http://experimentationlab.berkeley.edu/sites/default/files/AFMImages/butt_AFM.pdf

Force measurements with the atomic force microscope: Technique, interpretation and applications

Gut microbiota is an assortment of microorganisms inhabiting the length and width of the mammalian gastrointestinal tract. The composition of this microbial community is host specific, evolving throughout an individual's lifetime and susceptible to both exogenous and endogenous modifications. Recent renewed interest in the structure and function of this "organ" has illuminated its central position in health and disease. The microbiota is intimately involved in numerous aspects of normal host physiology, from nutritional status to behavior and stress response. Additionally, they can be a central or a contributing cause of many diseases, affecting both near and far organ systems. The overall balance in the composition of the gut microbial community, as well as the presence or absence of key species capable of effecting specific responses, is important in ensuring homeostasis or lack thereof at the intestinal mucosa and beyond. The mechanisms through which microbiota exerts its beneficial or detrimental influences remain largely undefined, but include elaboration of signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and mucosal immune cells. The advances in modeling and analysis of gut microbiota will further our knowledge of their role in health and disease, allowing customization of existing and future therapeutic and prophylactic modalities.

Gut Microbiota in Health and Disease

Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences

https://gut.bmj.com/content/gutjnl/45/suppl_2/II1.full.pdf

The functional gastrointestinal disorders and the Rome III process.

Mucins — large extracellular proteins that are heavily glycosylated with complex oligosaccharides — establish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. Mucins are used as diagnostic markers in cancer, and are under investigation as therapeutic targets for cancer.

Mucins in cancer: Protection and control of the cell surface

Nine mucin genes have been identified in non-ocular tissues. The many mucin gene products share a common structure with highly glycosylated variable number tandem repeat (VNTR) regions separated by naked nonVNTR regions [ 1,2]. How mucin structure relates to their function in the tear film awaits definitive characterisation of the mucins. Also to be elucidated is the relationship between mucins within the preocular mucus gel layer and mucins in aqueous tears over the gel. It is not yet clear whether these phases exist as separate entities. If they do then they might have shared and specific functions including lubrication, hydration of the gel and general and specific mucosal defence [ 1,3]. Gel mucins may require prior biochemical change or degradation for solubilisation to occur, or they may be secreted as a distinct mucin subgroup which makes its way through the gel to the aqueous phase. We aimed to assess the presence of soluble mucins in buman aqueous tears overlaying the preocular gel and to identify a source of these mucins. Unstimulated tears were carefully collected by touching a micro-capillary tube against the lower aqueous tear meniscus. These tears were from 4 normal volunteers aged 16, 31, 12 and 48 years. They were stored in guanidine hydrochloride with protease inhibitors [4] until a volume of 200 pl had been collected. Conjunctival mucins were extracted into guanidme hydrochloride with inhibitors from tissue obtained from the Bristol Eye Bank. Following caesium chloride density gradient centrifugation, native and reduced-alkylated tears and tissue extracts were gel filtrated on Sepharose CL2B then the excluded volume (V,) electrophoresed in 1% agarose gels [5]. Vacuum blots of the gels onto polyvinylidine difluoride membranes were probed with HRP-linked wheatgerm agglutinin (WGA) or antibodies antiMUC2 or anti-MUC5AC against non-VNTR regions of the mucin peptide cores [a]. Antibody binding was detected with HRP-linked second-layer antibody. Serial sections of formalin-fixed wax-embedded bulbar or eyelid conjunctiva were hybridised in situ with "SadATP-labelled 48mer antisense oligonucleotides complementary to messenger RNA for MUC 1-8 genes (VNTR regions). These probes have been applied successfully to several other mucosae [7]. Known positive control tissues and competition controls with the addition of unlabelled probe (50 x excess) were run at the same time. Slides were dipped in photographic emulsion auto-exposed, developed, counter-stained and examined with a microscope to localise MUC gene message. Some sections, and conjunctival impression cytology specimens on nitrocellulose membranes, were probed with the antibodies anti-MUC2 or anti-MUC5AC. Second-layer-antibody only served as a negative control. Tear mucins cross-reacted with aM1 only in the range 1.3-1.4 g/ml (Figure I), a more restricted range than found for tissue mucins. Sepbarose CLZB V, mucins of buoyant density 1.3-1.35 g/ml in native tears were found to migrate with subunit mucins from conjunctival tissue extract (Figure 2). This pattern is not repeated in the range 1.35-1.4 g/ml.

