Inquiry-Driven Bioinformatics Laboratory Research Module: Metagenomic Study of Student Oral Microbes.

American Society for Microbiology Curriculum Guidelines highlight the importance of enabling students to think critically and learn by doing research. Moreover, information in biology, especially genetics and biotechnology, increases too rapidly for instructors to teach everything. To increase students' interest and comprehension of important core genetic concepts and to encourage students to practice scientific investigation, we designed a research module for upper-level biology/genetics students to examine oral bacteria. Students extracted their own oral microbial DNA and amplified and analyzed with general genus- and species-specific 16S rRNA PCR primers. The microbial DNA samples were also amplified with conserved bacteria 16S rRNA primers and the amplicons TOPO cloned (topoisomerase-based cloning) and Sanger sequenced. Lastly, the metagenomic microbial DNA samples were also sequenced by Illumina next-generation sequencing and analyzed with bioinformatics tools. We have implemented the module in three iterations of an undergraduate class at a small, liberal arts college. The project culminates in a poster presentation that the students on average performed in a high B range. Pre- and postsurvey analysis of student learning gains revealed significant student learning (P < 0.05 one-tailed, paired Wilcoxon signed ranked test, n = 23). Next, we surveyed student perceptions of the activity by a self-assessment. Significantly more than the medians, the students enjoyed the inquiry-driven module and considered it more effective in teaching about PCR and other molecular genetics concepts than the traditional prescribed laboratory exercises. We conclude that this microbe laboratory module induces research interest and is useful in teaching important genetics concepts.

Role of calcineurin in activity-dependent pattern formation in the dorsal lateral geniculate nucleus of the ferret.

In the retinogeniculate pathway of the ferret, in addition to the separation of the inputs from the two eyes to form eye-specific layers, there is also an anatomical segregation of the terminal arbors of on-center retinal ganglion cells from the terminal arbors of off-center retinal ganglion cell axons to form on/off sublaminae. Sublamination normally occurs during postnatal weeks 3-4 and requires the activity of retinal afferents, N-methyl-D-aspartate receptors, nitric oxide synthase, and a target of nitric oxide, cyclic guanosine monophosphate. Calcineurin is a calcium/calmodulin dependent serine, threonine protein phosphatase suggested to mediate NMDA-receptor dependent synaptic plasticity in the hippocampus. We have examined whether calcineurin plays a role during on/off sublamination in the dorsal lateral geniculate nucleus (dLGN) of the ferret. Immunohistochemistry showed that calcineurin expression is transiently up-regulated in dLGN cells and neuropil during the period of on/off sublamination. A functional role for calcineurin during sublamination was investigated by blocking the enzyme locally via intracranial infusion of FK506. Treatment with FK506 during postnatal weeks 3-4 disrupted the appearance of sublaminae. These results suggest that calcineurin may play a role during this process of activity-dependent pattern formation in the visual pathway.

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation.

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.