Passage Of Macromolecules Research Articles

Compliant DNA Origami Nanoactuators as Size-Selective Nanopores.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

Loss of Glomerular Permselectivity in Type 2 Diabetes Associates With Progression to Kidney Failure.

We examined whether defects in glomerular size selectivity in type 2 diabetes (T2D) associated with progressive kidney disease. Glomerular filtration rate (GFR) and fractional clearances of dextrans of graded sizes were measured in 185 Pima Indians. The permselectivity model that best fit the dextran sieving data represented the glomerular capillary as being perforated by small restrictive pores and a parallel population of larger non-restrictive pores characterized by ωo, the fraction of total filtrate volume passing through this shunt. Hazard ratio (HR) for kidney failure was expressed per 1 standard deviation (SD) increase of ωo by Cox regression after adjusting for age, sex, mean arterial pressure, HbA1c, GFR, and the urine albumin-to-creatinine ratio (ACR). Baseline mean age (±SD) was 43±10 years, HbA1c 8.9±2.5%, GFR 147±46 ml/min, and median (IQR) ACR 41 (11-230) mg/g. During a median follow-up of 17.7 years, 70 participants developed kidney failure. After adjustment, each 1 SD increment in ωo was associated with a higher risk of kidney failure (HR=1.49, 95% CI 1.13, 1.96). Enhanced transglomerular passage of test macromolecules associated with progression to kidney failure, independent of albuminuria and GFR, suggesting that mechanisms associated with impaired glomerular permselectivity are important determinants of progressive kidney disease.

The Local Activation of Toll-like Receptor 7 (TLR7) Modulates Colonic Epithelial Barrier Function in Rats.

Toll-like receptors (TLRs)-mediated host-bacterial interactions participate in the microbial regulation of gastrointestinal functions, including the epithelial barrier function (EBF). We evaluated the effects of TLR7 stimulation on the colonic EBF in rats. TLR7 was stimulated with the selective agonist imiquimod (100/300 µg/rat, intracolonic), with or without the intracolonic administration of dimethyl sulfoxide (DMSO). Colonic EBF was assessed in vitro (electrophysiology and permeability to macromolecules, Ussing chamber) and in vivo (passage of macromolecules to blood and urine). Changes in the expression (RT-qPCR) and distribution (immunohistochemistry) of tight junction-related proteins were determined. Expression of proglucagon, precursor of the barrier-enhancer factor glucagon-like peptide 2 (GLP-2) was also assessed (RT-qPCR). Intracolonic imiquimod enhanced the EBF in vitro, reducing the epithelial conductance and the passage of macromolecules, thus indicating a pro-barrier effect of TLR7. However, the combination of TLR7 stimulation and DMSO had a detrimental effect on the EBF, which manifested as an increased passage of macromolecules. DMSO alone had no effect. The modulation of the EBF (imiquimod alone or with DMSO) was not associated with changes in gene expression or the epithelial distribution of the main tight junction-related proteins (occludin, tricellulin, claudin-2, claudin-3, junctional adhesion molecule 1 and Zonula occludens-1). No changes in the proglucagon expression were observed. These results show that TLR7 stimulation leads to the modulation of the colonic EBF, having beneficial or detrimental effects depending upon the state of the epithelium. The underlying mechanisms remain elusive, but seem independent of the modulation of the main tight junction-related proteins or the barrier-enhancer factor GLP-2.

Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Probing the leak pathway: Live-cell imaging of macromolecule passage through epithelia.

Epithelia compartmentalize multicellular organisms and provide interfacing between the inside and outside. Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune-competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore. These bind to cell-adherent avidin carrying donor fluorophore at the basolateral membranes of single-layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical-to-basolateral tracer passage as a Förster resonance energy transfer signal of the fluorescent dextran-avidin pair over time. Stimulated macromolecule passage using barrier-perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.

Ocular Barriers and Their Influence on Gene Therapy Products Delivery.

