The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103(+) type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Evaluation of a corrected implementation of a method of simulating pulmonary nodules in chest tomosynthesis.

Background A method of simulating pulmonary nodules in tomosynthesis images has previously been developed and evaluated. An unknown feature of a rounding function included in the computer code was later found to introduce an artifact, affecting simulated nodules in low-signal regions of the images. The computer code has now been corrected. Purpose To perform a thorough evaluation of the corrected nodule-simulation method, comparing the detection rate and visual appearance of artificial nodules with those of real nodules in an observer performance experiment. Material and Methods A cohort of 64 patients with a total of 129 pulmonary nodules was used in the study. Artificial nodules, each matching a corresponding real nodule by size, attenuation, and anatomical location, were generated and simulated into the tomosynthesis images of the different patients. The detection rate and visual appearance of artificial nodules generated using both the corrected and uncorrected computer code were compared to those of real nodules. The results were evaluated using modified receiver operating characteristic (ROC) analyses. Results The difference in detection rate between artificial and real nodules slightly increased using the corrected computer code (uncorrected code: area under the curve [AUC], 0.47; 95% CI, 0.43-0.51; corrected code: AUC, 0.42; 95% CI, 0.38-0.46). The visual appearance was however substantially improved using the corrected computer code (uncorrected code: AUC, 0.70; 95% CI, 0.63-0.76; corrected code: AUC, 0.49; 95% CI, 0.29-0.65). Conclusion The computer code including a correct rounding function generates simulated nodules that are more visually realistic than simulated nodules generated using the uncorrected computer code, but have a slightly different detection rate compared to real nodules.

Adolescents selling sex: exposure to abuse, mental health, self-harm behaviour and the need for help and support--a study of a Swedish national sample.

BACKGROUND: Selling sex is not uncommon among adolescents and we need to increase our knowledge of how this affects them. AIM: The aim of this study was to investigate adolescents who sell sex regarding sexual, mental and physical abuse, mental health as estimated by using the Hopkins Symptom Check List-25 (HSCL-25), self-harm behaviour and the adolescents' experience of receiving help and support. METHODS: The study was carried out on a national representative sample of adolescents (mean age 18.3 years) in Swedish high schools in the final year of their 3-year programme. The study had 3498 participants and a response rate of 60.4%. RESULTS: Of the adolescents, 1.5% stated that they had sold sexual services. The selling of sex was associated with a history of sexual, mental and physical abuse. Poorer mental health and a higher degree of self-harm behaviour were reported among the adolescents who had sold sex. Help and support was sought to a greater extent by adolescents who had sold sex but these adolescents were not as satisfied with this help and support as the other adolescents. CONCLUSIONS: Adolescents that sell sex are a group especially exposed to sexual, mental and physical abuse. They have poorer mental health and engage in more self-harm behaviour than other adolescents. They are in need of more help and support than other adolescents and it is reasonable to assert that more resources, research and attention should be directed to this group to provide better help and support in the future.

Adolescents' lifetime experience of selling sex: development over five years.

Lifetime experience of selling sex among adolescents was investigated together with sociodemographic correlates, parent-child relationship, and the existence of people to confide in. Changes over time regarding the selling of sex were investigated through a comparison of data from 2004 and 2009. This study was carried out using 3,498 adolescents from a representative sample of Swedish high school students with a mean age 18.3 years. Of these adolescents, 1.5% stated that they had given sexual services for reimbursement and both male and female buyers existed. The adolescents who had sold sex had a poorer parent-child relationship during childhood and had fewer people to confide in about problems and worries. Changes over time were found especially regarding the Internet as a contact source and also immigrant background.

Tryptophan orientations in membrane-bound gramicidin and melittin-a comparative linear dichroism study on transmembrane and surface-bound peptides.

In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.

Effects of chirality on the intracellular localization of binuclear ruthenium(II) polypyridyl complexes.

Interest in binuclear ruthenium(II) polypyridyl complexes as luminescent cellular imaging agents and for biomedical applications is increasing rapidly. We have investigated the cellular localization, uptake, and biomolecular interactions of the pure enantiomers of two structural isomers of [µ-bipb(phen)(4)Ru(2)](4+) (bipb is bis(imidazo[4,5-f]-1,10-phenanthrolin-2-yl)benzene and phen is 1,10-phenanthroline) using confocal laser scanning microscopy, emission spectroscopy, and linear dichroism. Both complexes display distinct enantiomeric differences in the staining pattern of fixed cells, which are concluded to arise from chiral discrimination in the binding to intracellular components. Uptake of complexes in live cells is efficient and nontoxic at 5 µM, and occurs through an energy-dependent mechanism. No differences in uptake are observed between the structural isomers or the enantiomers, suggesting that the interactions triggering uptake are rather insensitive to structural variations. Altogether, these findings show that the complexes investigated are promising for future applications as cellular imaging probes. In addition, linear dichroism shows that the complexes exhibit DNA-condensing properties, making them interesting as potential gene delivery vectors.

