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Background Often in simulated settings, quantitative analysis of technical skill relies largely on specially tagged instruments or tracers on surgeons’ hands. We investigated a novel, marker-less technique for evaluating technical skill during open operations and for differentiating tasks and surgeon experience level. Methods We recorded the operative field via in-light camera for open operations. Sixteen cases yielded 138 video clips of suturing and tying tasks ≥5 seconds in duration. Video clips were categorized based on surgeon role (attending, resident) and task subtype (suturing tasks: body wall, bowel anastomosis, complex anastomosis; tying tasks: body wall, superficial tying, deep tying). We tracked a region of interest on the hand to generate kinematic data. Nested, multilevel modeling addressed the nonindependence of clips obtained from the same surgeon. Results Interaction effects for suturing tasks were seen between role and task categories for average speed ( P = .04), standard deviation of speed ( P = .05), and average acceleration ( P = .03). There were significant differences across task categories for standard deviation of acceleration ( P = .02). Significant differences for tying tasks across task categories were observed for maximum speed ( P = .02); standard deviation of speed ( P = .04); and average ( P = .02), maximum ( P P = .03) of acceleration. Conclusion We demonstrated the ability to detect kinematic differences in performance using marker-less tracking during open operative cases. Suturing task evaluation was most sensitive to differences in surgeon role and task category and may represent a scalable approach for providing quantitative feedback to surgeons about technical skill.

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Genistein, a naturally occurring isoflavone, augments in vitro epithelial anion transport via activation of the cystic fibrosis transmembrane conductance regulator chloride channel. In this study, we examined whether chronic dietary exposure to 600 mg/kg genistein (600 G) for 1 mo would stimulate anion secretion across wild-type (Wt, normal) murine intestine. Anion secretion was assessed in freshly excised segments of murine jejuna by measuring short circuit current (I(sc)) and comparing with jejunal segments from mice fed 0 mg/kg genistein (0 G). Basal and forskolin-stimulated anion secretions were augmented (P < 0.05) in female but not in male mice fed 600 G, compared with their counterparts fed 0 G. Serum genistein concentrations were greater in both female and male mice fed 600 G (approximately 3.5-6.9 micromol/L) than those fed 0 G (approximately 100 nmol/L). Anion substitution experiments and bumetanide-sensitivity demonstrated that chloride was the major anion mediating the increased secretion. A smaller bicarbonate component was not augmented by consumption of the genistein diet. These data indicate that chronic exposure to dietary genistein stimulates a sex-dependent increase in basal and forskolin-stimulated chloride secretion across murine intestine.

Background & Aims: Unlike the intestine of normal subjects, small-intestinal epithelia of cystic fibrosis patients and cystic fibrosis transmembrane conductance regulator protein-null (CFTR − ) mice do not respond to stimulation of intracellular cyclic adenosine monophosphate with inhibition of electroneutral NaCl absorption. Because CFTR-mediated anion secretion has been associated with changes in crypt cell volume, we hypothesized that CFTR-mediated cell volume reduction in villus epithelium is required for intracellular cyclic adenosine monophosphate inhibition of Na + /H + exchanger (primarily Na + /H + exchanger 3) activity in the proximal small intestine. Methods: Transepithelial 22 Na flux across the jejuna of CFTR + , CFTR − , the basolateral membrane Na + /K + /2Cl − co-transporter protein NKCC1 + , and NKCC1 − mice were correlated with changes in epithelial cell volume of the midvillus region. Results: Stimulation of intracellular cyclic adenosine monophosphate resulted in cessation of Na + /H + exchanger-mediated Na + absorption (J ms NHE ) in CFTR + jejunum but had no effect on J ms NHE across CFTR − jejunum. Cell volume indices indicated an approximately 30% volume reduction of villus epithelial cells in CFTR + jejunum but no changes in CFTR − epithelium after intracellular cyclic adenosine monophosphate stimulation. In contrast, cell shrinkage induced by hypertonic medium inhibited J ms NHE in both CFTR + and CFTR − mice. Bumetanide treatment to inhibit Cl − secretion by blockade of the Na + /K + /2Cl − co-transporter, NKCC1, of stimulated CFTR + jejunum prevented maximal volume reduction of villus epithelium and recovered approximately 40% of J ms NHE . Likewise, J ms NHE and cell volume were unaffected by intracellular cyclic adenosine monophosphate stimulation in NKCC1 − jejuna. Conclusions: These findings show a previously unrecognized role of functional CFTR expressed in villus epithelium: regulation of Na + /H + exchanger 3-mediated Na + absorption by alteration of epithelial cell volume.

