Douglas B. Luckie

Less teaching, more learning: 10-yr study supports increasing student learning through less coverage and more inquiry

In this study, we compared gains in student content learning over a 10-yr period in which the introductory biology laboratory curriculum was changed in two ways: an increase of inquiry and a reduction of content. Three laboratory formats were tested: traditional 1-wk-long cookbook laboratories, two 7-wk-long inquiry laboratories, and one 14-wk-long inquiry laboratory. As the level of inquiry increased, student learning gains on content exams trended upward even while traditional content coverage taught decreased. In a quantitative assessment of content knowledge, students who participated in the 14-wk-long inquiry laboratory format outscored their peers in both 7- and 1-wk-long lab formats on Medical College Admissions Test exam questions (scores of 64.73%, 61.97%, and 53.48%, respectively, P < 0.01). In a qualitative study of student opinions, surveys conducted at the end of semesters where traditional 1-wk laboratories (n = 167 students) were used had low response rates and predominately negative opinions (only 20% of responses were positive), whereas those who participated in 7-wk (n = 543) or 14-wk (n = 308) inquiry laboratories had high response rates and 71% and 96% positive reviews, respectively. In an assessment of traditional content coverage in courses, three indexes were averaged to calculate traditional forms of coverage and showed a decrease by 44% over the study period. We believe that the quantitative and qualitative data support greater student-driven inquiry in the classroom laboratory, which leads to deeper learning in fewer topic areas (less teaching) and can reap gains in scientific thinking and fundamental understanding applicable to a broader range of topic areas (more learning) in introductory biology.

CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells

Abstract. Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes of wet epithelia. Since CFTR is permeable to HCO−3 and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for CFTR in controlling pHo and complement recent evidence that HCO−3 dependent epithelial secretion may be reduced in amount and altered in composition in CF.

Ouabain- and Ca2+-sensitive ATPase activity of chimeric Na- and Ca-pump molecules

Chimeric ion‐pumps, consisting of the N‐terminal of the α1‐subunit of the ouabain‐sensitive chicken Na+, K+‐ATPase and the C‐terminal of the sarcoplasmic reticulum Ca2+‐ATPase, were expressed in ouabain‐insensitive mouse L cells. These chimeric molecules exhibited ouabain‐sensitive ATPase activity very similar to that of the wild‐type chicken Na+ K+‐ATPase. This ATPase activity could be stimulated by adding Ca2+ to the assay system. These results suggest that the sites for ouabain‐inhibition are restricted to the N‐terminal of the Na‐pump, and the C‐terminal of the Ca‐pump interacts with Ca3+.

Direct measurement of acid efflux from isolated guinea pig pancreatic ducts

Objectives: The current studies used the technique of microphysiometry to directly determine the effects of stimulators and inhibitors of pancreatic duct secretion on acid efflux from isolated pancreatic ducts. Methods: Main and interlobular ducts were isolated from guinea pig pancreata by collagenase digestion and manual selection. Segments were placed in the chambers of a microphysiometer, which uses a silicon chip-based, light-addressable potentiometric sensor to determine the proton concentration in the superfusing solution. Isolated ducts were superfused with a low buffer capacity Ringers solution at 37°C and the extracellular acidification rate (EAR) was determined by computer-directed protocols. Results: A survey of potential agonists demonstrated that both secretin and the cholinomimetic, carbachol, dramatically increased EAR, with EC50 of 3 nmol/L and 0.6 μmol/L, respectively. The changes in EAR induced by both secretagogues were rapid, peaking within 4-6 minutes, and then declining to a level below the peak but above basal EAR. The enhanced EAR was maintained for at least 30 minutes in the presence of either secretagogue. More modest increases in EAR were evoked by bombesin, substance P, and vasoactive intestinal peptide (VIP). Cholecystokinin and isoproterenol caused no significant change in pancreatic duct EAR. A combination of amiloride and bafilomycin A1, inhibitors, respectively, of Na+/H+ exchange and of vacuolar type H+-ATPase activity, caused a dramatic drop in EAR but did not fully inhibit the increase in EAR elicited by carbachol, suggesting that other mechanisms may contribute to agonist-stimulated EAR of pancreatic ducts. Conclusions: Thus, the results support the use of microphysiometry as a tool to study pancreatic duct physiology and in particular a method to measure acid efflux from the serosal surface.

Extracellular acidification parallels insulin secretion in INS-1 and HIT-T15 β-cell lines

As an alternative to manual assays that track insulin secretion, we tested a silicon-based biosensor that allows automated monitoring of extracellular acidification. Glucose stimulation of INS-1 and HIT-T15 cells resulted in a rapid increase in extracellular acidification in a biphasic and concentration-dependent fashion much like insulin secretion (EC(50) INS-1=5 mM and HIT-T15=1 mM). This response was attenuated by verapamil (10 microM) and stimulated by administration of glybenclamide (100 nM) or KCl-induced (40 mM) depolarization. These experiments suggest that automated monitoring of extracellular pH may be a useful assay and support the relevance of linking metabolic activity to insulin secretion.

