altered by BPA exposure. Together, these studies support a model whereby BPA acts on mesenchymal ER to reprogram the chromatin landscape of mammary fibroblasts, leading to their altered differentiation.

molecular biology↗

bioRxiv2026

Chronic ER Stress Disrupts Mitochondrial-Associated ER Membrane Integrity in Corneal Endothelial Cells.

PurposeFuchs endothelial corneal dystrophy (FECD) is an age-related degenerative disease of the corneal endothelium cells (CEnCs), affecting 4% of the US population over 40. While Endoplasmic reticulum (ER) and mitochondrial stress have been independently associated with FECD pathogenesis, few studies have examined ER-mitochondrial interactions/ER-mitochondrial contact sites/mitochondria-associated ER membrane (MAM), or MAM proteins, and their contribution to ER and mitochondrial stress in FECD. This study aims to characterize alterations in MAMs and identify key MAM proteins associated with ER and mitochondrial stress in FECD. MethodHuman corneal endothelial cell line (HCEnC-21T) and Fuchs corneal endothelial cell line (F35T) were cultured and subjected to ER stressor tunicamycin (1, 10 g/ml) for 6 and 24 hours. MAM proteins were isolated by subcellular fractionation, and key ER and mitochondrial-damage-sensor proteins, such as PERK and Parkin, respectively, were identified by immunoblotting. ER-mitochondrial contact sites were quantified using the MAM plasmid and transmission electron microscopy (TEM) in normal and Fuchs cell lines, as well as in human tissues under chronic ER stress. ResultsER-mitochondrial contact distance significantly increased in Fuchs tissues compared with normal tissues, and a similar increase was observed in 21T cell line after tunicamycin treatment. There was a significant increase in the intensity of the MAM plasmid upon tunicamycin treatment at 6 hours in the 21T cell line compared to the non-treated control. However, MAM plasmid intensity significantly decreased at 24 hours compared to 6 hours post-tunicamycin treatment in 21T cell line. Analysis of MAM function by quantifying phosphatidylserine synthase 1 (PSS1 [gene PTDSS1]) expression in 21T cells showed a reduction in PTDSS1 expression after 24 hours of tunicamycin treatment. ER stress protein PERK and mitochondria damage sensor protein (Parkin) significantly increased in the MAM fraction after tunicamycin at 24 hours in 21T cell line. ConclusionsFuchs cell lines and tissues demonstrate decreased ER-mitochondrial interactions/MAMs, which are also seen in 21T cell line after chronic ER stress. Under chronic ER stress, ER and mitochondrial stress mediator proteins are translocated to MAM. This study highlights the importance of MAMs as a potential mediator of ER-mitochondria crosstalk in degenerating corneal endothelial cells for FECD.

cell biology↗

bioRxiv2025

Administration of short-chain fatty acids reduces bacterial translocation to peripheral organs and the brain, preserves gut barrier integrity, and mitigates brain injury following stroke

Ischemic stroke leads to neuronal death, neuroinflammation, blood-brain barrier breakdown, and disruption of the brain-immune-gut (BIG) axis. Despite improvements in recanalization treatments, stroke remains the second leading cause of death globally. Post-stroke infections (PSIs) are the main life-threatening complication (30-45% of all patients and 20% of mortality) after stroke. Traditionally attributed to nosocomial infections /associated medical procedures, ground-breaking research has revealed that PSIs primarily originate from bacterial translocation (BT) following gut barrier disruption after stroke. Despite the high mortality associated with PSIs, current treatments, including antibiotic prophylaxis, are largely ineffective, underscoring the urgent need for a better understanding of their aetiology. Short-chain fatty acids (SCFAs), microbiota-derived metabolites produced by bacterial fermentation of fibres, are key regulators of the BIG axis. SCFAs exert neuroprotective, anti-inflammatory, and antimicrobial effects across models of neurodegenerative and infectious diseases. Using a preclinical stroke model (transient middle cerebral artery occlusion, MCAO) in 12-week-old male mice, we evaluated the effects of SCFA administration starting 24 h after stroke until sacrifice at day 4 on BT, gut integrity, and brain injury. Our findings confirmed that stroke induces BT to several organs (e.g., liver, heart, spleen) and, for the first time, demonstrated bacterial presence in the brainstem and the ischemic core of the injured brain. Importantly, we also showed that SCFA treatment significantly reduced BT to several organs. In addition, SCFAs modulated gut barrier integrity, reduced brain lesion size, and improved functional recovery. These findings highlight the crucial role of SCFAs in the BIG axis following stroke and their potential to mitigate not only gut barrier disruption and brain injury, but also infection-related complications, offering a promising therapeutic strategy for one of the least understood yet most lethal complications of stroke.