Erratum: Reliable phase-contrast flow volume magnetic resonance measurements are feasible without adjustment of the velocity encoding parameter.

[This corrects the article DOI: 10.1117/1.JMI.7.6.063502.].

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.

Reliable phase-contrast flow volume magnetic resonance measurements are feasible without adjustment of the velocity encoding parameter.

Purpose: To show that adjustment of velocity encoding (VENC) for phase-contrast (PC) flow volume measurements is not necessary in modern MR scanners with effective background velocity offset corrections. Approach: The independence on VENC was demonstrated theoretically, but also experimentally on dedicated phantoms and on patients with chronic aortic regurgitation ( n = 17 ) and one healthy volunteer. All PC measurements were performed using a modern MR scanner, where the pre-emphasis circuit but also a subsequent post-processing filter were used for effective correction of background velocity offset errors. Results: The VENC level strongly affected the velocity noise level in the PC images and, hence, the estimated peak flow velocity. However, neither the regurgitant blood flow volume nor the mean flow velocity displayed any clinically relevant dependency on the VENC level. Also, the background velocity offset was shown to be close to zero ( < 0.6 cm / s ) for a VENC range of 150 to 500 cm / s , adding no significant errors to the PC flow volume measurement. Conclusions: Our study shows that reliable PC flow volume measurements are feasible without adjustment of the VENC parameter. Without the need for VENC adjustments, the scan time can be reduced for the benefit of the patient.

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6-/- , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18-/- mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.

Luminescent dipyridophenazine-ruthenium probes for liposome membranes.

Ruthenium complexes with dipyridophenazine (dppz) type ligands have several characteristics that make them good candidates for use as luminescence probes for hydrophobic environments. Most studies have concerned DNA intercalation, but also lipid membrane fluidity and liposome orientation have been assessed. We report here dipyridophenazine derivatives ([Ru(phen)2dppz]2+) substituted with one or two alkyl ether chains of different lengths aimed at finding the optimum substitution for a high quantum yield when bound to a phospholipid membrane bilayer. The orientation of membrane bound molecules is studied using flow linear dichroism (LD) with phospholipid vesicles as membrane models. LD, excitation anisotropy, steady state luminescence and excited-state lifetime measurements are used to quantitatively investigate the insertion and orientation of the complexes in the vesicles. All complexes are inserted with their long axis of the dppz moiety mainly parallel to the lipid chains, and the degree of orientation is comparable to that of the orientation probe retinoic acid. The ruthenium "head group" with its positive charge functions as a buoy at the water-membrane interface while the hydrophobic chain part embeds the complex down into the bilayer. The complex with two hexyl ether substituents (named D6) has the optimal chain length regarding membrane insertion and orientation, and together with the highest quantum yield, is the best luminescence membrane probe in the two series.

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals.

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

Study of mucin turnover in the small intestine by in vivo labeling.

Mucins are highly glycosylated proteins which protect the epithelium. In the small intestine, the goblet cell-secreted Muc2 mucin constitutes the main component of the loose mucus layer that traps luminal material. The transmembrane mucin Muc17 forms part of the carbohydrate-rich glycocalyx covering intestinal epithelial cells. Our study aimed at investigating the turnover of these mucins in the small intestine by using in vivo labeling of O-glycans with N-azidoacetylgalactosamine. Mice were injected intraperitoneally and sacrificed every hour up to 12 hours and at 24 hours. Samples were fixed with preservation of the mucus layer and stained for Muc2 and Muc17. Turnover of Muc2 was slower in goblet cells of the crypts compared to goblet cells along the villi. Muc17 showed stable expression over time at the plasma membrane on villi tips, in crypts and at crypt openings. In conclusion, we have identified different subtypes of goblet cells based on their rate of mucin biosynthesis and secretion. In order to protect the intestinal epithelium from chemical and bacterial hazards, fast and frequent renewal of the secreted mucus layer in the villi area is combined with massive secretion of stored Muc2 from goblet cells in the upper crypt.

Characterization of complex flow patterns in the ascending aorta in patients with aortic regurgitation using conventional phase-contrast velocity MRI.

