Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B.

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions.

The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.

Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

Mucins: the frontline defence of the lung.

Mucus plays a vital role in protecting the lungs from environmental factors, but conversely, in muco-obstructive airway disease, mucus becomes pathologic. In its protective role, mucus entraps microbes and particles removing them from the lungs via the co-ordinated beating of motile cilia. This mechanism of lung defence is reliant upon a flowing mucus gel, and the major macromolecular components that determine the rheological properties of mucus are the polymeric mucins, MUC5AC and MUC5B. These large O-linked glycoproteins have direct roles in maintaining lung homeostasis. MUC5B is essential for interaction with the ciliary clearance system and MUC5AC is up-regulated in response to allergic inflammatory challenge. Mucus with abnormal biophysical properties is a feature of muco-obstructive respiratory disease and can result from many different mechanisms including alterations in mucin polymer assembly, mucin concentration and the macromolecular form in mucus, as well as changes in airway surface hydration, pH and ion composition. The abnormal mucus results in defective lung protection via compromised ciliary clearance, leading to infection and inflammation.

BIOTA CONNECT AQUATIC HABITATS THROUGHOUT FRESHWATER ECOSYSTEM MOSAICS.

Freshwater ecosystems are linked at various spatial and temporal scales by movements of biota adapted to life in water. We review the literature on movements of aquatic organisms that connect different types of freshwater habitats, focusing on linkages from streams and wetlands to downstream waters. Here, streams, wetlands, rivers, lakes, ponds, and other freshwater habitats are viewed as dynamic freshwater ecosystem mosaics (FEMs) that collectively provide the resources needed to sustain aquatic life. Based on existing evidence, it is clear that biotic linkages throughout FEMs have important consequences for biological integrity and biodiversity. All aquatic organisms move within and among FEM components, but differ in the mode, frequency, distance, and timing of their movements. These movements allow biota to recolonize habitats, avoid inbreeding, escape stressors, locate mates, and acquire resources. Cumulatively, these individual movements connect populations within and among FEMs and contribute to local and regional diversity, resilience to disturbance, and persistence of aquatic species in the face of environmental change. Thus, the biological connections established by movement of biota among streams, wetlands, and downstream waters are critical to the ecological integrity of these systems. Future research will help advance our understanding of the movements that link FEMs and their cumulative effects on downstream waters.

The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin.

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.

Biosynthesis of the polymeric gel-forming mucin MUC5B.

MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non-O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B (non-O-glycosylated monomer and dimer, and O-glycosylated polymers). Biophysical analysis of recombinant MUC5B COOH-terminus (CT5B; D4-B-C-CK) expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies.

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D'-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the ß-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.

Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D'D3; NT5B) and subdomains (D1, D1-D2, D2-D'-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.

Assessment of polymeric mucin-drug interactions.

Mucosal-delivered drugs have to pass through the mucus layer before absorption through the epithelial cell membrane. Although there has been increasing interest in polymeric mucins, a major structural component of mucus, potentially acting as important physiological regulators of mucosal drug absorption, there are no reports that have systematically evaluated the interaction between mucins and drugs. In this study, we assessed the potential interaction between human polymeric mucins (MUC2, MUC5B, and MUC5AC) and various drugs with different chemical profiles by simple centrifugal method and fluorescence analysis. We found that paclitaxel, rifampicin, and theophylline likely induce the aggregation of MUC5B and/or MUC2. In addition, we showed that the binding affinity of drugs for polymeric mucins varied, not only between individual drugs but also among mucin subtypes. Furthermore, we demonstrated that deletion of MUC5AC and MUC5B in A549 cells increased the cytotoxic effects of cyclosporin A and paclitaxel, likely due to loss of mucin-drug interaction. In conclusion, our results indicate the necessity to determine the binding of drugs to mucins and their potential impact on the mucin network property.

Biofuels: network analysis of the literature reveals key environmental and economic unknowns.

Despite rapid growth in biofuel production worldwide, it is uncertain whether decision-makers possess sufficient information to fully evaluate the impacts of the industry and avoid unintended consequences. Doing so requires rigorous peer-reviewed data and analyses across the entire range of direct and indirect effects. To assess the coverage of scientific research, we analyzed over 1600 peer-reviewed articles published between 2000 and 2009 that addressed 23 biofuels-related topics within four thematic areas: environment and human well-being, economics, technology, and geography. Greenhouse gases, fuel production, and feedstock production were well-represented in the literature, while trade, biodiversity, and human health were not. Gaps were especially striking across topics in the Southern Hemisphere, where the greatest potential socio-economic benefits, as well as environmental damages, may co-occur. There was strong asymmetry in the connectedness of research topics; greenhouse gases articles were twice as often connected to other topics as biodiversity articles. This could undermine the ability of scientific and economic analyses to adequately evaluate impacts and avoid significant unintended consequences. At the least, our review suggests caution in this developing industry and the need to pursue more interdisciplinary research to assess complex trade-offs and feedbacks inherent to an industry with wide-reaching potential impacts.

Students' experiences of a placement with health visitors during their adult nurse training.

This qualitative study explored the experiences of adult nursing students from one university who had undertaken a clinical placement with health visitors. A convenience sample of 10 students were recruited to the study and semistructured interviews used to gather data. Interviews were digitally recorded, transcribed verbatim and subject to thematic analysis. Three global themes emerged: the learning experience; mentors and mentorship; and the nursing and health visiting relationship.

The lipophilic cyclic peptide cyclosporin A induces aggregation of gel-forming mucins.