physiology↗

bioRxiv2025

Gait adaptation to asymmetric foot-ground compliance applied by robotic footwear

Perturbing foot-ground interaction dynamics has shown promise for eliciting adaptations to inter-limb weight-bearing symmetry, a critical target for rehabilitation of asymmetrical neuromotor deficits affecting gait. To date, this perturbation paradigm has been delivered via adjustable stiffness treadmills, in which the treadmill deck displaces under the walkers foot during the stance phase of gait. Recently, we developed robotic footwear capable of delivering asymmetrical ground stiffness perturbations during walking, offering a portable experimental platform for asymmetrical ground stiffness perturbations, allowing it to be executed overground and on conventional treadmills. In this study, we quantified kinetic and spatio-temporal gait adaptation to a foot-ground compliance perturbation delivered by the novel robotic footwear. We found that participants shifted their vertical ground reaction force impulse, vertical pushoff peak, and peak braking force toward the unperturbed limb after the perturbation was removed, indicating a shift in neuromotor control elicited by the perturbation. We conclude that the robotic footwear can elicit weight bearing adaptations similar to adjustable stiffness treadmills, and that the dissipative mechanical properties of the shoes likely play a key role in the direction of the adaptation.

neuroscience↗

bioRxiv2025

Towards active vaccination against tumour endothelial marker Robo4

Targeting tumour antigens is a major challenge in cancer-immunotherapy. We use active vaccination to induce antibodies targeting self-antigen Robo4, which is selectively expressed on tumour vascular endothelium, supporting vascular development. Our previous work showed that a conjugate of Robo4 with a foreign carrier protein can induce autoantibodies specific to Robo4, which inhibited angiogenesis and tumour growth. The current project aims to translate the vaccine protocol to exploit a carrier protein used in routine human vaccination schedules. The well-characterised, non-toxic fragment C of tetanus toxin (TTc) was selected as the carrier protein. Here we show that priming with the carrier TTc followed by boost with Robo4-TTc (R4-TTc) efficiently induces strong antibody responses to Robo4 and inhibits tumour growth in LLC1 and 4T1 tumour models. The growth inhibition was correlated with anti-Robo4 IgG1 titres. Furthermore, we observed decreased vessel formation and increased immune cell infiltration in tumours from R4-TTc vaccinated mice in the absence of detectable adverse effects on health. The data indicate that this vaccination strategy remodels tumour vessels and probably promotes immunogenic pathway activation, therefore repressing tumour growth. One Sentence SummaryA conjugate vaccine inducing antibody responses to tumour endothelial markers Robo4 can inhibit tumour growth.

immunology↗

bioRxiv2025

G protein-coupled receptor 35 (GPR35) stimulation reduces osteoclast activity in primary human bone cells

