Quantitative Analysis of Hedgehog Gradient Formation Using an Inducible Expression System: a Dissertation

The Hedgehog (Hh) family of proteins are secreted growth factors that play an essential role in the embryonic development of all organisms and the main components in the pathway are conserved from insects to humans. These proteins affect patterning and morphogenesis of multiple tissues. Therefore, mutations in the Hh pathway can result in a wide range of developmental defects and oncogenic diseases. Because the main components in the pathway are conserved from insects to humans, Drosophilahas been shown to provide a genetically tractable system to gain insight into the processes that Hh is involved in. In this study, the roles of Hh cholesterol modification and endocytosis during gradient fonnation are explored in the Drosophila larval wing imaginal disc. To exclude the possibility of looking at a redistribution of preexisting Hh instead of Hh movement, a spatially and temporally regulated system has been developed to induce Hh expression. Functional Hh-GFP with and without the cholesterol-modification was expressed in a wild-type or shi-tslendocytosis mutant background. The Gal80 system was used to temporally express (pulse) the Hh-GFP transgenes to look at the rate of Hh gradient formation over time and determine whether this process was affected by cholesterol modification and/or endocytosis. Hh with and without cholesterol were both largely detected in punctate structures and the spreading of the different forms of Hh was quantified by measuring distances of these particles from the expressing cells. Hh without cholesterol showed a greater range of distribution, but a lower percentage of particles near the source. Loss of endocytosis blocked formation of intracellular Hh particles, but did not dramatically alter its movement to target cells. Staining for Hh, its receptor Ptc and cortical actin revealed that these punctate structures could be classified into four types of Hh containing particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cholesterol is specifically required for the formation of cytoplasmic particles lacking Ptc. While previous studies have shown discrepancies in the localization of Hh following a block in endocytosis, Hh with and without cholesterol is detected at both apical and basolateral surfaces, but not at basal surfaces. In the absence of cholesterol and endocytosis, Hh particles can be observed in the extracellular space. Through three-dimensional reconstruction and quantitative analysis, this study concludes that the cholesterol modification is required to restrict Hh movement. In addition, the cholesterol modification promotes Ptc-independent internalization. This study also observes that Dynamin-dependent endocytosis is necessary for internalization but does not play an essential role in Hh distribution. The data in this thesis supports the model in which Hh movement occurs via planar diffusion

Connexins: Mechanisms regulating protein levels and intercellular communication

AbstractIntercellular communication can occur through gap junction channels, which are comprised of connexin proteins. Therefore, levels of connexins can directly correlate with gap junctional intercellular communication. Because gap junctions have a critical role in maintaining cellular homeostasis, the regulation of connexin protein levels is important. In the connexin life cycle, connexin protein levels can be modified through differential gene transcription or altered through trafficking and degradation mechanisms. More recently, significant attention has been directed to the pathways that cells utilize to increase or decrease connexin levels and thus indirectly, gap junctional communication. Here, we review the studies revealing the mechanisms that affect connexin protein levels and gap junctional intercellular communication

Drosophila Bruce Can Potently Suppress Rpr- and Grim-Dependent but Not Hid-Dependent Cell Death

Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2) 1, 2. BRUCE upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs [2]. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Here, we demonstrate, using gain-of-function and deletion alleles, that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that dBruce can regulate cell death at a novel point

Quantitative analysis of Hedgehog gradient formation using an inducible expression system

BACKGROUND: The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Mutations in human Hh and other Hh pathway components have been linked to human diseases. Analysis of Hh distribution during development indicates that cholesterol modification and receptor mediated endocytosis affect the range of Hh signaling and the cellular localization of Hh. RESULTS: We have used an inducible, cell type-specific expression system to characterize the three-dimensional distribution of newly synthesized, GFP-tagged Hh in the developing Drosophila wing. Following induction of Hh-GFP expression in posterior producing cells, punctate structures containing Hh-GFP were observed in the anterior target cells. The distance of these particles from the expressing cells was quantified to determine the shape of the Hh gradient at different time points following induction. The majority of cholesterol-modified Hh-GFP was found associated with cells near the anterior/posterior (A/P) boundary, which express high levels of Hh target genes. Without cholesterol, the Hh gradient was flatter, with a lower percentage of particles near the source and a greater maximum distance. Inhibition of Dynamin-dependent endocytosis blocked formation of intracellular Hh particles, but did not prevent movement of newly synthesized Hh to the apical or basolateral surfaces of target cells. In the absence of both cholesterol and endocytosis, Hh particles accumulated in the extracellular space. Staining for the Hh receptor Ptc revealed four categories of Hh particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Interestingly, mainly cholesterol-modified Hh is detected in the cytoplasmic particles lacking Ptc. CONCLUSION: We have developed a system to quantitatively analyze Hh distribution during gradient formation. We directly demonstrate that inhibition of Dynamin-dependent endocytosis is not required for movement of Hh across target cells, indicating that transcytosis is not required for Hh gradient formation. The localization of Hh in these cells suggests that Hh normally moves across both apical and basolateral regions of the target cells. We also conclude that cholesterol modification is required for formation of a specific subset of Hh particles that are both cytoplasmic and not associated with the receptor Ptc

