Excitotoxic stimulation activates distinct pathogenic and protective expression signatures in the hippocampus.

Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N-methyl-d-aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology-specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24-h post-insult. Egr-1 and NOR-1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA-induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up-regulated pathogenic genes, down-regulated pro-survival genes) and opposing compensatory responses (down-regulated pathogenic genes, up-regulated pro-survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF-κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF-κB inhibitor MG-132 indeed altered NMDA-mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine.

Mathematical model reveals role of nucleotide signaling in airway surface liquid homeostasis and its dysregulation in cystic fibrosis.

Mucociliary clearance is composed of three components (i.e., mucin secretion, airway surface hydration, and ciliary-activity) which function coordinately to clear inhaled microbes and other foreign particles from airway surfaces. Airway surface hydration is maintained by water fluxes driven predominantly by active chloride and sodium ion transport. The ion channels that mediate electrogenic ion transport are regulated by extracellular purinergic signals that signal through G protein-coupled receptors. These purinoreceptors and the signaling pathways they activate have been identified as possible therapeutic targets for treating lung disease. A systems-level description of airway surface liquid (ASL) homeostasis could accelerate development of such therapies. Accordingly, we developed a mathematical model to describe the dynamic coupling of ion and water transport to extracellular purinergic signaling. We trained our model from steady-state and time-dependent experimental measurements made using normal and cystic fibrosis (CF) cultured human airway epithelium. To reproduce CF conditions, reduced chloride secretion, increased potassium secretion, and increased sodium absorption were required. The model accurately predicted ASL height under basal normal and CF conditions and the collapse of surface hydration due to the accelerated nucleotide metabolism associated with CF exacerbations. Finally, the model predicted a therapeutic strategy to deliver nucleotide receptor agonists to effectively rehydrate the ASL of CF airways.

A model of threshold behavior reveals rescue mechanisms of bystander proteins in conformational diseases.

Conformational diseases result from the failure of a specific protein to fold into its correct functional state. The misfolded proteins can lead to the toxic aggregation of proteins. Protein misfolding in conformational diseases often displays a threshold behavior characterized by a sudden shift between nontoxic to toxic levels of misfolded proteins. In some conformational diseases, evidence suggests that misfolded proteins interact with bystander proteins (unfolded and native folded proteins), eliciting a misfolded phenotype. These bystander isomers would follow their normal physiological pathways in absence of misfolded proteins. In this article, we present a general mechanism of bystander and misfolded protein interaction which we have used to investigate how the threshold behavior in protein misfolding is triggered in conformational diseases. Using a continuous flow reactor model of the endoplasmic reticulum, we found that slight changes in the bystander protein residence time in the endoplasmic reticulum or the ratio of basal misfolded to bystander protein inflow rates can trigger the threshold behavior in protein misfolding. Our analysis reveals three mechanisms to rescue bystander proteins in conformational diseases. The results of our model can now help direct experiments to understand the threshold behavior and develop therapeutic strategies targeting the modulation of conformational diseases.

Building the lac Operon: A Guided-Inquiry Activity Using 3D-Printed Models.

Undergraduate biology courses rely heavily on visual representation of information. Students view images of plants, animals, and microbes, interpret data presented in graphs, and use drawings to understand how cells and molecules interact in three dimensions. Traditional teaching approaches exclude students with visual impairments and disadvantage students with disabilities that affect their interpretation and processing of visual and spatial information, and also students who simply do not identify as "visual learners." By using new technologies to develop tactile teaching tools (TTTs) that can be employed in classrooms, we aim to create inclusive learning environments and more effectively instruct diverse learners. The advent of affordable and accessible 3D printing technology makes it possible to create tactile models that represent molecules, cells, and entire organisms more accurately than traditional visual representations. We describe the assessment of a 3D gene expression puzzle as a guided inquiry learning activity in which students must correctly assemble a series of components in order to achieve an output. Upon completion of the puzzle, the TTT provides tactile feedback through vibration to signal transcriptional activation. Analysis of pre- and postassessment performance demonstrated statistically significant increases in individual students' paired assessment scores in two different classroom implementations, with a greater effect size at a rural minority-serving institution than an urban R1 university. These encouraging preliminary data suggest that TTTs with guided-inquiry learning disproportionately benefit disadvantaged student populations and could serve as a tool in leveling the playing field when teaching abstract biological concepts in diverse educational settings.

Sequence determinants of human microsatellite variability.

