Margaret S. Saha

A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students

ABSTRACT Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students’ interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations. Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

Vertebrate eye development

Vertebrate eye determination is mediated by a series of inductive interactions that have now been more precisely defined with the use of regional markers. Analyses of the genes responsible for eye mutations and the cloning of genes delimiting spatial domains within the developing eye have begun to elucidate the molecular basis of this process.

A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus

The process by which the vertebrate central nervous system acquires its regional properties remains a central problem in developmental biology. It is generally argued that at early gastrula stages the dorsal mesoderm possesses precise anterior-posterior positional information, which is subsequently imparted to the overlying ectoderm. However, using regionally specific gene probes to monitor regional responses in Xenopus embryos, we find that anterior-posterior properties are not fixed until early neurula stages. During gastrulation the regional inducing capacities of the dorsal mesoderm as well as the regional responses of the presumptive neural ectoderm are activated along the entire anterior-posterior axis when these properties are assayed in recombinant and explant experiments, respectively. Restriction of regional inducing capacity in the mesoderm and responsiveness in the neural ectoderm occur only at neural plate stages.

SPECT-CT system for small animal imaging

The Detector Group at the Thomas Jefferson National Accelerator Facility (Jefferson Lab) and the Biology, Physics, and Applied Sciences Departments at the College of William and Mary are collaborating on the development of a miniature dual modality SPECT-CT system for mouse imaging. The detector heads of the SPECT sub-system are designed to be capable of imaging the gamma- and X-ray emissions (28-35 keV) of the radioactive isotope iodine-125 (I-125). Two different sets of I-125 imaging detectors are configured on a gantry that has an open-barrel type design. One set of detector heads is based on the 1-in square Hamamatsu R5900-M64 position sensitive photomultiplier tube coupled to crystal scintillator arrays. The other detector heads configured on the gantry are two 5-in diameter Hamamatsu R3292-based compact gamma cameras. The X-ray radiographic projections are obtained using a LIXI Inc. model LF-85-503-OS X-ray imaging system that has an active area of 5.5 cm in diameter. The open-barrel shaped gantry facilitates the positioning of various mini gamma-ray imaging detectors and the X-ray system. The data acquisition and gantry control is interfaced through a Macintosh G3 workstation. Preliminary SPECT reconstruction results using the R5900 based detector are presented.

Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse preBötzinger complex.

Neurons of the preBötzinger complex (preBötC) form local excitatory networks and synchronously discharge bursts of action potentials during the inspiratory phase of respiratory network activity. Synaptic input periodically evokes a Ca2+‐activated non‐specific cation current (ICAN) postsynaptically to generate 10–30 mV transient depolarizations, dubbed inspiratory drive potentials, which underlie inspiratory bursts. The molecular identity of ICAN and its regulation by intracellular signalling mechanisms during inspiratory drive potential generation remains unknown. Here we show that mRNAs coding for two members of the transient receptor potential (TRP) family of ion channels, namely TRPM4 and TRPM5, are expressed within the preBötC region of neonatal mice. Hypothesizing that the phosphoinositides maintaining TRPM4 and TRPM5 channel sensitivity to Ca2+ may similarly influence ICAN and thus regulate inspiratory drive potentials, we manipulated intracellular phosphatidylinositol 4,5‐bisphosphate (PIP2) and measured its effect on preBötC neurons in the context of ongoing respiratory‐related rhythms in slice preparations. Consistent with the involvement of TRPM4 and TRPM5, excess PIP2 augmented the inspiratory drive potential and diminution of PIP2 reduced it; sensitivity to flufenamic acid (FFA) suggested that these effects of PIP2 were ICAN mediated. Inositol 1,4,5‐trisphosphate (IP3), the product of PIP2 hydrolysis, ordinarily causes IP3 receptor‐mediated ICAN activation. Simultaneously increasing PIP2 while blocking IP3 receptors intracellularly counteracted the reduction in the inspiratory drive potential that normally resulted from IP3 receptor blockade. We propose that PIP2 protects ICAN from rundown by interacting directly with underlying ion channels and preventing desensitization, which may enhance the robustness of respiratory rhythm.

Embryonic lens induction: more than meets the optic vesicle

The classic model of lens induction stipulated that the optic vesicle is both a necessary and sufficient inductor of the lens in amphibian development. Although this view has subsequently been modified to encompass the contributions of earlier inductors, such as the involuting endo-mesoderm, it is still widely reported that the optic vesicle alone is able to elicit lens formation from ectoderm. Recent work, which has employed a host- and donor-marking scheme, has shown the optic vesicle to be a weak inductor of the lens, involved only in the final rather than the initial phases of determination. In addition, a review of the literature substantiates this conclusion since many of the transplantation experiments arguing for the sufficiency of the optic vesicle are characterized by the lack of adequate criteria for judging the authenticity of the resulting lens responses, particularly the absence of a host- and donor-marking strategy. This analysis of the literature, together with our own results, lead us to propose a new model of lens determination in which tissue interactions during gastrulation are required to confer a lens-forming bias upon a large area of head ectoderm allowing the optic vesicle to induce lens formation in a defined area of this primed ectoderm. Data from studies on mesoderm and neural induction are also beginning to suggest a multistep model involving the initial establishment of bias and subsequent interactions resulting in determination, and we propose that this framework will serve as a general paradigm for embryonic induction.