Thromboprophylaxis Is Associated With Reduced Post-hospitalization Venous Thromboembolic Events in Patients With Inflammatory Bowel Diseases

Background & Aims Patients with inflammatory bowel diseases (IBDs) have increased risk for venous thromboembolism (VTE); those who require hospitalization have particularly high risk. Few hospitalized patients with IBD receive thromboprophylaxis. We analyzed the frequency of VTE after IBD-related hospitalization, risk factors for post-hospitalization VTE, and the efficacy of prophylaxis in preventing post-hospitalization VTE. Methods In a retrospective study, we analyzed data from a multi-institutional cohort of patients with Crohn's disease or ulcerative colitis and at least 1 IBD-related hospitalization. Our primary outcome was a VTE event. All patients contributed person-time from the date of the index hospitalization to development of VTE, subsequent hospitalization, or end of follow-up. Our main predictor variable was pharmacologic thromboprophylaxis. Cox proportional hazard models adjusting for potential confounders were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results From a cohort of 2788 patients with at least 1 IBD-related hospitalization, 62 patients developed VTE after discharge (2%). Incidences of VTE at 30, 60, 90, and 180 days after the index hospitalization were 3.7/1000, 4.1/1000, 5.4/1000, and 9.4/1000 person-days, respectively. Pharmacologic thromboprophylaxis during the index hospital stay was associated with a significantly lower risk of post-hospitalization VTE (HR, 0.46; 95% CI, 0.22–0.97). Increased numbers of comorbidities (HR, 1.30; 95% CI, 1.16–1.47) and need for corticosteroids before hospitalization (HR, 1.71; 95% CI, 1.02–2.87) were also independently associated with risk of VTE. Length of hospitalization or surgery during index hospitalization was not associated with post-hospitalization VTE. Conclusions Pharmacologic thromboprophylaxis during IBD-related hospitalization is associated with reduced risk of post-hospitalization VTE.National Institutes of Health (U.S.) (U54-LM008748

Improving Case Definition of Crohnʼs Disease and Ulcerative Colitis in Electronic Medical Records Using Natural Language Processing

available in PMC 2014 June 01Background: Previous studies identifying patients with inflammatory bowel disease using administrative codes have yielded inconsistent results. Our objective was to develop a robust electronic medical record–based model for classification of inflammatory bowel disease leveraging the combination of codified data and information from clinical text notes using natural language processing. Methods: Using the electronic medical records of 2 large academic centers, we created data marts for Crohn’s disease (CD) and ulcerative colitis (UC) comprising patients with ≥1 International Classification of Diseases, 9th edition, code for each disease. We used codified (i.e., International Classification of Diseases, 9th edition codes, electronic prescriptions) and narrative data from clinical notes to develop our classification model. Model development and validation was performed in a training set of 600 randomly selected patients for each disease with medical record review as the gold standard. Logistic regression with the adaptive LASSO penalty was used to select informative variables. Results: We confirmed 399 CD cases (67%) in the CD training set and 378 UC cases (63%) in the UC training set. For both, a combined model including narrative and codified data had better accuracy (area under the curve for CD 0.95; UC 0.94) than models using only disease International Classification of Diseases, 9th edition codes (area under the curve 0.89 for CD; 0.86 for UC). Addition of natural language processing narrative terms to our final model resulted in classification of 6% to 12% more subjects with the same accuracy. Conclusions: Inclusion of narrative concepts identified using natural language processing improves the accuracy of electronic medical records case definition for CD and UC while simultaneously identifying more subjects compared with models using codified data alone.National Institutes of Health (U.S.) (NIH U54-LM008748)American Gastroenterological AssociationNational Institutes of Health (U.S.) (NIH K08 AR060257)Beth Isreal Deaconess Medical Center (Katherine Swan Ginsburg Fund)National Institutes of Health (U.S.) (NIH R01-AR056768)Burroughs Wellcome Fund (Career Award for Medical Scientists)National Institutes of Health (U.S.) (NIH U01-GM092691)National Institutes of Health (U.S.) (NIH R01-AR059648

