Identifying new small proteins through a molecular biology course-based undergraduate research experience laboratory class.

We developed a curriculum for an upper-level molecular biology course-based undergraduate research laboratory class funded by a National Science Foundation CAREER grant that focuses on identifying new small proteins in the bacterium, Escherichia coli. Our CURE class has been continually offered each semester for the last 10 years, with multiple instructors collaboratively developing and implementing their own pedagogical approach while maintaining the same overall scientific goal and experimental strategy. In this paper, we delineate the experimental strategy for our molecular biology CURE laboratory class, describe a range of pedagogical approaches implemented by multiple instructors, and provide recommendations for teaching the class. The purpose of our paper is to share our experiences both in developing and teaching a molecular biology CURE laboratory class based on small protein identification and in creating a curriculum and support system that allows traditional, non-traditional, and under-represented students to participate in authentic research projects.

Heritability of respiratory infection with Pseudomonas aeruginosa in cystic fibrosis.

OBJECTIVE: To quantify the relative contribution of factors other than cystic fibrosis transmembrane conductance regulator genotype and environment on the acquisition of Pseudomonas aeruginosa (Pa) by patients with cystic fibrosis. STUDY DESIGN: Lung infection with Pa and mucoid Pa was assessed using a co-twin study design of 44 monozygous (MZ) and 17 dizygous (DZ) twin pairs. Two definitions were used to establish infection: first positive culture and persistent positive culture. Genetic contribution to infection (ie, heritability) was estimated based on concordance analysis, logistic regression, and age at onset of infection through comparison of intraclass correlation coefficients. RESULTS: Concordance for persistent Pa infection was higher in MZ (0.83; 25 of 30 pairs) than DZ twins (0.45; 5 of 11 pairs), generating a heritability of 0.76. Logistic regression adjusted for age corroborated genetic control of persistent Pa infection. The correlation for age at persistent Pa infection was higher in MZ twins (0.589; 95% CI, 0.222-0.704) than in DZ twins (0.162; 95% CI, -0.352 to 0.607), generating a heritability of 0.85. CONCLUSION: Genetic modifiers play a significant role in the establishment and timing of persistent Pa infection in individuals with cystic fibrosis.

Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients.

BACKGROUND: Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF. METHODS: Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories. RESULTS: Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile). CONCLUSIONS: Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

Use of a modeling framework to evaluate the effect of a modifier gene (MBL2) on variation in cystic fibrosis.

Variants in mannose-binding lectin (MBL2; protein MBL) have shown association with different aspects (eg, lung function, infection, survival) of cystic fibrosis (CF) in some studies but not others. Inconsistent results may be due to confounding among disease variables that were not fully accounted for in each study. To account for these relationships, we derived a modeling framework incorporating CFTR genotype, age, Pseudomonas aeruginosa (Pa) infection, and lung function from 788 patients in the US CF Twin and Sibling Study. This framework was then used to identify confounding variables when testing the effect of MBL2 variation on specific CF traits. MBL2 genotypes corresponding to low levels of MBL associated with Pa infection 1.94 years earlier than did MBL2 genotypes corresponding to high levels of MBL (P=0.0034). In addition, Pa-infected patients with MBL2 genotypes corresponding to low levels of MBL underwent conversion to mucoid Pa 2.72 years earlier than did patients with genotypes corresponding to high levels of MBL (P=0.0003). MBL2 was not associated with the time to transition from infection to conversion or with lung function. Thus, use of a modeling framework that identified confounding among disease variables revealed that variation in MBL2 associates with age at infection with Pa and age at conversion to mucoid Pa in CF.

Variation in the lymphotoxin-alpha/tumor necrosis factor locus modifies risk of erythema nodosum in sarcoidosis.

Sarcoidosis is a multi-system inflammatory disease with organ involvement that varies by race and sex. Family studies indicate that genes play a role in the etiology and extent of organ involvement in sarcoidosis. In this study, we evaluated whether 25 variants distributed in 19 genes with a known role in inflammation were associated with erythema nodosum status in 659 sarcoidosis patients and 658 controls from A Case-Control Etiologic Study of Sarcoidosis (ACCESS). We found no association with affectation status; however, a variant in the promoter of tumor necrosis factor (TNF) at position -308 was found to be associated with erythema nodosum in Caucasian sarcoidosis patients (study-wide P=0.027). When separated by sex, a variant in intron 1 of lymphotoxin-alpha (LTA), a gene adjacent to TNF, was associated with erythema nodosum in female Caucasian sarcoidosis patients (study-wide P=0.027). These DNA variants frequently occur together in Caucasians, and each variant has individually been associated with erythema nodosum in sarcoidosis patients. These results confirm that variation in the LTA/TNF gene cluster modifies a major skin manifestation of sarcoidosis and may explain the higher rate of erythema nodosum in females with sarcoidosis.

Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.

Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, '-509' and rs1982073, 'codon 10') in the 5' region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455 and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3' SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.

Interactions between secondhand smoke and genes that affect cystic fibrosis lung disease.

CONTEXT: Disease variation can be substantial even in conditions with a single gene etiology such as cystic fibrosis (CF). Simultaneously studying the effects of genes and environment may provide insight into the causes of variation. OBJECTIVE: To determine whether secondhand smoke exposure is associated with lung function and other outcomes in individuals with CF, whether socioeconomic status affects the relationship between secondhand smoke exposure and lung disease severity, and whether specific gene-environment interactions influence the effect of secondhand smoke exposure on lung function. DESIGN, SETTING, AND PARTICIPANTS: Retrospective assessment of lung function, stratified by environmental and genetic factors. Data were collected by the US Cystic Fibrosis Twin and Sibling Study with missing data supplemented by the Cystic Fibrosis Foundation Data Registry. All participants were diagnosed with CF, were recruited between October 2000 and October 2006, and were primarily from the United States. MAIN OUTCOME MEASURES: Disease-specific cross-sectional and longitudinal measures of lung function. RESULTS: Of 812 participants with data on secondhand smoke in the home, 188 (23.2%) were exposed. Of 780 participants with data on active maternal smoking during gestation, 129 (16.5%) were exposed. Secondhand smoke exposure in the home was associated with significantly lower cross-sectional (9.8 percentile point decrease; P < .001) and longitudinal lung function (6.1 percentile point decrease; P = .007) compared with those not exposed. Regression analysis demonstrated that socioeconomic status did not confound the adverse effect of secondhand smoke exposure on lung function. Interaction between gene variants and secondhand smoke exposure resulted in significant percentile point decreases in lung function, namely in CFTR non-DeltaF508 homozygotes (12.8 percentile point decrease; P = .001), TGFbeta1-509 TT homozygotes (22.7 percentile point decrease; P = .006), and TGFbeta1 codon 10 CC homozygotes (20.3 percentile point decrease; P = .005). CONCLUSIONS: Any exposure to secondhand smoke adversely affects both cross-sectional and longitudinal measures of lung function in individuals with CF. Variations in the gene that causes CF (CFTR) and a CF-modifier gene (TGFbeta1) amplify the negative effects of secondhand smoke exposure.

Variants in mannose-binding lectin and tumour necrosis factor alpha affect survival in cystic fibrosis.

BACKGROUND: Patients with cystic fibrosis with the same mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene differ widely in survival suggesting other factors have a substantial role in mortality. OBJECTIVE: To determine if the genotype distribution of variants in three putative cystic fibrosis modifier genes (tumour necrosis factor alpha (TNFalpha), transforming growth factor beta1 (TGFbeta1) or mannose-binding lectin (MBL2)) differed among patients with cystic fibrosis grouped according to age and survival status. METHODS: Genotypes of four variants (TNFalpha-238, TNFalpha-308, TGFbeta1-509 and MBL2 O) were determined in three groups of Caucasians from a single medical centre: 101 children with cystic fibrosis (aged <17 years; mean age 9.4 years), 115 adults with cystic fibrosis (aged > or =17 years; mean age 30.8 years) and 38 non-surviving adults with cystic fibrosis (21 deceased and 17 lung transplant after 17 years of age). Genotypes of 127 healthy Caucasians in the same geographical region were used as controls. Kaplan-Meier and Cox hazard regression were used to evaluate the genotype effect on cumulative survival. RESULTS: Genotype frequencies among adults and children with cystic fibrosis differed for TNFalpha-238 (G/G vs G/A; p = 0.022) and MBL2 (A/A vs O/O; p = 0.016). When adults with cystic fibrosis were compared to non-surviving adults with cystic fibrosis, genotype frequencies of both genes differed (TNFalpha-238G/G vs G/A; p = 0.0015 and MBL2: A/A vs O/O; p = 0.009). The hazard ratio for TNFalpha-238G/G vs G/A was 0.25 (95% CI 0.06 to 1.0, p = 0.04) and for MBL2 O/O vs A/A or A/O was 2.5 (95% CI 1.3 to 4.9, p = 0.007). CONCLUSIONS: TNFalpha-238 G/A and MBL2 O/O genotypes appear to be genetic modifiers of survival of cystic fibrosis.