The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Action of reactive oxygen species on colonic mucus secretions

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many colonic diseases. Mucus is the colon's first line of defence against luminal agents. This study has therefore characterised ROS action on colonic mucus secretions. ROS were produced using peroxide-based systems of different concentrations. The effects of these systems were tested on native colonic mucus gels, isolated colonic mucins, and in vivo models. Colonic mucus gels were resistant to ROS breakdown. Mucins were susceptible to ROS attack, causing loss of terminal sugars and protein and mucin fragmentation. The in vivo thickness of the mucus barrier was reduced by up to 50% by ROS (above 5 mM peroxide). A 5 mM peroxide caused a significant increase in resting mucus thickness of ca. 15%. All ROS-generating systems caused mucosal damage once the loosely adherent mucus had been removed. As native mucus gel is more resistant to ROS damage than purified mucin, nonmucin components of mucus may have extensive ROS-scavenging properties. Low levels of luminal colonic ROS increase the protection afforded by the mucus barrier in vivo. Higher levels of ROS significantly reduce this protection. In vitro modeling of mucus degradation by ROS does not necessarily correlate with the dynamic, in vivo situation.     

Survival of nisin activity in intestinal environment

The sensitivity of nisin to proteolytical breakdown in intestinal environment was studied in an ex vivo model using jejunal chyme from fistulated dogs. Sixty six percentage of the added nisin retained induction activity after 30 min incubation in jejunal chyme, indicating that nisin has potential to be used as an inducing agent in in situ delivery systems of bioactive peptides and proteins by genetically modified bacteria in the intestine.     

Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Evaluation of the ability of Bifidobacterium longum to metabolize human intestinal mucus

The ability of Bifidobacterium longum to use intestinal mucus as a metabolizable source was characterized. Bifidobacterium longum biotype longum NCIMB8809 was grown in a chemically semi-defined medium supplemented with human intestinal mucus, and the cytoplasmic protein profiles and several glycosyl hydrolase activities were analysed and compared with those obtained from the same bacterium grown in the absence of mucus. We were able to identify 22 different proteins in the cytoplasmic fraction, of which nine displayed a different concentration in the presence of mucus. Among the proteins whose concentrations varied, we found specific enzymes that are involved in the response to different environmental conditions, and also proteins that mediate interaction with mucus in bacteria. Significant changes in some glycoside-hydrolysing activities were also detected. In addition, stable isotope labelling of amino acids in cell culture demonstrated that B. longum incorporates leucine from the glycoprotein matrix of mucin within its proteins. This study provides the first proteomic data regarding the interaction of B. longum with intestinal mucus, and contributes to the understanding of the behaviour of this intestinal species in its natural ecological niche.     

An adherent mucus layer attenuates the genotoxic effect of colibactin

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase‐island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide‐nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA‐DSBs. Removal of the adherent mucus layer restored the occurrence of DNA‐DSBs.     

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Hyperglycemic and Hyperlipidemic Conditions Alter Cardiac Cell Biomechanical Properties

Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.     

Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon

Background: Short chain fatty acids (SCFAs) affect various intestinal functions. Mucus is an important physiological component of the intestinal mucosal barrier. However, the effect of SCFAs or other organic acids on the intestinal mucus release is poorly understood. The aim of this study was to investigate whether lumen SCFA stimulates mucus release into the rat colon. Methods: A solution of SCFA, lactate or succinate was infused into the colon of anesthetized rats, and we then measured the hexose content of the effluent. We also examined the influence of cholinergic antagonists on the effects of SCFA. Results: A SCFA mixture (75 mM acetate, 35 mM propionate and 20 mM butyrate) or individual SCFAs (130 mM) increased the mucus release into the colon in a similar manner. The individual SCFAs, but not lactate or succinate, stimulated colonic mucus secretion in similar concentration-dependent manners. Butyrate stimulated colonic mucus secretion at 20 mM, but acetate, propionate, lactate and succinate at this concentration did not. Pretreatment with an anti-cholinergic agent diminished the stimulatory effects of SCFAs on mucus secretion. Conclusions: Lumen SCFAs, but not lactate or succinate, stimulate mucus release from the rat colon via a cholinergic nerve mechanism.     

Correlation between in vitro mucus adhesion and the in vivo colonization ability of lactic acid bacteria: screening of new candidate carp probiotics

We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.     