G protein-coupled receptor 35 (GPR35) is an orphan receptor that is widely expressed in tissues including human osteoblasts and osteoclasts. Expression of the GPR35 gene and protein are downregulated in osteoporosis patients and in mouse models of the disease. Gpr35-knockout mice have reduced bone mass, while GPR35 agonism rescues bone loss in rodent osteoporosis models indicating that GPR35 has an important role in bone. Our previous studies demonstrated GPR35 is expressed in human osteoclasts, and we sought to determine the receptors function in these cells. We differentiated human peripheral blood mononuclear cells to mature osteoclasts and assessed effects of the GPR35 synthetic agonists, TCG1001 and Zaprinast on osteoclast activity and differentiation. Both agonists stimulated significant reductions in osteoclast bone resorption and TRAP activity, and downregulated expression of MMP9, a gene that regulates osteoclast bone resorption. These effects were prevented by pre-incubation of cells with a GPR35-specific antagonist. To understand GPR35 signaling pathways, we measured the phosphorylation of secondary messengers known to have important roles in osteoclast activity using AlphaLISA assays. Upon GPR35 stimulation, we observed reduced phosphorylation of cSrc, which stimulates actin ring formation necessary for bone resorption, and decreased phosphorylation of Akt, CREB and NF{kappa}B that drive transcription of genes required for bone resorption. Additionally, we used chemical inhibitors and siRNA knockdown to show that GPR35 couples to Gi/o and G12/13 to stimulate these signaling pathways. Finally, we compared the ability of GPR35 agonists to suppress osteoclast activity to that of current osteoporosis drugs, denosumab and alendronic acid, and showed TRAP activity was similar suppressed under all conditions. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments. Lay summaryExpression of G protein-coupled receptor 35 (GPR35) is reduced in osteoporosis and Gpr35-knockout mice have reduced bone mass. Here we showed stimulation of GPR35 activates Gi/o and G12/13 signaling pathways to reduce bone resorption in human osteoclast. The anti-resorptive activity of GPR35 agonists was comparable to current osteoporosis drugs, denosumab and alendronic acid. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments.

pharmacology and toxicology↗

bioRxiv2025

Adjustable Compliance Footwear Technology to Investigate Gait Adaptation

Kinetic asymmetries in gait caused by neurological impairments are a major factor in mobility deficiency. Gait perturbation training has shown promise for rehabilitating neurological gait asymmetry, but the transfer of benefits from treadmill training to overground walking is limited. To enable kinetic gait perturbation training on treadmills and overground, we developed novel adjustable compliance footwear technology. The system consists of a wearable pneumatic actuation module which controls the pressure of custom air pockets embedded in a soft lattice midsole, and is capable of independently and wirelessly adjusting the midsole compliance under each foot from 300 kN/m and 60% energy absorption to 18 kN/m and 75% energy absorption in under 4 seconds. Hindfoot and forefoot sole deflection data are measured via Hall effect sensors and paired magnets characterized to 1.7 mm accuracy. To demonstrate the experimental capability of the system, we successfully triggered multiple compliance changes during overground walking and recorded ground reaction forces and embedded sensor values during a prolonged asymmetric compliance walking bout on a treadmill. Future work will include a full experimental study of gait adaptation to asymmetric footwear compliance perturbations.

bioengineering↗

bioRxiv2025

Lactuchelins: New lipopeptide siderophores from Pseudomonas lactucae inhibit Xanthomonas campestris pv. campestris 8004

Seeds harbor diverse microbial communities, including beneficial microbes that play a vital role in protecting plants from seed-borne pathogens. Despite their critical importance, the molecular mechanisms driving intermicrobial competition within the seed microbiome remain poorly understood, limiting the potential to optimize seed inoculation strategies. In this study, we evaluated the inhibitory potential of 30 seed-borne bacterial strains against the phytopathogen Xanthomonas campestris pv. campestris 8004 (Xcc8004). We identified Pseudomonas lactucae CFBP13502 as a potent inhibitor of Xcc8004, mediated by exometabolites specifically induced in the presence of Lysobacterales (formerly Xanthomonadales). Transcriptomic analysis of CFBP13502 revealed upregulation of a gene cluster involved in the biosynthesis of a lipopeptide siderophore biosynthesis. Gene deletion confirmed that this cluster is essential for the growth inhibition of Xcc8004. Furthermore, iron supplementation abolished this inhibitory effect, providing strong evidence for the role of iron chelation. Through comparative metabolomics, we elucidated the structure of a novel family of lipopeptide siderophores, which we named lactuchelins, produced by CFBP13502. Our findings provide the first molecular evidence of competitive exclusion mechanisms at the seed microbiome interface, highlighting lactuchelins as a promising avenue for the development of seed-based biocontrol strategies against seed-borne phytopathogens.

