In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and super-resolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of Vibrio polysaccharide (VPS), RbmC, and Bap1 formed dynamic, flexible and ordered envelopes that encased the cell clusters.

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of Vibrio polysaccharide (VPS), RbmC, and Bap1 formed dynamic, flexible and ordered envelopes that encased the cell clusters. Microbes within biofilms are more resistant to antibiotics, to immune clearance, and to osmotic, acid and oxidative stresses compared to planktonic cells (1-7). Despite advances in identifying the polysaccharide and proteinaceous constituents of the biofilm extracellular matrix, the mechanisms by which these factors yield a mechanically defined and spatially organized biofilm are largely unknown (8-10). The small size of most microbes has precluded multi-scale optical investigation of living biofilms. Vibrio cholerae biofilm formation involves the production of Vibrio polysaccharide (VPS) and three matrix proteins (RbmA, RbmC, and Bap1) predicted to contain carbohydrate-binding domains (fig. S1A) (11-13). To investigate the molecular mechanisms of biofilm development, we used a V. cholerae rugose variant with increased capacity to form biofilms (11). We inserted Myc, FLAG, and HA (Human influenza hemagglutinin) epitopes into its genome at the 3' ends of * To whom correspondence should be addressed.

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Objective: To investigate the effects of Ramadan fasting on serum concentrations of immunoglobulin (Ig)G and IgM, and salivary IgA concentrations. Methods: Blood and saliva samples were collected one week before and during the last week of Ramadan from healthy male volunteers. Albumin, total lymphocyte count, electrolytes, and IgG and IgM concentrations were determined in serum; salivary IgA concentrations were measured. Anthropometric measurements were also recorded. Results: Samples were collected from 35 subjects (mean age 35.86 years, range 20-59 years). Weight, body mass index, albumin levels and the nutritional risk index decreased significantly during Ramadan fasting compared with before fasting. In addition, Na þ and Cl À electrolyte levels were significantly decreased during Ramadan. Serum IgG concentrations decreased significantly during Ramadan compared with before fasting, but were still within the normal range. Salivary IgA concentrations also decreased significantly, whereas serum IgM levels did not change. Lymphocyte numbers increased significantly, but there was no correlation between Ig levels and lymphocyte count. Conclusion: Ramadan fasting did not result in severe immunological disturbances.

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