BACKGROUND: Microsatellite loci are frequently used in genomic studies of DNA sequence repeats and in population studies of genetic variability. To investigate the effect of sequence properties of microsatellites on their level of variability we have analyzed genotypes at 627 microsatellite loci in 1,048 worldwide individuals from the HGDP-CEPH cell line panel together with the DNA sequences of these microsatellites in the human RefSeq database. RESULTS: Calibrating PCR fragment lengths in individual genotypes by using the RefSeq sequence enabled us to infer repeat number in the HGDP-CEPH dataset and to calculate the mean number of repeats (as opposed to the mean PCR fragment length), under the assumption that differences in PCR fragment length reflect differences in the numbers of repeats in the embedded repeat sequences. We find the mean and maximum numbers of repeats across individuals to be positively correlated with heterozygosity. The size and composition of the repeat unit of a microsatellite are also important factors in predicting heterozygosity, with tetra-nucleotide repeat units high in G/C content leading to higher heterozygosity. Finally, we find that microsatellites containing more separate sets of repeated motifs generally have higher heterozygosity. CONCLUSIONS: These results suggest that sequence properties of microsatellites have a significant impact in determining the features of human microsatellite variability.

PedMine--a simulated annealing algorithm to identify maximally unrelated individuals in population isolates.

UNLABELLED: In family-based genetic studies, it is often useful to identify a subset of unrelated individuals. When such studies are conducted in population isolates, however, most if not all individuals are often detectably related to each other. To identify a set of maximally unrelated (or equivalently, minimally related) individuals, we have implemented simulated annealing, a general-purpose algorithm for solving difficult combinatorial optimization problems. We illustrate our method on data from a genetic study in the Old Order Amish of Lancaster County, Pennsylvania, a population isolate derived from a modest number of founders. Given one or more pedigrees, our program automatically and rapidly extracts a fixed number of maximally unrelated individuals. AVAILABILITY: http://www.hg.med.umich.edu/labs/douglaslab/software.html (version 1.0.0).

Pyrimethamine-resistant dihydrofolate reductase enzymes of Plasmodium falciparum are not enzymatically compromised in vitro.

Plasmodium falciparum, the protozoan that causes the most lethal form of human malaria, has been controlled principally by two safe, affordable drugs, chloroquine and sulfadoxine-pyrimethamine (SP). Studies in the laboratory and in the field have demonstrated that resistance to SP depends on non-synonymous point mutations in the dihydrofolate reductase (DHFR), and dihydropteroate synthase (DHPS) coding regions. Parasites that carry dhfr genes with 3 or 4 point mutations (51I/59R/108N triple mutation or 51I/59R/108N/164L quadruple mutation) are resistant to pyrimethamine in vitro and patients infected with these parasites respond poorly to SP treatment. The wide spread of these pyrimethamine-resistant alleles demonstrates the increased fitness over drug-sensitive alleles in the presence of the drug. However, it is not clear whether these alleles might reduce the fitness of parasites in the absence of drug pressure. As a first step, we compared the kinetic properties of the wild type, and three mutant alleles to determine whether the native DHFR-thymidylate synthase form of the mutant proteins showed compromised activity in vitro. The mutant enzymes had K(m) values for their substrate, dihydrofolate that were significantly lower than the wild type, k(cat) values in the same range as the wild type enzyme, and k(cat)/K(m) values higher than wild type. In contrast, the K(m) values for the NADPH cofactor were higher than wild type for the mutant enzymes. These observations suggest that the fitness of these parasites may not be compromised relative to those that carry the wild type allele, even without sustained SP drug pressure.

Undergraduate journal club as an intervention to improve student development in applying the scientific process.

Active learning improves student performance in STEM courses. Exposure to active learning environments generally occurs through traditional laboratory courses and independent research, both of which require access to resources that are limited at many universities. A previously reported active learning-based undergraduate journal club improved student achievement in communicating science. Here, we expanded on this previous journal club to improve student performance in the process of science. We developed and implemented a series of workshops and seminars referred to as "CASL Club," an undergraduate journal club targeted at improving student development in applying the scientific process. Students were surveyed before and after CASL club about their confidence in accessing, analyzing, and reporting scientific research. Post-CASL club, the students reported increases in confidence in their abilities to access and present scientific articles and write scientific abstracts. Additionally, the students reported improved confidence and performance in their courses. Compared to the previous journal club study, the majority of sampled journal club participants were not exposed to primary literature as part of their general coursework. Our results illustrate active-learning based undergraduate journal clubs as a way to expose students to primary literature and improve students' ability to apply scientific process in an active-learning environment at resource-limited universities.

Network representations and methods for the analysis of chemical and biochemical pathways.

Systems biologists increasingly use network representations to investigate biochemical pathways and their dynamic behaviours. In this critical review, we discuss four commonly used network representations of chemical and biochemical pathways. We illustrate how some of these representations reduce network complexity but result in the ambiguous representation of biochemical pathways. We also examine the current theoretical approaches available to investigate the dynamic behaviour of chemical and biochemical networks. Finally, we describe how the critical chemical and biochemical pathways responsible for emergent dynamic behaviour can be identified using network mining and functional mapping approaches.