A first attempt to bring computational biology into advanced high school biology classrooms

Computer science has become ubiquitous in many areas of biological research, yet most high school and even college students are unaware of this. As a result, many college biology majors graduate without adequate computational skills for contemporary fields of biology. The absence of a computational element in secondary school biology classrooms is of growing concern to the computational biology community and biology teachers who would like to acquaint their students with updated approaches in the discipline. We present a first attempt to correct this absence by introducing a computational biology element to teach genetic evolution into advanced biology classes in two local high schools. Our primary goal was to show students how computation is used in biology and why a basic understanding of computation is necessary for research in many fields of biology. This curriculum is intended to be taught by a computational biologist who has worked with a high school advanced biology teacher to adapt the unit for his/her classroom, but a motivated high school teacher comfortable with mathematics and computing may be able to teach this alone. In this paper, we present our curriculum, which takes into consideration the constraints of the required curriculum, and discuss our experiences teaching it. We describe the successes and challenges we encountered while bringing this unit to high school students, discuss how we addressed these challenges, and make suggestions for future versions of this curriculum.We believe that our curriculum can be a valuable seed for further development of computational activities aimed at high school biology students. Further, our experiences may be of value to others teaching computational biology at this level. Our curriculum can be obtained at http://ecsite.cs.colorado.edu/?page_id=149#biology or by contacting the authors.     

Identification and Validation of Urinary Biomarkers for Differential Diagnosis and Evaluation of Therapeutic Intervention in Anti-neutrophil Cytoplasmic Antibody-associated Vasculitis

Renal activity and smoldering disease is difficult to assess in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) because of renal scarring. Even repeated biopsies suffer from sampling errors in this focal disease especially in patients with chronic renal insufficiency. We applied capillary electrophoresis coupled to mass spectrometry toward urine samples from patients with active renal AAV to identify and validate urinary biomarkers that enable differential diagnosis of disease and assessment of disease activity. The data were compared with healthy individuals, patients with other renal and non-renal diseases, and patients with AAV in remission. 113 potential biomarkers were identified that differed significantly between active renal AAV and healthy individuals and patients with other chronic renal diseases. Of these, 58 could be sequenced. Sensitivity and specificity of models based on 18 sequenced biomarkers were validated using blinded urine samples of 40 patients with different renal diseases. Discrimination of AAV from other renal diseases in blinded samples was possible with 90% sensitivity and 86.7–90% specificity depending on the model. 10 patients with active AAV were followed for 6 months after initiation of treatment. Immunosuppressive therapy led to a change of the proteome toward “remission.” 47 biomarkers could be sequenced that underwent significant changes during therapy together with regression of clinical symptoms, normalization of C-reactive protein, and improvement of renal function. Proteomics analysis with capillary electrophoresis-MS represents a promising tool for fast identification of patients with active AAV, indication of renal relapses, and monitoring for ongoing active renal disease and remission without renal biopsy.Systemic vasculitides are a heterogeneous group of disorders with inflammation of the blood vessel wall as their common hallmark. These disorders often pose difficulties with regard to diagnosis and monitoring of disease activity both at the initial presentation and during follow-up. In one subgroup of small vessel vasculitides, the advent of anti-neutrophil cytoplasmic antibodies (ANCAs)¹ in the 1980s not only provided a new pathogenetic concept but also a diagnostic marker (1, 2). In this group, the granulomatous ANCA-associated vasculitis (GAAV) (previously named Wegener granulomatosis) and the microscopic polyangiitis (MPA) share several common features, including pauci-immune focal crescentic necrotizing glomerulonephritis and often a pulmonary capillaritis (3). Because of the association with ANCA, these diseases (together with the Churg-Strauss syndrome) are sometimes collectively referred to as ANCA-associated vasculitis (AAV). Recently circulating endothelial cells have emerged as an important marker correlating with severity and activity of the systemic vasculitic disease, and their clinical use in small vessel AAV has been demonstrated (4, 5). Regarding renal involvement, which is found in up to 80–90% of the patients with AAV, activity is defined by kidney biopsy with pauci-immune necrotizing glomerulonephritis. Renal involvement may occur or recur at every point of the disease and the follow-up even if other organ involvement is controlled by immunosuppressive therapy. Early detection is important as renal prognosis depends on early administration of immunosuppressive treatment (6), and scarring and relapses increase the risk for terminal renal failure, which itself is a risk factor for patient survival. As kidney biopsy is invasive and the risk of bleeding increases with chronic renal damage, surrogate markers, such as rising creatinine, increasing proteinuria, and most importantly erythrocytes and erythrocyte cast in the urinary sediment, are used. However, these markers have limitations. Microhematuria might persist despite remission, proteinuria might increase despite improvement in renal function, and other renal diseases can also develop (7). Therefore, new markers for renal disease activity are eagerly awaited.Recently proteome analysis of urine has presented itself as a promising tool in the definition of chronic renal diseases (8, 9). We have developed an analytical platform for human urine analysis using capillary electrophoresis (CE) coupled on line to an ESI-TOF mass spectrometer (8, 10, 11). This approach permits the rapid analysis of the low molecular weight urinary proteome/peptidome in a single step and has enabled identification and validation of several urinary biomarkers in patients with different renal diseases (11–14).In this study, we aimed toward identification of biomarkers for active AAV and response to immunosuppressive treatment. The results indicate that urinary proteome analysis enables differential diagnosis and monitoring of renal disease activity of AAV.     

Immunochemical studies on the metabolism of nitrosamines by ethanol-inducible cytochrome P-450

The ethanol-induced rabbit liver microsomal cytochrome P-450, P-450LM3a, has been shown previously to efficiently catalyze the demethylation of N-nitrosodimethylamine (NDMA) with a Km of 2.9 mM. Since the predominant Km in hepatic microsomes from ethanol-treated rabbits is 0.07 mM, the role of P-450LM3a in the activation of this carcinogen has been uncertain. In the present study, antibodies to P-450LM3a were shown to almost completely inhibit NDMA demethylation by the purified P-450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the high-Km NDMA demethylase activity in the microsomes. These results indicate that P-450LM3a is the major P-450 responsible for the low-Km NDMA demethylase activity. In addition, evidence is provided for the existence of a cytochrome immunochemically similar to P-450LM3a in liver microsomes from rats, mice, and guinea pigs that effectively catalyzes the demethylation of NDMA.     

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.     

Brain-penetrating 2-aminobenzimidazole H1-antihistamines for the treatment of insomnia

The benzimidazole core of the selective non-brain-penetrating H₁-antihistamine mizolastine was used to identify a series of brain-penetrating H₁-antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H₁-antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.