Synchrotron x-ray microdiffraction reveals intrinsic structural features of amyloid deposits in situ

Amyloidoses are increasingly recognized as a major public health concern in Western countries. All amyloidoses share common morphological, structural, and tinctorial properties. These consist of staining by specific dyes, a fibrillar aspect in electron microscopy and a typical cross-β folding in x-ray diffraction patterns. Most studies that aim at deciphering the amyloid structure rely on fibers generated in vitro or extracted from tissues using protocols that may modify their intrinsic structure. Therefore, the fine details of the in situ architecture of the deposits remain unknown. Here, we present to our knowledge the first data obtained on ex vivo human renal tissue sections using x-ray microdiffraction. The typical cross-β features from fixed paraffin-embedded samples are similar to those formed in vitro or extracted from tissues. Moreover, the fiber orientation maps obtained across glomerular sections reveal an intrinsic texture that is correlated with the glomerulus morphology. These results are of the highest importance to understanding the formation of amyloid deposits and are thus expected to trigger new incentives for tissue investigation. Moreover, the access to intrinsic structural parameters such as fiber size and orientation using synchrotron x-ray microdiffraction, could provide valuable information concerning in situ mechanisms and deposit formation with potential benefits for diagnostic and therapeutic purposes.     

In Vitro Cellular Division of Trypanosoma abeli Reveals Two Pathways for Organelle Replication

Since the observation of the great pleomorphism of fish trypanosomes, in vitro culture has become an important tool to support taxonomic studies investigating the biology of cultured parasites, such as their structure, growth dynamics, and cellular cycle. Relative to their biology, ex vivo and in vitro studies have shown that these parasites, during the multiplication process, duplicate and segregate the kinetoplast before nucleus replication and division. However, the inverse sequence (the nucleus divides before the kinetoplast) has only been documented for a species of marine fish trypanosomes on a single occasion. Now, this previously rare event was observed in Trypanosoma abeli, a freshwater fish trypanosome. Specifically, from 376 cultured parasites in the multiplication process, we determined the sequence of organelle division for 111 forms; 39% exhibited nucleus duplication prior to kinetoplast replication. Thus, our results suggest that nucleus division before the kinetoplast may not represent an accidental or erroneous event occurring in the main pathway of parasite reproduction, but instead could be a species‐specific process of cell biology in trypanosomes, such as previously noticed for Leishmania. This “alternative” pathway for organelle replication is a new field to be explored concerning the biology of marine and freshwater fish trypanosomes.     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

Oxymatrine exerts protective effects on osteoarthritis via modulating chondrocyte homoeostasis and suppressing osteoclastogenesis

Osteoarthritis (OA) is a common degenerative disease characterized by the progressive destruction both articular cartilage and the subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone loss are crucial for the prevention and treatment of OA. Oxymatrine (OMT) is a natural compound with anti‐inflammatory and antitumour properties. We found that OMT exhibited a strong inhibitory effect on LPS‐induced chondrocyte inflammation and catabolism. To further support our results, fresh human cartilage explants were treated with LPS to establish an ex vivo degradation model, and the results revealed that OMT inhibited the catabolic events of LPS‐stimulated human cartilage and substantially attenuated the degradation of articular cartilage ex vivo. As subchondral bone remodelling is involved in OA progression, and osteoclasts are a unique cell type in bone resorption, we investigated the effects of OMT on osteoclastogenesis, and the results demonstrated that OMT suppresses RANKL‐induced osteoclastogenesis by suppressing the RANKL‐induced NFATc1 and c‐fos signalling pathway in vitro. Further, we found that the anti‐inflammatory and anti‐osteoclastic effects of oxymatrine are mediated via the inhibition of the NF‐κB and MAPK pathways. In animal studies, OMT suppressed the ACLT‐induced cartilage degradation, and TUNEL assays further confirmed the protective effect of OMT on chondrocyte apoptosis. MicroCT analysis revealed that OMT had an attenuating effect on ACLT‐induced subchondral bone loss in vivo. Taken together, these results show that OMT interferes with the vicious cycle associated with OA and may be a potential therapeutic agent for abnormal subchondral bone loss and cartilage degradation in osteoarthritis.     

Multiple shoot induction and plant regeneration of the endangered species <Emphasis Type="Italic">Crepis novoana</Emphasis>

In vitro culture is a useful tool in the ex situ conservation of rare, endemic, and threatened plant species. Crepis novoana (Compositae) is an endangered endemic in northwestern Spain. Use of in vitro culture tools is necessary due to the poor conservation status of populations of the species. The systems of in vitro propagation developed for this species in the present study were caulogenesis from leaf explants and growth of axillary buds from shoots. Explants were produced by placing fragments of leaves on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzyladenine (BA) and 2.69 μM naphthaleneacetic acid (NAA); caulogenesis was induced in 80% of explants, with development of a mean number of 2.48 shoots per explant. Axillary bud development from shoots was highest with MS supplemented with 4.44 μM BA and 0.54 μM NAA, resulting in production of a mean number of 49.77 shoots per explant. Immersion of the basal side of shoots in a solution of 5.37 mM NAA for 30 s yielded 90% success in the production of rooted shoots. Plantlets were well acclimatized, and almost 100% of plants transferred to soil recovered successfully.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.     

Quantifying Intracellular Particle Flows by DIC Object Tracking

Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.