Normalization of Plasma 25-Hydroxy Vitamin D Is Associated with Reduced Risk of Surgery in Crohn’s Disease

available in PMC 2014 August 01AB Background: Vitamin D may have an immunologic role in Crohn's disease (CD) and ulcerative colitis (UC). Retrospective studies suggested a weak association between vitamin D status and disease activity but have significant limitations. Methods: Using a multi-institution inflammatory bowel disease cohort, we identified all patients with CD and UC who had at least one measured plasma 25-hydroxy vitamin D (25(OH)D). Plasma 25(OH)D was considered sufficient at levels >=30 ng/mL. Logistic regression models adjusting for potential confounders were used to identify impact of measured plasma 25(OH)D on subsequent risk of inflammatory bowel disease-related surgery or hospitalization. In a subset of patients where multiple measures of 25(OH)D were available, we examined impact of normalization of vitamin D status on study outcomes. Results: Our study included 3217 patients (55% CD; mean age, 49 yr). The median lowest plasma 25(OH)D was 26 ng/mL (interquartile range, 17-35 ng/mL). In CD, on multivariable analysis, plasma 25(OH)D =30 ng/mL. Similar estimates were also seen for UC. Furthermore, patients with CD who had initial levels <30 ng/mL but subsequently normalized their 25(OH)D had a reduced likelihood of surgery (odds ratio, 0.56; 95% confidence interval, 0.32-0.98) compared with those who remained deficient. Conclusion: Low plasma 25(OH)D is associated with increased risk of surgery and hospitalizations in both CD and UC, and normalization of 25(OH)D status is associated with a reduction in the risk of CD-related surgery. (C) Crohn's & Colitis Foundation of America, Inc

Similar Risk of Depression and Anxiety Following Surgery or Hospitalization for Crohn's Disease and Ulcerative Colitis

OBJECTIVES: Psychiatric comorbidity is common in Crohn's disease (CD) and ulcerative colitis (UC). Inflammatory bowel disease (IBD)-related surgery or hospitalizations represent major events in the natural history of the disease. The objective of this study is to examine whether there is a difference in the risk of psychiatric comorbidity following surgery in CD and UC. METHODS: We used a multi-institution cohort of IBD patients without a diagnosis code for anxiety or depression preceding their IBD-related surgery or hospitalization. Demographic-, disease-, and treatment-related variables were retrieved. Multivariate logistic regression analysis was performed to individually identify risk factors for depression and anxiety. RESULTS: Our study included a total of 707 CD and 530 UC patients who underwent bowel resection surgery and did not have depression before surgery. The risk of depression 5 years after surgery was 16% and 11% in CD and UC patients, respectively. We found no difference in the risk of depression following surgery in the CD and UC patients (adjusted odds ratio, 1.11; 95% confidence interval, 0.84–1.47). Female gender, comorbidity, immunosuppressant use, perianal disease, stoma surgery, and early surgery within 3 years of care predicted depression after CD surgery; only the female gender and comorbidity predicted depression in UC patients. Only 12% of the CD cohort had ≥4 risk factors for depression, but among them nearly 44% subsequently received a diagnosis code for depression. CONCLUSIONS: IBD-related surgery or hospitalization is associated with a significant risk for depression and anxiety, with a similar magnitude of risk in both diseases.National Institutes of Health (U.S.) (U54-LM008748

Psychiatric co-morbidity is associated with increased risk of surgery in Crohn's disease

Psychiatric co-morbidity, in particular major depression and anxiety, is common in patients with Crohn's disease (CD) and ulcerative colitis (UC). Prior studies examining this may be confounded by the co-existence of functional bowel symptoms. Limited data exist examining an association between depression or anxiety and disease-specific endpoints such as bowel surgery.National Institutes of Health (U.S.) (NIH U54-LM008748)American Gastroenterological AssociationNational Institutes of Health (U.S.) (NIH K08 AR060257)Beth Isreal Deaconess Medical Center (Katherine Swan Ginsburg Fund)National Institutes of Health (U.S.) (NIH R01-AR056768)National Institutes of Health (U.S.) (NIH U01-GM092691)National Institutes of Health (U.S.) (NIH R01-AR059648)Burroughs Wellcome Fund (Career Award for Medical Scientists)National Institutes of Health (U.S.) (NIH K24 AR052403)National Institutes of Health (U.S.) (NIH P60 AR047782)National Institutes of Health (U.S.) (NIH R01 AR049880