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Background & AimClostridioides difficileinfection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced byC. difficileare the major determinants of disease. However, the physiological cause of diarrhea associated with CDI is not well understood. We investigated the effects of CDI on paracellular permeability and apical ion transporters.MethodsWe studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure regional differences in paracellular permeability and ion transporter function in intestinal mucosa. Intestinal tissues were collected from mice and analyzed by immunofluorescence microscopy and RNA-sequencing.ResultsCDI increased intestinal permeability through the size-selective leak pathwayin vivo, but permeability was not increased at the sites of pathological damage. Chloride secretion was reduced in the cecum during infection by decreased CaCC function. Infected mice had decreased SGLT1 (also called SLC5A1) activity in the cecum and colon along with diminished apical abundance and an increase in luminal glucose. SGLT1 and DRA (also called SLC26A3) expression was ablated by either TcdA or TcdB, but NHE3 (also called SLC9A3) was decreased in a TcdB-dependent manner. Finally, expression of these three ion transporters was drastically reduced at the transcriptional level.ConclusionsCDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination may cause a dysfunction in water and solute absorption in the lower gastrointestinal tract, leading to osmotic diarrhea. These findings may open novel pathways for attenuating CDI-associated diarrhea.GRAPHICAL ABSTRACT

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626–633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474–4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota, et al. first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin (H&E) for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore-formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared with slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474–4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Clostridioides difficile is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous sglucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a “cytopathic” disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of “hit compounds” with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.

SUMMARY Clostridioides difficile is the leading cause of nosocomial intestinal infections in the United States. Ingested C. difficile spores encounter host bile acids and other cues that are necessary for germinating into toxin-producing vegetative cells. While gut microbiota disruption (often by antibiotics) is a prerequisite for C. difficile infection (CDI), the mechanisms C. difficile employs for colonization remain unclear. Here, we pioneered the application of imaging mass spectrometry to study how enteric infection changes gut metabolites. We find that CDI induces an influx of bile acids into the gut within 24 h of the host ingesting spores. In response, the host reduces bile acid biosynthesis gene expression. These bile acids drive C. difficile outgrowth, as mice receiving the bile acid sequestrant cholestyramine display delayed colonization and reduced germination. Our findings indicate that C. difficile may facilitate germination upon infection and suggest that altering flux through bile acid pathways can modulate C. difficile outgrowth in CDI-prone patients., Graphical abstract, In brief To illuminate how the gut environment changes during enteric infection, Wexler, Guiberson et al. measured the abundances and distributions of gut metabolites during C. difficile infection using imaging mass spectrometry. They find that C. difficile rapidly alters host bile acid physiology during colonization, which facilitates spore germination and outgrowth.

Currently, Hawaii is almost totally dependent for energy on imported oil. The island state has a wide variety of renewable energy resources, however, and for the past decade has supported the development of these resources as substitutes for seaborne petroleum. Sufficient progress has been made to date in commercializing a number of these alternative energy sources to give cause for optimism tIhat Hawaii will be able to achieve energy self-sufficiency with its indigenous renewable resources.

Hawaii is the state most vulnerable to dislocation in the global energy market, with more than 91% of its total energy coming into the state as imported oil-two thirds of which is foreign crude. This near-total dependence on seaborne petroleum is a paradox, since Hawaii has a wide variety of renewable energy resources which can serve as alternatives to oil. These renewable resources have the potential of being less polluting than conventional energy supplies and so are consistent with Hawaii’s concern for the quality of life. For the past decade Hawaii has been engaged in a comprehensive program of resource assessment, research, development, demonstration, and commercialization of its indigenous energy resources: direct solar, wind, biomass, ocean thermal, geothermal. Sufficient progress has been made to date with a number of these energy alternatives, so that Hawaii should be able to maintain its momentum toward achieving energy self-sufficiency—even in the face of drastic reductions in federal support for renewable energy research, development and demonstration and the present declining cost of oil.