A 1953 telescopic photograph of a flash on the Moon is the only unequivocal record of the rare crash of an asteroid-sized body onto the lunar surface. We estimate that this event would create an impact feature up to several km in size and that the diameter of the impacting body would be about 20 m. Such an event would cause regional devastation if it occurred on Earth. Although not detectable with ground- based telescopes, the lunar crater should be visible in space-based images of the Moon. A search of images from the Clementine mission reveals an ∼1.5-km high-albedo, blue, fresh-appearing crater with an associated ejecta blanket at the location of the flash. The identification of this crater offers an opportunity to investigate subsurface unaltered lunar soils.

In comparing product bioavailability across animal species, it is not unusual to observe marked interspecies differences. For many compounds, these differences reflect presystemic drug metabolism. However, a host of other variables must also be considered such as in vivo drug solubility, gastric transit time, intestinal permeability, diet, and species-by-formulation interactions. By combining information on drug solubility and intestinal permeability with an understanding of the interrelationship between pH, product dissolution and gastrointestinal physiology, we attempt to define those conditions under which in vitro dissolution data may be used as a surrogate for data on in vivo bioavailability. We consider the likely physiological causes for species-related differences in the absolute and relative bioavailability of orally administered pharmaceuticals, and examine the potential for these normal interspecies differences to reflect bioavailability changes that can occur with various human pathologies.

We report the cloning of a murine ClC-2 chloride channel cDNA from duodenal epithelium by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers and by rapid amplification of cDNA ends (RACE)-PCR. Other than CFTR, this represents the first cloned chloride channel from intact intestine. The ClC-2 cDNA predicts encoding of a 908 amino acid polypeptide with a calculated M(r) of 99,373. The amino acid sequence of the murine ClC-2 chloride channel is over 94% identical to the ClC-2 chloride channel proteins of other species. Of interest is the finding that the ClC-2 mRNA is expressed about the same level in duodena from both CFTR knockout and wild-type mice. This is in keeping with the suggestion that ClC-2 might be a therapeutic target in cystic fibrosis.

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

The association of feeding practices with the development of digestive disorders in horses has long been recognized, although the underlying mechanisms had been barely considered. The physiologic consequences of meal frequency may help to explain the relationship and prove to be of major significance in the induction of many conditions. Many Equidae kept for performance and leisure activities are fed high-energy, low-forage rations twice daily, with limited access to hay or grazing. Rapid ingestion of such meals stimulates a copious outpouring of upper alimentary secretions and results in transient hypovolemia (15% plasma volume loss). Subsequent activation of the renin-angiotensin-aldosterone system (RAAS) contributes to the preservation of circulatory status. Large meals may accelerate digesta passage to the cecum and, thereby, increase soluble carbohydrate availability for large intestinal fermentation. Intense periods of fermentation develop that require significant shifts of fluid into the colonic lumen. This is followed by net fluid absorption, which, in part, is dependent on postprandial increases of aldosterone. Potential consequences of these events include (1) imbalances in the RAAS response, which may promote conditions favorable to gastrointestinal disturbance, notably large intestinal impaction, and (2) changes in the gastrointestinal microflora, which may affect the intraluminal endotoxin pool and the population of enterotoxin-producing bacteria. In contrast to episodic feedings, similar changes are absent or greatly attenuated under simulated grazing conditions (for example, small, frequent meals). Thus, modification of management practices to facilitate a more continuous feeding pattern may significantly reduce the incidence of digestive problems in the stabled horse.