The eye is formed by tissues and cavities that contain liquids whose compositions are highly regulated to ensure their optical properties and their immune and metabolic functions. The integrity of the ocular barriers, composed of different elements that work in a coordinated fashion, is essential to maintain the ocular homeostasis. Specialized junctions between the cells of different tissues have specific features which guarantee sealing properties and selectively control the passage of drugs from the circulation or the outside into the tissues and within the different ocular compartments. Tissues structure also constitute selective obstacles and pathways for various molecules. Specific transporters control the passage of water, ions, and macromolecules, whilst efflux pumps reject and eliminate toxins, metabolites, or drugs. Ocular barriers, thus, limit the bioavailability of gene therapy products in ocular tissues and cells depending on the route chosen for their administration. On the other hand, ocular barriers allow a real local treatment, with limited systemic side-effects. Understanding the different barriers that limit the accessibility of different types of gene therapy products to the different target cells is a prerequisite for the development of efficient gene delivery systems. This review summarizes actual knowledge on the different ocular barriers that limit the penetration and distribution of gene therapy products using different routes of administration, and it provides a general overview of various methods used to bypass the ocular barriers.

Endothelial Zeb2 preserves the hepatic angioarchitecture and protects against liver fibrosis.

AimsHepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis.Methods and resultsTo study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called ‘capillarization’. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC–HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis.ConclusionEndothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

Expression of tricellular tight junction proteins and the paracellular macromolecule barrier are recovered in remission of ulcerative colitis

BackgroundUlcerative colitis (UC) has a relapsing and remitting pattern, wherein the underlying mechanisms of the relapse might involve an enhanced uptake of luminal antigens which stimulate the immune response. The tricellular tight junction protein, tricellulin, takes charge of preventing paracellular passage of macromolecules. It is characterized by downregulated expression in active UC and its correct localization is regulated by angulins. We thus analyzed the tricellulin and angulin expression as well as intestinal barrier function and aimed to determine the role of tricellulin in the mechanisms of relapse.MethodsColon biopsies were collected from controls and UC patients who underwent colonoscopy at the central endoscopy department of Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin. Remission of UC was defined basing on the clinical appearance and a normal Mayo endoscopic subscore. Intestinal barrier function was evaluated by electrophysiological and paracellular flux measurements on biopsies mounted in Ussing chambers.ResultsThe downregulated tricellulin expression in active UC was recovered in remission UC to control values. Likewise, angulins were in remission UC at the same levels as in controls. Also, the epithelial resistance which was decreased in active UC was restored in remission to the same range as in controls, along with the unaltered paracellular permeabilities for fluorescein and FITC-dextran 4 kDa.ConclusionsIn remission of UC, tricellulin expression level as well as intestinal barrier functions were restored to normal, after they were impaired in active UC. This points toward a re-sealing of the impaired tricellular paracellular pathway and abated uptake of antigens to normal rates in remission of UC.

Local Pressure Drives Low-Density Lipoprotein Accumulation and Coronary Atherosclerosis in Hypertensive Minipigs

BackgroundThe mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. ObjectivesThis study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. MethodsUsing inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. ResultsUnder hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. ConclusionsIncreased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins.

Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine.

An increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls.

The Immature Gut Barrier and Its Importance in Establishing Immunity in Newborn Mammals.