microbiology↗

bioRxiv2024

Lack of cortistatin worsens neurological outcome and exacerbates aging-related glial and vascular dysfunction in stroke

BackgroundIschemic stroke remains a leading cause of death and disability, with limited therapies addressing its pathophysiology. Neuroinflammation, blood-brain barrier disruption, and immune dysregulation critically contribute to both acute neuronal injury and chronic functional decline. Elucidating the endogenous factors that govern these processes and understanding the intricate interplay among them is essential for developing effective therapeutic strategies. Cortistatin, a neuropeptide expressed in the nervous and immune systems, exhibits potent immunomodulatory actions, but its role in neuroinflammatory diseases remains unclear. MethodsIn this study, we combined human and murine transcriptomic analyses with a preclinical stroke model in cortistatin-deficient mice to investigate the endogenous and therapeutic roles of cortistatin during the acute (48 h) and subacute (7 days) phases of ischemic stroke. ResultsWe demonstrate for the first time that cortistatin deficiency amplified ischemia-induced transcriptional programs, heightening neuroinflammation and glial/neurovascular dysfunction, and worsening neurological outcomes. Interestingly, cortistatin deficiency accelerated aging-associated pathological features, underscoring its essential role in neuroimmune and cerebrovascular homeostasis. Conversely, cortistatin administration improved neuronal survival, mitigated immune dysfunction, and preserved myelin and vascular integrity during both the acute and subacute stages. ConclusionsThese findings provide the first evidence identifying cortistatin as an endogenous neuroimmune regulator in ischemic stroke and a promising multimodal therapeutic agent for improving acute and long-term outcomes. Trial registrationNot applicable

immunology↗

bioRxiv2023

Intracranial tumors elicit systemic sympathetic hyperactivity that limits immunotherapeutic responses

Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression whose mechanistic underpinnings are incompletely understood. Here, we reveal that tumors harbored intracranially elicit systemic increases to circulating catecholamine levels, with the resultant chronic sympathetic hyperactivity driving T cell dysfunction and limiting immunotherapeutic success. Conversely, treatment with {beta}-adrenergic blockade increases NF-{kappa}B activity in immune cells, restores T cell polyfunctionality, modifies the tumor microenvironment, and licenses immune-based therapies in murine models of glioblastoma (GBM) to extend survival. Extended survival is also observed in GBM patients having received {beta}-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received {beta}-blockade alongside concomitant immune checkpoint inhibition. While {beta}-blockade also impacts outcomes in the setting of extracranial disease, the benefits are especially pronounced in patients harboring intracranial disease burdens. These data suggest that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors, specifically and advance a role for {beta}-adrenergic blockade in licensing immunotherapeutic responses within the intracranial compartment.

immunology↗

bioRxiv2023

Gait adaptation to asymmetric hip stiffness applied by a robotic exoskeleton

Wearable exoskeletons show significant potential for improving gait impairments, such as interlimb asymmetry. However, a more profound understanding of whether exoskeletons are capable of eliciting neural adaptation is needed. This study aimed to characterize how individuals adapt to bilateral asymmetric joint stiffness applied by a hip exoskeleton, similar to split-belt treadmill training. Thirteen unimpaired individuals performed a walking trial on the treadmill while wearing the exoskeleton. The right side of the exoskeleton acted as a positive stiffness torsional spring, pulling the thigh towards the neutral standing position, while the left acted as a negative stiffness spring pulling the thigh away from the neutral standing position. The results showed that this intervention applied by a hip exoskeleton elicited adaptation in spatiotemporal and kinetic gait measures similar to split-belt treadmill training. These results demonstrate the potential of the proposed intervention for retraining symmetric gait.