Early opsin expression in Xenopus embryos precedes photoreceptor differentiation

The visual pigment which serves as the first step in the phototransduction cycle in vertebrate rod cells consists of a retinal chromophore which is linked to the transmembrane protein, opsin. Opsin genes have been isolated from a number of different organisms and studies have shown opsin to be developmentally regulated with both mRNA and protein expression associated with the morphological differentiation of photoreceptor cells. Due to its potential utility as a marker for rod photoreceptor determination in studies of retinal tissue interactions, and because no amphibian opsin genes have as yet been cloned, we isolated cDNA clones of the Xenopus laevis opsin gene. Sequence analysis shows that within the coding region Xenopus opsin shares a high degree of identity with other rod opsin genes, except at the C-terminal where it more closely resembles the mammalian color opsins. A developmental analysis, on the other hand, reveals that Xenopus opsin transcripts are detectable in a retina-specific fashion early in retinal development. Using in situ hybridization we find that Xenopus opsin mRNA is initially restricted to a few isolated cells in the presumptive photoreceptor layer which express the gene at relatively high levels. This suggests that rod photoreceptor determination occurs in single cells, and that the mechanisms controlling opsin expression in Xenopus are initiated well before any evidence of morphological differentiation.

Anthropogenic noise is associated with reductions in the productivity of breeding Eastern Bluebirds (Sialia sialis).

Although previous studies have related variations in environmental noise levels with alterations in communication behaviors of birds, little work has investigated the potential long-term implications of living or breeding in noisy habitats. However, noise has the potential to reduce fitness, both directly (because it is a physiological stressor) and indirectly (by masking important vocalizations and/or leading to behavioral changes). Here, we quantified acoustic conditions in active breeding territories of male Eastern Bluebirds (Sialia sialis). Simultaneously, we measured four fitness indicators: cuckoldry rates, brood growth rate and condition, and number of fledglings produced (i.e., productivity). Increases in environmental noise tended to be associated with smaller brood sizes and were more strongly related to reductions in productivity. Although the mechanism responsible for these patterns is not yet clear, the breeding depression experienced by this otherwise disturbance-tolerant species indicates that anthropogenic noise may have damaging effects on individual fitness and, by extraction, the persistence of populations in noisy habitats. We suggest that managers might protect avian residents from potentially harmful noise by keeping acoustically dominant anthropogenic habitat features as far as possible from favored songbird breeding habitats, limiting noisy human activities, and/or altering habitat structure in order to minimize the propagation of noise pollution.

Design features and performance of a CsI(Na) array based gamma camera for small animal gene research

The authors report on the development of a radiation imaging system based on a matrix of CsI(Na) scintillating crystals. This detector system has been optimized for high resolution radiation imaging of the distribution of iodine 125 (/sup 125/I) in a laboratory animal. The radioisotope /sup 125/I is readily available as a label to molecular biology probes that can be used in gene research. Iodine 125 decays via electron capture emitting a 35 keV gamma-ray with the prompt emission of several 27-32 keV X-rays. A coincidence condition can be set to detect the /sup 125/I decays thus reducing background radiation contribution to the image. The system utilizes the Hamamatsu 5 inch diameter 3292 position sensitive photomultiplier tube (PSPMT) coupled to the CsI(Na) matrix and a custom built high resolution copper-beryllium collimator. A conventional photomultiplier coupled to a NaI(Ti) crystal scintillator is used to detect the coincident radiation resulting from the decay of /sup 125/I.

Development of a novel radiation imaging detector system for in vivo gene imaging in small animal studies

We report preliminary results from a prototype of radiation imaging technology which takes advantage of the emission properties of the radioisotope iodine /sup 125/I as the probe. The detector system utilizes crystal scintillators and a position sensitive photomultiplier tube. Iodine 125 decays via electron capture emitting a 35-keV gamma ray with the prompt emission of several 27-32-keV K/spl alpha/ and K/spl beta/ shell X-rays. Because of this, a coincidence condition can be set to detect the /sup 125/I decay, thus reducing background radiation contribution to the image. The prototype detector we report has a limited sensitivity and detection area because of the size of the scintillators and photomultiplier tubes, yet it performed well enough to demonstrate the viability of this method for imaging /sup 125/I in a mouse. Mouse imaging studies of iodine uptake by the thyroid and melatonin binding have been done with this detector system using doses of /sup 125/I alone or attached to the melatonin. Many studies in molecular biology follow the expression and regulation of a gene at different stages of an organisms development or under different physiological conditions. Molecular biology research could benefit from this detection system by utilizing /sup 125/I-labeled gene probes.