The gut is an efficient barrier which protects against the passage of pathogenic microorganisms and potential harmful macromolecules into the body, in addition to its primary function of nutrient digestion and absorption. Contrary to the restricted macromolecular passage in adulthood, enhanced transfer takes place across the intestines during early life, due to the high endocytic capacity of the immature intestinal epithelial cells during the fetal and/or neonatal periods. The timing and extent of this enhanced endocytic capacity is dependent on animal species, with a prominent non-selective intestinal macromolecular transfer in newborn ungulates, e.g., pigs, during the first few days of life, and a selective transfer of mainly immunoglobulin G (IgG), mediated by the FcRn receptor, in suckling rodents, e.g., rats and mice. In primates, maternal IgG is transferred during fetal life via the placenta, and intestinal macromolecular transfer is largely restricted in human neonates. The period of intestinal macromolecular transmission provides passive immune protection through the transfer of IgG antibodies from an immune competent mother; and may even have extra-immune beneficial effects on organ maturation in the offspring. Moreover, intestinal transfer during the fetal/neonatal periods results in increased exposure to microbial and food antigens which are then presented to the underlying immune system, which is both naïve and immature. This likely stimulates the maturation of the immune system and shifts the response toward tolerance induction instead of activation or inflammation, as usually seen in adulthood. Ingestion of mother's milk and the dietary transition to complex food at weaning, as well as the transient changes in the gut microbiota during the neonatal period, are also involved in the resulting immune response. Any disturbances in timing and/or balance of these parallel processes, i.e., intestinal epithelial maturation, luminal microbial colonization and mucosal immune maturation due to, e.g., preterm birth, infection, antibiotic use or nutrient changes during the neonatal period, might affect the establishment of the immune system in the infant. This review will focus on how differing developmental processes in the intestinal epithelium affect the macromolecular passage in different species and the possible impact of such passage on the establishment of immunity during the critical perinatal period in young mammals.

Genetic Disorders of the Glomerular Filtration Barrier.

The glomerular filtration barrier is a highly specialized capillary wall comprising fenestrated endothelial cells, podocytes, and an intervening basement membrane. In glomerular disease, this barrier loses functional integrity, allowing the passage of macromolecules and cells, and there are associated changes in both cell morphology and the extracellular matrix. Over the past 3 decades, there has been a transformation in our understanding about glomerular disease, fueled by genetic discovery, and this is leading to exciting advances in our knowledge about glomerular biology and pathophysiology. In current clinical practice, a genetic diagnosis already has important implications for management, ranging from estimating the risk of disease recurrence post-transplant to the life-changing advances in the treatment of atypical hemolytic uremic syndrome. Improving our understanding about the mechanistic basis of glomerular disease is required for more effective and personalized therapy options. In this review, we describe genotype and phenotype correlations for genetic disorders of the glomerular filtration barrier, with a particular emphasis on how these gene defects cluster by both their ontology and patterns of glomerular pathology.

Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

P015 Tricellulin overexpression protects against elevated macromolecule uptake in DSS colitis mice

Abstract Background In Inflammatory bowel disease, intestinal homeostasis cannot be sustained due to an overreaction of the immune system and a disturbed epithelial barrier due to an impaired tight junction (TJ). At tricellular TJs (tTJs), which are formed where three cells meet, the TJ network is basolaterally expanded and forms a central tube which would be potentially large enough to allow for a paracellular passage of macromolecules. Under normal conditions, the tTJ protein tricellulin (Tric) seals the central tube against luminal uptake. However, in ulcerative colitis (UC), Tric is downregulated, resulting in increased uptake of macromolecules including antigens which may further support the inflammatory processes. Thus, we aimed to identify whether or not overexpression of Tric has a protective effect against inflammation. Methods We generated intestinal epithelial cell-specific overexpression of Tric in mice (B6.C-Tg(Vil-cre) Rosa26tgGFP-Tric). In Tric-overexpressing (Tric-OE) and in wild-type (wt) mice experimental colitis was induced by 3% dextran sodium sulphate (DSS) for 6 days. The effect was assessed by determining the disease activity index (DAI: weight loss, stool consistency, and stool bleeding) and the colon dimensions. Using chamber experiments were performed to determine permeabilities for ions and macromolecules of different sizes (4 and 10 kDa). Results No difference in weight loss was observed between wt and Tric-OE mice upon DSS treatment. However, Tric-OE mice suffered less from diarrhoea and had less bloody stools compared with wt mice resulting in a lower DAI. In addition, DSS caused a reduced colon length which was less prominent in Tric-OE mice than in wt mice. Functional analysis revealed higher permeabilities for Na+ and Cl− after DSS treatment with no significant differences between Tric-OE and wt mice. In contrast, permeabilities for macromolecules of both sizes were unchanged after DSS treatment in Tric-OE mice but elevated in wt-mice. Conclusion Tric-overexpression prevents the epithelial barrier from elevated paracellular uptake of macromolecules during DSS colitis without affecting ion permeability. This protects against DSS colitis as indicated by less affected clinical and structural outcome parameters, most likely via a reduction in a load of immunologically active macromolecules in the subepithelium.