neuroscience↗

bioRxiv2022

Receptor for Hyaluronan-Mediated Motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant metabolism and subsequent signaling of hyaluronan (HA). Hyaluronan Mediated Motility Receptor (RHAMM, HMMR) is a HA receptor that enables tumor cells to sense and respond to the TME during breast cancer progression. Focused gene expression analysis of an internal breast cancer patient cohort demonstrates increased RHAMM expression correlates with aggressive clinicopathological features. We also develop a 27-gene RHAMM-dependent signature (RDS) by intersecting differentially expressed genes in lymph node positive cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validate in an independent cohort. We demonstrate RDS predicts for poor survival and associates with invasive pathways. Further analyses using CRISPR/Cas9 generated RHAMM -/- breast cancer cells provide direct evidence that RHAMM promotes invasion in vitro and in vivo. Additional immunohistochemistry studies highlight heterogeneous RHAMM expression, and spatial transcriptomics confirms the RDS emanates from RHAMM-high invasive niches. We conclude RHAMM upregulation leads to the formation of invasive niches, which are enriched in RDS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes.

cancer biology↗

bioRxiv2021

Chronic Alcohol Dysregulates Glutamatergic Function in the Basolateral Amygdala in a Projection- and Sex-Specific Manner

Chronic intermittent ethanol (CIE) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections. The BLA receives information from two distinct input pathways. Afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE induces input- and sex-specific adaptations to glutamatergic function in the BLA. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative reward and aversion BLA neurons, and showed that CIE alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.

neuroscience↗

bioRxiv2021

Forward simulations of walking on a variable surface-impedance treadmill: A comparison of two methods

Recent experiments with a variable stiffness tread-mill (VST) suggest that modulating foot-ground contact dynamics during walking may offer an effective new paradigm for gait rehabilitation. How gait adapts to extended perturbations of asymmetrical surface stiffness is still an open question. In this study, we simulated human gait with prolonged asymmetrical changes in ground stiffness using two methods: (1) forward simulation of a muscle-reflex model and (2) optimal control via direct collocation. Simulation results showed that both models could competently describe the biomechanical trends observed in human experiments with a VST which altered the walking surface stiffness for one step. In addition, the simulations revealed important considerations for future experiments studying the effect of asymmetric ground stiffness on gait behavior. With the muscle-reflex model, we observed that although subtle, there was a difference between gait biomechanics before and after the prolonged asymmetric stiffness perturbation, showing the behavioral signature of an aftereffect despite the lack of supraspinal control in the model. In addition, the optimal control simulations showed that damping has a large effect on the overall lower-body muscle activity, with the muscle effort cost function used to optimize the biomechanics increasing 203% between 5 Ns/m and 2000 Ns/m at a stiffness of 10 kN/m. Overall, these findings point to new insights and considerations for advancing our understanding of human neuromotor control of locomotion and enhancing robot-aided gait rehabilitation.

neuroscience↗

bioRxiv2019

Curated BLAST for Genomes

"Curated BLAST for Genomes" finds candidate genes for a process or an enzymatic activity within a genome of interest. In contrast to annotation tools, which usually predict a single activity for each protein, Curated BLAST asks if any of the proteins in the genome are similar to characterized proteins that are relevant. Given a query such as an enzymes name or an EC number, Curated BLAST searches the curated descriptions of over 100,000 characterized proteins, and it compares the relevant characterized proteins to the predicted proteins in the genome of interest. In case of errors in the gene models, Curated BLAST also searches the six-frame translation of the genome. Curated BLAST is available at http://papers.genomics.lbl.gov/curated.

bioinformatics↗

bioRxiv2017

Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

microbiology↗

bioRxiv2017

Magic pools: parallel assessment of transposon delivery vectors in bacteria

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a parts-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term \"magic pools\". Within a magic pool, each transposon vector has a different combination of promoters and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use the best vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes.

genomics↗

bioRxiv2017

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

microbiology↗