Ascending aorta (AA) flow displacement (FD) is a surrogate for increased wall shear stress. We prospectively studied the flow profile in the AA in patients with aortic regurgitation (AR), to identify predictors of FD and investigate whether magnetic resonance imaging (MRI) phase-contrast flow rate curves (PC-FRC) contain quantitative information related to FD. Forty patients with chronic moderate (n = 14) or severe (n = 26) AR (21 (53%) with bicuspid aortic valve) and 22 controls were investigated. FD was determined from phase-contrast velocity profiles and defined as the distance between the center of the lumen and the "center of velocity" of the peak systolic forward flow or the peak diastolic negative flow, normalized to the lumen radius. Forward and backward volume flow was determined separately for systole and diastole. Seventy percent had systolic backward flow and 45% had diastolic forward flow in large areas of the vessel. AA dimension was an independent predictor of systolic FD while AA dimension and regurgitant volume were independent predictors of diastolic FD. Valve phenotype was not an independent predictor of systolic or diastolic FD. The linear relationships between systolic backward flow and systolic FD and diastolic forward flow and diastolic FD were strong (R = 0.77 and R = 0.76 respectively). Systolic backward flow and diastolic forward flow identified marked systolic and diastolic FD (≥0.35) with a positive likelihood ratio of 6.0 and 10.8, respectively. In conclusion, conventional PC-FRC data can detect and quantify FD in patients with AR suggesting the curves as a research and screening tool in larger patient populations.

Pulsed-Wave Doppler Recordings in the Proximal Descending Aorta in Patients with Chronic Aortic Regurgitation: Insights from Cardiovascular Magnetic Resonance.

BACKGROUND: The pulsed-wave Doppler recording in the descending aorta (PWDDAO) is one of the parameters used in grading aortic regurgitation (AR) severity. The aim of the present study was to investigate the assessment of chronic AR by PWDDAO with insights from cardiovascular magnetic resonance (CMR). METHODS: This prospective study comprised 40 patients investigated with echocardiography and CMR within 4 hours either prior to valve surgery (n = 23) or as part of their follow-up (n = 17) due to moderate or severe AR. End-diastolic flow velocity (EDFV) and the diastolic velocity time integral (dVTI) were measured. The appearance of diastolic forward flow (DFF) was noted. Phase-contrast flow rate curves were obtained in the DAO. RESULTS: Twenty-five patients had severe and eight had moderate AR by echocardiography (seven were indeterminate). The EDFV was below the recommended threshold (>20 cm/sec) in 13 patients (52%) with severe AR. Lowering the EDFV threshold (>13 cm/sec) and with a dVTI threshold >13 cm showed negative likelihood ratios of 0.27 and 0.09, respectively. Detection of DFF with PWDDAO identified a nonuniform velocity profile by CMR with positive and negative likelihood ratios of 7.0 and 0.19, respectively. The relation between EDFV and DAO regurgitant volume (DAO-RVolCMR) was strong in patients without (R = 0.88) and weak in patients with DFF (R = 0.49). The DAO-RVolCMR as a percent of the total RVolCMR decreased with increasing ascending aorta (AAO) size and increased with increasing AR severity. CONCLUSIONS: Our findings suggest that PWDDAO provides semiquantitative parameters useful to assess chronic AR severity. The limitations are related to nonuniform velocity contour and variable degree of lower body contribution, which depends on AR severity but also on the AAO size.

Retinoid chromophores as probes of membrane lipid order.

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.

Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization.

The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus organization of GF mice was similar to that of conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required 5 weeks to be normally detached and colonic inner mucus 6 weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until 3 weeks, when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.

Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell-protective device.

MUC1 and other membrane-associated mucins harbor long, up to 1 µm, extended highly glycosylated mucin domains and sea urchin sperm protein, enterokinase and agrin (SEA) domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is highly conserved and undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. To date, no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nN·s(-1). Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.

Correlation between cellular localization and binding preference to RNA, DNA, and phospholipid membrane for luminescent ruthenium(II) complexes.

Because of their unique photophysical properties, sensitively depending on environment, ruthenium dipyridophenazine (dppz) complexes are interesting as probes for cellular imaging with fluorescence microscopy. Here three complexes derivatized with alkyl ether chains of varied length, which exhibit distinctly different cellular staining patterns by confocal laser scanning microscopy, are studied regarding their binding preference for rRNA compared with calf thymus DNA (ct-DNA) and phospholipid membranes. Co-staining with commercial RNA and membrane-specific dyes shows that whereas the least lipophilic complex exclusively stains DNA inside the nucleus, the most lipophilic complex preferentially stains membrane-rich parts of the cell. Interestingly, only the intermediate lipophilic complex shows intense staining of the RNA-rich nucleoli. The intracellular localizations of the probes correlate with their binding preferences concluded from spectroscopy measurements.

Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy.

Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.