Soluble mucins in human aqueous tears.

Physiological functions for the preocular mucus gel are suggested by bulk properties of the gel and the general structure of mucins, i.e. alternating highly glycosylated (rigid) regions and poorly glycosylated (flexible) peptide domains. Eye-specific adaptations, e.g. short oligosaccharides, highly sialylated epitopes, suggest that these molecules are active at the ocular surface. Short oligosaccharides1 might be imposed by the transparency requirements of the preocular fluid or they might represent an adaptation to fast movement of resident entities through the preocular gel. Sialylated epitopes are an embodiment of the pro-2 and anti-adhesive3 properties of ocular mucins.

Functional processing of ocular mucins.

Metabolic labeling methods for the preparation and biosynthetic study of mucin.

Neuraminidase from Vibrio cholerae was immobilized on Sepharose 4B using the cyanogen bromide technique. The properties of the bound enzyme were found to be similar to those of the soluble form, except for an appreciably improved stability on storage at equivalent dilution and a reduction in recovered activity. Evidence was obtained that the binding of large molecular weight substrates to the bound enzyme is modified due to the immobilized state of the enzyme. The use of the enzyme gel for desialylation of glycoconjugates in a closed circuit system was investigated and optimal conditions delineated using fetuin as a model substrate. The bound enzyme could be used repeatedly with only low loss of activity. The value of the desialylation circuit in the preparation of partially and completely desialylated glycoconjugates is discussed. The use of immobilized neuraminidase for the desialylation of cells is described.

Desialylation of glycoconjugates using immobilized vibrio cholerae neuraminidase. Preparation, properties and use of the bound enzyme.

Restorative proctocolectomy is regarded as a standard surgical procedure for patients who require a proctocolectomy for ulcerative colitis and familial adenomatous polyposis. The ileal mucosa undergoes colonic phenotypic change with time, but the extent and relevance of these changes to the long-term safety of the ileoanal pouch are unclear. The aim of this study was to study the mucin biology of this adaptive process in order to assess its extent and possible impact on pouch safety. Ileoanal pouch biopsies from a cohort of patients and normal ileal and colonic controls were subjected to histological, biochemical, histochemical, and immunohistochemical mucin analysis. Mucin sulphation and sialic acid O-acetylation were studied as parameters of colonic phenotypic change. Fifty-one patients, 16 ileal, and 22 colonic controls were studied. Seventy per cent of biopsies retained villous mucosal architecture, with no cases of dysplasia detected. Ileoanal pouch mucosal sulphation and sialic acid O-acetylation did not reach colonic levels, thus indicating limited evidence for a more colonic phenotype. The data from this study suggest that colonic phenotypic change within the ileoanal reservoir is incomplete, with no cases of dysplasia detected. The degree of phenotypic change is less than in previous studies, which may support, but not prove, our hypothesis that there may be a process of reversion to an ileal type mucosa in the ileoanal reservoir with time.

Anthony P. Corfield

Papers

Soluble mucins in human aqueous tears.

Functional processing of ocular mucins.

Metabolic labeling methods for the preparation and biosynthetic study of mucin.

Desialylation of glycoconjugates using immobilized vibrio cholerae neuraminidase. Preparation, properties and use of the bound enzyme.

Mucin expression in the ileoanal reservoir reflects incomplete mucosal adaptation