Biology subjects

Crane, A.

Publications and source records attributed to Crane, A..

14 recordsLinked to original sources

bioRxiv2026

Modeling Risk Group 4 virus infection and antiviral treatment in microfluidic lung organ-on-chips in maximum containment laboratories

Development of candidate countermeasures against human pathogens frequently includes nonhuman animal experimentation. Preclinical animal pathogen exposure studies are conducted to model diseases and accumulate preliminary and hypothetically translatable data to inform and justify the design of clinical trial evaluation of countermeasure safety and efficacy. In addition to frequent ethical critiques, challenges associated with animal experimentation include considerable resources needed to achieve statistical power and robustness, replicability and reproducibility concerns, potentially compromised objectivity through lack of blinding, fundamental species-specific biological differences, and risk of unpredictable pathogen adaptation to the experimental animal. Recent U.S. and U.K. government initiatives aim to reduce animal experimentation by complementing or potentially replacing them with new approach methodologies (NAMs), i.e., increasingly sophisticated in silico, in chemico, and in vitro approaches. We piloted development of one type of NAM, organ-on-chips (OOCs), in the highly challenging environment of a maximum (biosafety level 4) containment laboratory. Using a Risk Group 4 virus, Nipah virus (NiV), and two types of lung OOCs seeded with human or porcine cells, we demonstrated the recapitulation of key features of NiV lung infection, including viral infection, replication, and translocation, that are associated with proinflammatory cytokine secretion, immune cell recruitment, and disruption of the air-liquid interface barrier. We reproduced the known anti-NiV activity of remdesivir and evaluated that of another potential antiviral, zotatifin. Our results pave the way for similar applications of advanced microphysiological systems for modeling infections caused by high-consequence viruses.

microbiology↗

bioRxiv2025

Sleep loss suppresses the motivation to prepare for sleep

In the period preceding sleep, humans and other animals display a stereotyped repertoire of behaviors-including hygiene-related activities and preparing a place to sleep. Evidence suggests that this pre-sleep phase actively contributes to sleep initiation and quality. Nonetheless, individuals can sometimes fall asleep without preparation, even under undesirable circumstances. These abrupt transitions into sleep can have severe consequences, particularly in high-risk environments. Although progress has been made in identifying neuronal populations controlling sleep/wake states and mechanisms regulating cortical oscillations during sleep, little is known about the natural processes that govern the pre-sleep phase, under baseline conditions and following sleep loss. Here, we examine factors regulating pre-sleep behaviors using environmental and behavioral manipulations, video recordings, machine-learning-based tracking, and EEG-EMG analysis in freely-behaving mice. We focus on nest-building-a key pre-sleep behavior-and assess its modulation by time of day and sleep deprivation. We find that mice are highly motivated to build nests during the light phase but show reduced motivation during most of the dark phase. Sleep deprivation significantly suppresses pre-sleep nest-building and promotes the direct initiation of sleep. Varying amounts of sleep deprivation, from 2-6 hours, uniformly suppress nest-building. This suppression is not due to stress, as mice exposed to acute restraint stress engage robustly in nest-building. Our findings provide insight into processes regulating the transition from wakefulness to sleep. Understanding pre-sleep regulation has important implications for treating sleep-onset difficulties-prevalent in insomnia and predictive of cognitive decline-and for mitigating risks associated with uncontrolled sleep onset in high-stakes situations. STATEMENT OF SIGNIFICANCEPrior to sleep, animals-including humans-engage in stereotyped behaviors such as preparing a sleep space, thought to facilitate sleep initiation and quality. However, sleep can occur abruptly without preparation, posing serious risks, especially in high-stakes environments. Despite progress in understanding sleep/wake circuitry, the regulation of pre-sleep behaviors remains poorly understood. Here, we used multiple techniques in mice to examine nest-building, a key pre-sleep behavior. We found that mice are more motivated to build nests during the light phase, but this behavior is strongly suppressed by sleep loss and is not affected by acute stress. These findings highlight pre-sleep behaviors as an active, regulated component of sleep onset, with implications for understanding and treating sleep initiation difficulties.