Pseudomonas or LPS exposure alters CFTR iodide efflux in 2WT2 epithelial cells with time and dose dependence

The most common heritable genetic disease in the United States, cystic fibrosis (CF), is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride channel that interacts with and regulates a number of other proteins. The bacteria Pseudomonas aeruginosa infects 80% of patients causing decreased pulmonary function and life expectancy. It is not known how malfunction of the chloride channel allows for preferential colonization of patients by a single pathogen. The hypothesis that CFTR interacts with toll-like receptor 4 (TLR4) to phagocytize bacteria was tested. A competitive antagonist of TLR4, MKLPS, was studied for its effect in gentamicin-protection-based bacterial invasion assays. Pre-incubation (15 min 50 microg/mL) with MKLPS did not alter the rate of phagocytosis of P. aeruginosa by cultured epithelia. However, further studies with GFP-transfected P. aeruginosa revealed prominent antibiotic resistant microcolonies were formed. If CFTR is involved in phagocytosis of the bacteria, then internalization was predicted to decrease in iodide efflux. Surprisingly, cultured epithelia exposed to P. aeruginosa for 15 min showed increased cAMP-activated iodide efflux through CFTR. In addition, 15-min exposure to bacterial cell wall component, LPS, purified from P. aeruginosa also increased CFTR iodide efflux in a dose-dependent manner (50, 100 and 200 microg/mL LPS had 25%, 37% and 47% increase). In a reversal of this phenomenon, shorter 5-min exposure to 100 microg/mL LPS resulted in a 25% decrease in forskolin-activated CFTR channel activity compared to controls. This data is consistent with a model in which CFTR is removed from the plasma membrane during phagocytosis of P. aeruginosa followed by recruitment of channels to the membrane to replace those removed during phagocytosis. More studies are needed to confirm this model, but this is the first report of a bacterial product causing a biphasic time-dependent and a dose-dependent alteration of CFTR channel activity.

Integrating Concepts in Biology Textbook Increases Learning: Assessment Triangulation Using Concept Inventory, Card Sorting, and MCAT Instruments, Followed by Longitudinal Tracking

This study examined the educational impact of an inquiry-focused textbook, Integrating Concepts in Biology (ICB). Our findings support those of another study that found that performance of an ICB cohort also surpassed that of peers and suggest that the ICB textbook enables learning gains beyond those found using traditional content-focused textbooks.

Verbal Final Exam in Introductory Biology Yields Gains in Student Content Knowledge and Longitudinal Performance

The authors studied gains in student learning when curriculum was changed to include an optional verbal final exam (VF). Students who passed the VF outscored peers on MCAT questions (66.4% [n=160] and 62% [n=285], respectively; p < 0.001), and passing the VF also correlated with higher performance in a range of upper-level science courses.

Chemical rescue of Δf508-CFTR in C127 epithelial cells reverses aberrant extracellular pH acidification to wild-type alkalization as monitored by microphysiometry

Cystic fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel expressed in apical membranes of wet epithelia. Since CFTR is permeable to HCO3(-), and may regulate bicarbonate exchangers, it is not surprising evidence of changes in extracellular pH (pHo) have been found in CF. Previously we have shown that tracking pHo can be used to differentiate cells expressing wild-type CFTR from controls in mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. In this study we characterized forskolin-stimulated extracellular acidification rates in epithelia where chemical correction of mutant ΔF508-CFTR converted an aberrant response in acidification (10%+ increase) to wild-type (25%+ decrease). Thus treatment with corrector (10% glycerol) and the resulting increased expression of ΔF508-CFTR at the surface was detected by microphysiometry as a significant reversal from acidification to alkalization of pHo. These results suggest that CFTR activation as well as correction can be detected by carefully monitoring pHo and support findings in the field that extracellular pH acidification may impact the function of airway surface liquid in CF.

Cystic Fibrosis: Does CFTR Malfunction Alter pH Regulation?

“Woe to the child kissed on the forehead who tastes salty, for it is cursed and soon must die,” is a form of diagnosis for cystic fibrosis that dates back to the Middle Ages in Eastern Europe [1]. This observation was rediscovered and made relevant in 1948 during a heat wave in New York City. Paul di Sant’Agnese at Columbia University noticed that CF pa‐ tients had a larger incidence of heat prostration than others, due to excessive salt loss [2]. This ultimately led to the use of the “sweat test” (measurement of sweat electrolytes) as the definitive test for diagnosing cystic fibrosis. Originally, Dorothy Andersen in 1938 created the term cystic fibrosis [4] in describing the appearance of the pancreas from certain individ‐ uals. Later she went on to show that CF was a genetic disease [5].