Island communities have energy-supply problems that differ appreciably from those of major land masses. There is usually an absence of local fossil fuel reserves, such as oil, natural gas and coal, as well as a limited infrastructure for delivering energy. This lack of flexibility requires island communities to satisfy their energy demands with indigenous resources, plus whatever energy supply is imported by sea. In recent years, the energy needs of many island communities have been met almost entirely by seaborne petroleum. As imported oil has become increasingly more expensive and less secure, greater effort has been directed by some island communities toward the development of their local energy resources. A case study illustrates the progress made to date in one island area - Hawaii - in minimizing its near-total dependence on seaborne petroleum. However, there should be some spin-off of this experience to regional energy planning, not only to other island communities, but also to regions within developing countries that share many of these characteristics. 34 references, 6 figures, 6 tables.

"Presented to the Circum-Pacific Energy and Mineral Resources Conference in Honolulu, Hawaii, August 2, 1978." Includes bibliography.

This report lists the desirable features that should be included in a laboratory method for evaluating the skidding resistance of different types of pavement surfaces, and discusses them in light of previous research. The laboratory skid-test apparatus, resulting from an endeavor to incorporate these desirable features into the design, is described. In order to use this skid-test apparatus with some degree of confidence, a field correlation study was performed on 18 different bituminous pavement surfaces. A procedure, with associated instrumentation, was also developed for simulating the wear and polishing effect that a pavement surface receives under the action of traffic. The skidding resistance after various degrees of wear was determined for bituminous mixtures made with 22 different aggregates. A petrographic analysis was performed on each of the 22 aggregates, as well as a partial chemical analysis on each of the 12 limestones. The skidding characteristics X are discussed with respect to the basic properties of the aggregates. The effect of surface texture, primarily as determined by gradation, on the skidding resistance of bituminous mixtures was evaluated for four different aggregate types. The effect of initial aggregate shape was also evaluated by comparing the skidding characteristics of mixtures containing freshly-crushed aggregate with those made with the same type of aggregate after it had been rounded in a Los Angeles abrasion machine. A series of skid-resistance tests was also performed on selected dense-graded mixtures. In general, the results of this study substantiated the prevailing opinion as to the high skidding resistance of dense surfaces containing hard, angular quartz particles. The final phase of this study investigated the effectiveness of different blending procedures on improving the skidding resistance of specimens containing a polish-susceptible limestone. Of the six blending ingredients investigated, silica sand was the most effective in improving the anti-skid characteristics of a bituminous mixture made with a polish-susceptible aggregate.

The majority of the highways which have been constructed recently, whether of portland cement or of bituminous materials, have possessed adequate wet skidding resistance when new. Unfortunately, on many of these surfaces this initial anti-skid resistance has been short-lived. As a result, it has become increasingly important to give some consideration to the change in skidding resistance of pavement surfaces due to the polishing effects of traffic. There is little existing knowledge, other than experience on specific surfaces, with which to predict the long term behavior of the many different surface types. Those contributions that have been made have resulted, for the most part, from field investigations. It is impossible, however, in a field investigation to evaluate the separate effect of many inter-related variables. It was felt that the control and consistency inherent in a laboratory investigation would permit a more accurate determination of the basic factors contributing to slipperiness of pavement surfaces. Therefore, the laboratory equipment and testing procedure were developed with this end in mind. Although this paper relates entirely to bituminous mixtures, many phases of the testing procedure pertain equally well to portland cement concrete surfaces, which currently are being investigated in a similar study.