neuroscience↗

bioRxiv2025

Characterization of the intraspecies chimeric mouse brain at embryonic day 12.5

Incidence of neurodegenerative diseases such as Alzheimers, Parkinsons, Huntingtons, and amyotrophic lateral sclerosis have increased dramatically as life expectancy at birth has risen year-over-year and the population ages. Neurological changes within the central nervous system, specifically the brain, include cell loss and deterioration that impact motor function, memory, executive function, and mood. Available treatments are limited and often only address symptomatic manifestations of the disease rather than disease progression. Cell transplantation therapy has shown promise for treating neurodegenerative diseases, but a source of autologous cells is required. Blastocyst complementation provides an innovative method for generating those autologous neural cells. By injecting mouse induced pluripotent stem cells (iPSCs) into a wild type (WT) mouse blastocyst, we generated a chimeric mouse brain derived of both donor and host neuronal and non-neuronal cells. An embryonic day 12.5 (E12.5), automated image analysis of mouse-mouse chimeric brains showed the presence of GFP-labeled donor-derived dopaminergic and serotonergic neuronal precursors. GFP-labeled donor-derived cholinergic precursor neurons and non-neuronal microglia-like and macrophage-like cells were also observed using more conventional imaging analysis software. This work demonstrates that the generation of mouse-mouse chimeric neural cells is possible; and that characterization of early neuronal and non-neuronal precursors provides a first step towards utilizing these cells for cell transplantation therapies for neurodegenerative diseases.

developmental biology↗

bioRxiv2024

Rapamycin increases murine lifespan but does not reduce mineral volume in the Matrix GLA Protein (MGP) knockout mouse model of medial arterial calcification.

Peripheral artery disease (PAD) is the narrowing of the arteries that carry blood to the lower extremities. PAD has been traditionally associated with atherosclerosis. However, recent studies have found that thrombotic events triggered by medial arterial calcification (MAC) is the primary cause of chronic limb ischemia below the knee. MAC is localized around the elastic fibers surrounding smooth muscle cells (SMCs) in arteries. Matrix GLA protein (MGP) binds circulating calcium and prevents hydroxyapatite mineral deposition, while also modulating proosteogenic signaling by attenuating BMP-2-mediated activation of Runx2 gene expression. Mgp-/- mice develop severe MAC and die around 8 weeks after birth due to aortic rupture or heart failure. We previously discovered a rare genetic disease Arterial Calcification due to Deficiency of CD73 (ACDC), in which patients present with extensive MAC in their lower extremity arteries. Using a patient-specific induced pluripotent stem cell model, we found that rapamycin inhibited calcification. Here we investigated whether rapamycin could reduce MAC in vivo using the Mgp-/- murine model. Mgp+/+ and Mgp-/- mice received 5mg/kg rapamycin or vehicle. Calcification content was assessed via microCT, and vascular morphology and extracellular matrix content were assessed histologically. Immunostaining and western blot analysis were used to examine SMC phenotype and extracellular matrix content. Rapamycin prolonged Mgp-/- mice lifespan, decreased mineral density in the arteries, maintained SMC contractile phenotype, and improved vessel structure, however, calcification volume was unchanged. Mgp-/- mice with SMC-specific deletion of Raptor or Rictor did not recapitulate treatment with rapamycin. These findings suggest rapamycin promotes beneficial vascular remodeling in vessels with MAC. NEWS AND NOTEWORTHYPeripheral artery disease (PAD) is associated with medial arterial calcification (MAC), which involves calcification of arterial elastic fibers and smooth muscle cells (SMCs). Matrix GLA protein (MGP) inhibits vascular calcification, and Mgp-/- mice develop severe MAC. Using this model, we found rapamycin prolonged lifespan, reduced arterial mineral density, maintained SMC contractile phenotype, and improved vessel structure, though calcification volume remained unchanged. Findings highlight rapamycins potential for vascular remodeling in MAC.

physiology↗

bioRxiv2024

Oral cannabidiol administration in mice during pregnancy and lactation affects early postnatal body weight, fasting glucose, ingestive behavior, anxiety- and obsessive compulsive-like behaviors, and long-term object-memory in adult offspring in a sex-dependent manner