The “HDL receptor” scavenger receptor class B type 1 finesses the uptake of low-density lipoproteins into the subendothelial space of arteries

Atherosclerosis is a chronic inflammation initiated by the accumulation of low-density lipoproteins (LDL) in the subendothelial space of the artery wall in spatially selected regions of the artery. The LDL is derived from circulating plasma lipoproteins. The passage of macromolecules across the endothelial barrier may involve one of three mechanisms—diffusional, paracellular or transcellular.

Occludin knockdown is not sufficient to induce transepithelial macromolecule passage

ABSTRACTOccludin, a tight junction protein, has been reported to regulate barrier function – particularly the leak pathway for larger solutes – in epithelia. Therefore, we aimed to precisely define its role in macromolecule passage at single cell–cell junctions. A combination of varying occludin expression by transient and stable knockdown including systematic seeding strategies was employed to achieve a broad and defined pattern of variance in occludin expression over epithelia. This variance model enabled us to examine occludin function in the leak pathway using global and local analysis, i.e. to analyze macromolecule flux across epithelia and macromolecule passage at single-cell level. Macromolecular flux was found not to correlate with occludin expression in intestinal epithelial cells. In fact, by spatially resolving macromolecular permeation sites using a recently developed method we uncovered leaky cell junctions at the edge of Transwells resulting in increased passage. This demonstrates that rare leaks can determine net flux of macromolecules across epithelia while the vast majority of cellular junctions do not contribute significantly. Hence, concomitant local analysis of macromolecule passage across epithelial barriers is indispensable for interpretation of global flux data. By combining this new approach with cell culture models of the leak pathway, we can present evidence that lack of occludin is not sufficient to stimulate the epithelial leak pathway.

Nucleocytoplasmic transport: Mechanisms and involvement in neurodegenerative disease.

There is a bidirectional communication between the nucleus and the cytoplasm through highly regulated nucleocytoplasmic transport. This process allows the transport of proteins, RNAs, and ribonucleoproteins across the nuclear envelope via the nuclear pore complexes. The nuclear pore complexes are large macromolecular complexes composed of highly conserved proteins called nucleoporins (Nups). Proteins and RNAs are transported across the nuclear pore after binding of cargo transport receptors that interact with specific hydrophobic amino acid repeats of Nups that form a meshwork within the nuclear pore. These transporters include importins that recognize nuclear localization signals (NLS) in their cargo and mediate transport from the cytoplasm to the nucleoplasm and exportins that recognize nuclear export signals (NES) in their cargos and transport their cargo from the nucleus to the cytoplasm. Whereas nucleocytoplasmic transport largely involves protein–protein interactions between transport proteins and nuclear pore Nups, the directionality of transport is determined by a gradient of the small Ras-related nuclear protein (Ran) guanosine triphosphatase (Ran-GTPase). Whereas proteins and most RNA species are transported by these mechanisms, the transport of messenger RNA ribunucleoprotein particles (mRNPs) utilize an independent transport system. Overall, normal nucleocytoplasmic transport mechanisms ensure that transported cargos are retained in their respective compartments and prevent the passage of nonspecific macromolecules. Impaired nucleocytoplasmic transport is a major disease mechanism in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington disease (HD), and tauopathies. Studies in fly and mouse models, in patient-derived induced pluripotent stem cells (iPSCs), and in brain obtained from autopsy have elucidated pathogenic mechanisms that affect nucleocytoplasmic transport in these disorders. The mechanisms of nucleocytoplasmic transport and its involvement on neurodegenerative disorders have been reviewed recently.1–10

HDAC inhibitors promote intestinal epithelial regeneration via autocrine TGFβ1 signalling in inflammation.

Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory boweldisease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.