Standard methods such as western blot and flow-cytometry (protein expression); quantitative real time polymerase chain reaction (qPCR) (mRNA levels) and TCF/LEF reporter assay (βcatenin transcription) were used. Chromatin immunoprecipitation assay was performed with anti-CtBP2 antibody on spheroids. Results: Spheroid showed significant up regulation of CSCs factors (CD133, CXCR4, LGR5 and C-MYC), TCF/LEF activity as well as increased NADH/NAD ratio and enhanced binding of CtBP on p21 (a known CtBP target) promoter compared to monolayer controls. Depletion of CtBP2 inhibited, while its overexpression enhanced CSC growth (1° spheroid) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of CSC growth and self-renewal in a dose dependent manner (IC50: 250-700μM) in 4/5 cells tested. The corresponding IC50 for monolayer growth was 6-fold higher, suggesting selective targeting of CSCs. Moreover, MTOB treatment (1mM) caused robust apoptosis induction only in spheroids. Additionally, MTOB inhibited expression of CSCmarkers (CD133, CD44 and LGR5) and self-renewal factor (C-MYC) in spheroids. Moreover, MTOB inhibited basal as well as induced (GSK-3β inhibitor) TCF/LEF activity while inhibiting mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5) and significantly reduced CtBP binding to LGR5 (a TCF/LEF target) and p21 promoters. Lastly, CtBP physically interacted with β-catenin. Conclusion: We have discovered a novel role of CtBP in promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a paradigm-shifting treatment approach for colorectal cancer.

Dra is the major apical membrane Cl/HCO3 exchanger in the murine small intestine. Studies of recombinant DRA have proposed an electrogenic 2Cl:1HCO3 stoichiometry (J. Gen. Physiol. 127: 511, 2006). In Dra knockout jejunum (DraKO), loss of Clabsorption is associated with an increase in the baseline short-circuit current (Isc) that may result from loss of an inward anion current due to electrogenic Cl/HCO3 exchange. Therefore, we investigated the ionic basis of the increased Isc in isolated DraKO jejunal preparations using intracellular pH (pHi) microfluorimetry and Ussing chamber studies. As described previously (Gastroenterology 136: 1645, 2008), pHi of the DraKO villous epithelium was alkaline relative to wild type (WT), ostensibly due to loss of Cl/HCO3 exchange. Studies also revealed little Cl-/OHexchange in villous epithelium by measurements performed in the nominal absence of extracellular HCO3. In Ussing chamber studies, removal of extracellular Cleliminated the elevated baseline Isc whereas removal of extracellular HCO3 had no effect in DraKO jejunum, indicating that Cl/HCO3 exchange was not responsible for the elevated Isc. The contribution of the cystic fibrosis transmembrane conductance regulator (Cftr) anion channel was investigated usingDra/Cftr double knockout micewhich demonstrated a baseline Isc that was reduced by 40% relative to the DraKO jejunum. Epithelial alkalinity in the DraKO jejunal epithelium may increase the electrical gradient for Clsecretion by hyperpolarization secondary to activation of basolateral K+ channels (Am. J. Physiol. 258:C336, 1990). Therefore, epithelial acidification was induced by exposing the luminal surface to pH 6.4 by replacing HCO3 with TES buffer in the presence of 5% CO2: 95% O2. Under this condition, the elevated baseline Isc in the DraKO was abolished. On-going studies are investigating the effect of basolateral K+ channel blockers in the DraKO jejunum. We conclude that the elevated baseline Isc in the DraKO intestine does not result from loss of electrogenic Cl-/ HCO3 exchange but from increased anion secretion associated with epithelial alkalinity that may activate basolateral membrane K+ channels. Supported by NIDDK and the Cystic Fibrosis Foundation.

Background & Aims Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl − /HCO 3 − exchanger(s) primarily with Na + /H + exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl − /HCO 3 − exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl − /HCO 3 − exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1). Methods Intracellular pH (pH i ) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial 22 Na 36 Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction. Results Basal pH i of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pH i in the Nhe3KO was acidic relative to wild-type. Altered pH i was associated with robust Na + /H + and Cl − /HCO 3 − exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pH i but coordinately inhibited Dra. Flux studies revealed that Cl − absorption was essentially abolished (>80%) in the DraKO and little changed ( + absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters. Conclusions These studies provide functional evidence that Dra is the major Cl − /HCO 3 − exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.