RationaleThe consequences of perinatal cannabidiol (CBD) exposure are severely understudied, but are important, given its widespread use and believed safety as a natural supplement. ObjectiveThe objective of this study was to test the health, metabolic, and behavioral consequences of perinatal CBD exposure on dams and their offspring raised to adult. MethodsPrimiparous female C57BL/6J mice were orally administered 100 mg/kg CBD in strawberry jam to expose offspring during gestation, lactation, or both using a cross-fostering design. Adult offspring were metabolically profiled using indirect calorimetry and intraperitoneal glucose tolerance testing. Adults were behaviorally phenotyped, video recorded, and mouse position tracked using DeepLabCut. ResultsCBD was detected in maternal plasma using LC-MS 10-min post consumption (34.2 {+/-} 1.7 ng/ul) and peaked within 30 min (371.0 {+/-} 34.0 ng/ul). Fetal exposure to CBD significantly decreased survival of the pups, and decreased male postnatal development, but did not alter litter size, maternal body weight or pup birth weight. We observed many sex-dependent effects of perinatal CBD exposure. Exposure to CBD during gestation and lactation increased meal size, caloric intake, and respiratory exchange ratio for adult male offspring, while exposure during lactation decreased fasting glucose, but had no effect on clearance. Adult female offspring exposed to CBD during lactation showed increased drink size. Perinatal CBD exposure increased obsessive compulsive- and decreased anxiety-like behaviors (marble burying, light-dark box, elevated-plus maze) in female mice, decreased long-term object memory in male mice, and had no effect on attention tasks for either sex. ConclusionsWe conclude that orally-administered CBD during pregnancy affects behavior and metabolism in a sex-dependent manner, and mice are differentially sensitive to exposure during gestation vs. lactation, or both. Because long-term changes are observed following perinatal exposure to the drug, and exposure significantly decreases survival to weaning, more research during development is warranted. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/602955v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a9341dorg.highwire.dtl.DTLVardef@19c7356org.highwire.dtl.DTLVardef@5833baorg.highwire.dtl.DTLVardef@ac153c_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIMice can be trained to orally consume CBD using strawberry jam as the vehicle. C_LIO_LICBD administration to pregnant dams decreases pup survival to weaning age without significantly affecting maternal behavior. C_LIO_LIPerinatal CBD exposure decreases developmental body weight in males. C_LIO_LIGestational or lactational CBD increases the respiratory exchange ratio (RER), increases mean meal and drink size, and reduces fasting glucose in a sex-dependent manner. C_LIO_LICBD increases obsessive-compulsive like behavior in adult offspring, which could be eliminated in females by cross-fostering to a drug-free dam. C_LIO_LIPerinatal CBD selectively decreases anxiety-like behavior in females and decreases long-term object memory in males. C_LI

neuroscience↗

bioRxiv2023

A sequence context-based germline filter for structural variant calling from tumor samples without paired normal

Although several recent studies have characterized structural variants (SVs) in germline and cancer genomes, the features of SVs in these different contexts have not been directly compared. We examined similarities and differences between 2 million germline and 115 thousand tumor SVs from a cohort of 963 patients from The Cancer Genome Atlas (TCGA). We found significant differences in features related to their genomic sequences and localization that suggest differences between SV-generating processes and selective pressures. For example, we found that transposon-mediated processes shape germline much more than somatic SVs, while somatic SVs more frequently show features characteristic of chromoanagenesis. These differences were extensive enough to enable us to develop a classifier-"the great GaTSV"-that accurately distinguishes between germline and cancer SVs in tumor samples that lack a matched normal sample.

bioinformatics↗

bioRxiv2023

Taphonomic damage obfuscates interpretation of the retroarticular region of the Asteriornis mandible

Asteriornis maastrichtensis, from the latest Cretaceous of Belgium, is among the oldest known crown bird fossils, and its three-dimensionally preserved skull provides the most substantial insights into the cranial morphology of early crown birds to date. Phylogenetic analyses recovered Asteriornis as a total-group member of Galloanserae (the clade uniting Galliformes and Anseriformes. One important feature supporting this placement was enlargement of the retroarticular processes, which form elongate caudal extensions of the mandible in extant Galloanserae. Here, we reinterpret the jaw of Asteriornis and illustrate that the caudalmost portion of the mandibles are in fact not preserved. Instead, the caudal extremities of both the left and right mandibular rami extend to the surface of the fossil block containing the holotype skull, where they have eroded away. The originally identified retroarticular process of the right mandible--which exhibits a morphology and orientation strikingly similar to the retroarticular processes of certain extant and fossil galloanserans, including the early Palaeogene total-clade anseriforms Conflicto and Nettapterornis--instead represents a twisted and caudally displaced medial process. Nonetheless, anatomical comparisons with extant taxa reveal that we are unable to exclude the possibility that Asteriornis exhibited robust retroarticular processes comparable to those of extant Galloanserae. In light of the reinterpreted morphology of the Asteriornis mandible, we update the original anatomical character matrix used to investigate its phylogenetic relationships, and our revised phylogenetic analyses continue to support its position as a total-group galloanseran, as initially interpreted. We demonstrate additional morphological traits of the mandible supporting this phylogenetic position and provide new data on the nature and distribution of retroarticular processes among early crown birds.