Cyclic peptides are good candidates for orally delivered therapeutics, however, issues remain in their development due to low intestinal permeability. Although some of the biological factors have been reported that regulate intestinal permeation of cyclic peptides, the influence of the mucus barrier, a major hurdle to epithelial drug delivery, on cyclic peptide bioavailability is unclear. In this study, we show that the lipophilic cyclic peptide, cyclosporin A (CsA), interacted with, and likely induced aggregation, of polymeric, gel-forming mucins (MUC2, MUC5AC and MUC5B) which underpin the mucus gel-networks in the gastrointestinal tract. Under similar conditions, two other cyclic peptides (daptomycin and polymyxin B) did not cause mucin aggregation. Using rate-zonal centrifugation, purified MUC2, MUC5AC and MUC5B mucins sedimented faster in the presence of CsA, with a significant increase in mucins in the pellet fraction. In contrast, mucin sedimentation profiles were largely unaltered after treatment with daptomycin or polymyxin B. CsA increased MUC5B sedimentation was concentration-dependent, and sedimentation studies using recombinant mucin protein domains suggests CsA most likely causes aggregation of the relatively non-O-glycosylated N-terminal and C-terminal regions of MUC5B. Furthermore, the aggregation of the N-terminal region, but not the C-terminal region, was affected by pH. CsA has partially N-methylated amide groups, this unique molecular structure, not present in daptomycin and polymyxin B, may potentially be involved in interaction with gel-forming mucin. Taken together, our results indicate that the interaction of gel-forming mucins with the cyclic peptide CsA is mediated at the N- and C-terminal domains of mucin polymers under physiological conditions. Our findings demonstrate that the mucus barrier is an important physiological factor regulating the intestinal permeation of cyclic peptides in vivo.

Defining the early stages of intestinal colonisation by whipworms.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.

The experiences of nursing students with dyslexia.

AIM: To explore the experiences of pre-registration nursing students with dyslexia at one university. METHOD: Qualitative methodology was used and semi-structured interviews were carried out with a convenience sample of seven students with formally diagnosed dyslexia. Data were analysed using a thematic network approach. FINDINGS: Fear of ridicule and discrimination exist for nursing students with dyslexia, and delays in identification, referral and testing may adversely affect learning. A sense of duty to disclose dyslexia is linked to students' determination to safeguard those in their care, although there is confusion about the nature of disclosure by the university to lecturers and practice colleagues. Students are acutely self-aware although not all feel disabled by dyslexia. Requirements for support relate to personal attributes, knowledge and perception. A perceived lack of caring in the profession for nurses with dyslexia is of concern. CONCLUSION: Early diagnosis of dyslexia enables the provision of appropriate support. Support should be tailored to individuals' needs. Disclosure can cause anxiety, and the attitude of educators and clinical colleagues towards students with dyslexia affects students' experiences. Students with dyslexia are aware of their professional responsibilities. Professional and legislative guidance provides information for those working with students who have a disability.

Mucus.

Mucus is a slimy hydrogel that lines the mucosal surfaces in our body, including the intestines, stomach, eyes, lungs and urogenital tract. This glycoprotein-rich network is truly the jack of all trades. As a barrier, it lubricates surfaces, protects our cells from physical stress, and selectively allows the passage of nutrients while clearing out pathogens and debris. As a home to our microbiota, it supports a level of microbial diversity that is unattainable with most culture methods. As a reservoir of complex carbohydrate structures called glycans, it plays critical roles in controlling cell adhesion and signaling, and it alters the behavior and spatial distribution of microbes. On top of all this, mucus regulates the passage of sperm during fertilization, heals wounds, helps us smell, and prevents the stomach from digesting itself, to name just a few of its functions. Given these impressive features, it is no wonder that mucus crosses boundaries of species and kingdoms - mucus gels are made by organisms ranging from the simplest metazoans to corals, snails, fish, and frogs. It is also no surprise that mucus is exploited in everyday applications, including foods, cosmetics, and other products relevant to medicine and industry.

Fibulin 5 forms a compact dimer in physiological solutions.

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.

Longitudinal analysis of Plantago: age-by-environment interactions reveal aging.

We know very little about aging (senescence) in natural populations, and even less about plant aging. Demographic aging is identified by an increasing rate of mortality following reproductive maturity. In natural populations, quantifying aging is often confounded because changes in mortality may be influenced by both short- and long-term environmental fluctuations as well as age-dependent changes in performance. Plants can be easily marked and monitored longitudinally in natural populations yet the age-dependent dynamics of mortality are not known. This study was designed to determine whether a plant species, Plantago lanceolata, shows demographic aging in its natural environment. A large, multiple-cohort design was used to separate age-independent and age-dependent processes. Seven years of results show environmental influences on mortality as evidenced by synchronous changes in mortality across four cohorts over time. Age-dependent mortality was found through an age-by-environment interaction when the oldest cohorts had significantly higher mortality relative to the younger cohorts during times of stress. Neither size nor quantity of reproduction could explain this variation in mortality across cohorts. These results demonstrate demographic senescence in a natural population of plants.

Effective communication with older people.

Effective communication with older people is an important aspect of nursing practice. Ineffective communication can lead to older people feeling inadequate, disempowered and helpless. Nurses have a duty to ensure that older people think they are being listened to and that their concerns are being validated in a non-judgemental way. Central to effective communication is the ability of nurses to be self-aware, and monitor their thoughts and feelings about, for example, negative stereotypes associated with the ageing process. Effective communication can sometimes be difficult to achieve due to the effects of ageing, but nurses can overcome some barriers through thoughtful interventions. It is important to treat older people as individuals, and to monitor and adapt communication accordingly. By doing so, nurses can ensure older people feel empowered, respected and able to maintain their independence.