Experience indicates that highways which are constructed to conform to current design standards may become dangerously slippery when wet after a relatively short period of wear. As the polishing effect of traffic continues to become more intensified, the incidence of slippery sections of pavement will tend to increase. In order to minimize the occurrence of these skidding hazards, the highway engineer must include permanency of skid resistance as a design parameter in selecting a suitable paving mixture. A laboratory testing procedure was developed at Purdue University to investigate the slipperiness potential of different highway materials, and to predict the resistance of paving mixtures to the polishing effect of simulated traffic. The laboratory testing method, a field correlation study, and an accelerated wear and polish procedure are summarized in this report, along with the results of the initial phases of the research which have been reported upon previously. Included in this summary are 1) a report of the polishing characteristics of aggregates in both portland-cement and bituminous mixtures and 2) a study of the effect of surface texture, or degree of openness, and initial aggregate shape on the skid resistance of bituminous mixtures. A more complete discussion is presented of subsequent research in which the effect of blending a polish-resistance material with a polish-susceptible aggregate in both portland-cement and bituminous mixtures was determined, and of a study in which the anti-skid characteristics of fine-grained surface treatments were investigated. A final summary is presented which includes the authors' recommendations with regard to design and construction practices that tend to minimize pavement slipperiness.

The majority of the highways which have been constructed recently, whether of portland cement or of bituminous materials, have possessed adequate wet skidding resistance when new. Unfortunately, on many of these surfaces this initial anti-skid resistance has been short-lived. As a result, it has become increasingly important to give some consideration to the change in skidding resistance of pavement surfaces due to the polishing effects of traffic. There is little existing knowledge, other than experience on specific surfaces, with which to predict the long term behavior of the many different surface types. Those contributions that have been made have resulted, for the most part, from field investigations. It is impossible, however, in a field investigation to evaluate the separate effect of many inter-related variables. It was felt that the control and consistency inherent in a laboratory investigation would permit a more accurate determination of the basic factors contributing to slipperiness of pavement surfaces. Therefore, the laboratory equipment and testing procedure were developed with this end in mind. Although this paper relates entirely to bituminous mixtures, many phases of the testing procedure pertain equally well to portland cement concrete surfaces, which currently are being investigated in a similar study.

The majority of the highways which have been constructed recently, whether of portland cement or of bituminous materials, initially have exhibited good resistance to skidding for the critical design condition, which occurs when the surface is wet. Unfortunately, for many of these pavements, this initial skid resistance has been short-lived. The anti-skid properties of a well-designed pavement surface are dependent to a large degree upon the polishing characteristics of the mineral aggregate or aggregates of which the paving mixture is composed. Although the type of cementing agent, the gradation, and the openness of the surface have some effect upon the rate at which polishing progresses, the ultimate state of slipperiness is determined by the nature of wear of the pavement which, in turn, is directly related to the resistance to polishing of the surface aggregate. If highways are to be built which possess adequate anti-skid characteristics for a reasonable period of wear under the ever-increasing polishing effect of traffic, consideration must be given to the slipperiness potential of highway materials of which paving mixtures are composed, so that adequate skid resistance ray be included as a design parameter. A laboratory investigation was undertaken at Purdue University in an endeavor to contribute to a better understanding of some of the variables involved in pavement slipperiness. This paper is a report of a phase of the Purdue study which includes the following factors: 1) The polishing characteristics of different mineral aggregates, in both portland-cement and bituminous-concrete mixtures, and their relation to basic aggregate properties; 2) The effect of texture, as determined by gradation, on the anti-skid characteristics of bituminous mixtures; and 3) The effect of initial aggregate shape, or degree of harshness, on the skid resistance of bituminous mixtures.

The lift and drag forces on a model of a pipeline resting on the ocean floor in a uniform current were measured in a wind tunnel. Measurements of lift and drag were made on the upper cylinder for a Reynolds number range of 5X10 to the 4th power to 1.3X10 to the 5th power. They were obtained using eight strain gages mounted on each of two strain rings, with the output read from a calibrated strip-chart recording. Lift and drag coefficients were calculated from the force measurements. The lift coefficient maintained a value of approximately 1.4 over the Reynolds number range tested. The drag coefficient, however, rose gradually from about 0.8 to 0.9. These results apply to moderate offshore conditions.