paleontology↗

bioRxiv2022

Aberrant DNA repair is a vulnerability in histone H3.3-mutant brain tumors

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent oncogenic mechanism, which fosters genome instability and tumor cell growth in H3.3 mutant pHGG, thus opening new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme Polynucleotide Kinase 3-Phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

cancer biology↗

bioRxiv2022

Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in valve leaflet osteogenic reprogramming are not fully understood. As telomerase reverse transcriptase (TERT) overexpression primes mesenchymal stem cells to differentiate into osteoblasts, we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). MethodsHuman control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/-and Terc-/- mice were used for mechanistic studies. Calcification was assessed in Tert+/+ and Tert-/- mice ex vivo and in vivo. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. ResultsTERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic or inflammatory stimuli, and knock-down or genetic deletion of TERT prevented calcification in vitro and in vivo. Mechanistically, TERT was upregulated via NF-{kappa}B and required to initiate osteogenic reprogramming, independent of its canonical reverse transcriptase activity and the lncRNA TERC. TERT exerts non-canonical osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion or inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Lastly, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue compared to control tissue. ConclusionsTERTs non-canonical activity is required to initiate calcification. TERT is upregulated via inflammatory signaling pathways and partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Novelty and SignificanceWhat is known? Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients. Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its activity in aortic valve cells are poorly defined. In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage. What new information does this article contribute? TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence. Genetic loss or depletion of TERT prevented calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells in vitro and the vasculature in vivo. Early in the osteogenic reprogramming inflammatory signaling promotes TERT to co-localize with SMARCA4 and STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs. STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells. What are the clinical implications? We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT/STAT5 interaction or their activity may be leveraged for the development of therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and or perhaps other cardiovascular tissues. Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.

pathology↗

bioRxiv2020

The complete genome sequence of the Staphylococcus bacteriophage Metroid

Phages infecting bacteria of the genus Staphylococcus play an important role in their hosts ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities - including a conservation of the lysis cassette - with other Staphylococcus cluster C1 bacteriophages, several of which were previously characterized for therapeutic applications.

genomics↗

bioRxiv2020

Zika virus-based immunotherapy enhances long-term survival of rodents with brain tumors through upregulation of memory T-cells

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an upregulation in the levels of T-cells, including CD4+ and tissue-resident memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naive untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

cancer biology↗

bioRxiv2020

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

The derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells ("iBCs"), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker NGFR. However, in response to primary basal cell medium, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.

developmental biology↗

bioRxiv2018

Vasculature-associated adipose tissue macrophages dynamically adapt to inflammatory and metabolic challenges

Tissue-resident macrophages comprise the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophage populations change during metabolic stress and ageing, and are thought to contribute to the pathogenesis of obesity. Here, we studied adipose tissue macrophage subpopulations in the steady state, and in response to nutritional and infectious challenges.\n\nUsing comprehensive cell-surface-based and gene expression analyses, we found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying a very high endocytic capacity. We refer to these cells as Vasculature-associated Adipose tissue Macrophages (VAMs). Chronic high fat diet (HFD) feeding results in the accumulation of a monocyte-derived CD11c+CD64+ double positive (DP) macrophage eWAT population with a predominant anti-inflammatory gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that adipose tissue macrophage populations adapt to metabolic stress and inflammation, suggesting an important role for these cells in restoring homeostasis.

immunology↗

bioRxiv2017

Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than pre-Haptoglobin 2

BACKGROUNDThere is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.\n\nMETHODSSerum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.\n\nRESULTSAs zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.\n\nCONCLUSIONSOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

molecular biology↗