Student Self-perception on Digital Literacy in STEM Blended Learning Environments.

As students transition into tertiary blended learning environments, their digital literacy in terms of technical capabilities have potential to impact on their access to digital resources. The first foundational year of STEM degrees includes compulsory courses across a broad range of scientific areas, each of which incorporates online technology in a discipline-specific manner. Given the diversity of online resources that STEM students need to access across their first-year coursework, this study applies learning analytical methods to determine whether students' perceived level of digital literacy has an effect on their navigation of learning management systems (LMS) and overall academic performance. The frequency and nature of LMS interactivity were examined across four first-year STEM courses offered in the same semester at a single institution, using a K-means cluster analysis to group student responses. It was observed that high achieving students accessed LMS resources more frequently than mid or low-achieving students across all four STEM courses. Students' perceived level of digital literacy was collected via survey (n = 282), and students were sorted high (n = 106) and low-level (n = 176) of perceived digital literacy-HDL and LDL, respectively. HDL students were not consistently found in the high-achieving academic group and did not perform better in their overall grade when compared to LDL students. LDL students were observed to perform better in specific online assessment tasks, which may be attributed to their increased frequency of LMS interactivity. These findings highlight the delicate balance between students' perceived level of digital literacy, motivation for engaging with online learning environments, and academic performance.

Local axonal protection by WldS as revealed by conditional regulation of protein stability.

The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment.

Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella.

3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.

Statistical and visual differentiation of subcellular imaging.

BACKGROUND: Automated microscopy technologies have led to a rapid growth in imaging data on a scale comparable to that of the genomic revolution. High throughput screens are now being performed to determine the localisation of all of proteins in a proteome. Closer to the bench, large image sets of proteins in treated and untreated cells are being captured on a daily basis to determine function and interactions. Hence there is a need for new methodologies and protocols to test for difference in subcellular imaging both to remove bias and enable throughput. Here we introduce a novel method of statistical testing, and supporting software, to give a rigorous test for difference in imaging. We also outline the key questions and steps in establishing an analysis pipeline. RESULTS: The methodology is tested on a high throughput set of images of 10 subcellular localisations, and it is shown that the localisations may be distinguished to a statistically significant degree with as few as 12 images of each. Further, subtle changes in a protein's distribution between nocodazole treated and control experiments are shown to be detectable. The effect of outlier images is also examined and it is shown that while the significance of the test may be reduced by outliers this may be compensated for by utilising more images. Finally, the test is compared to previous work and shown to be more sensitive in detecting difference. The methodology has been implemented within the iCluster system for visualising and clustering bio-image sets. CONCLUSION: The aim here is to establish a methodology and protocol for testing for difference in subcellular imaging, and to provide tools to do so. While iCluster is applicable to moderate (<1000) size image sets, the statistical test is simple to implement and will readily be adapted to high throughput pipelines to provide more sensitive discrimination of difference.

Disease gene candidates revealed by expression profiling of retinal ganglion cell development.

To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.

A role for SNX5 in the regulation of macropinocytosis.

BACKGROUND: The mechanisms and components that regulate macropinocytosis are poorly understood. Here we have investigated the role of sorting nexin 5 (SNX5) in the regulation of macropinocytic activity. RESULTS: SNX5 is abundantly expressed in macrophages, cells very active in macropinocytosis, and is recruited onto newly-formed macropinosomes. LPS treatment of bone marrow-derived macrophages resulted in a 2.5 fold decrease in macropinosome formation that correlates with a reduction in the levels of SNX5. To investigate the relationship between SNX5 levels and macropinocytic activity we examined the formation of macropinosomes in HEK-FlpIn cells stably expressing GFP-SNX5. Constitutive macropinocytosis was increased approximately 2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Furthermore, EGF stimulation resulted in a significant increase in macropinocytosis and there was also a 2.0 fold increase in the generation of macropinosomes in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. SNX5, which interacts specifically with PtdIns(3)P and PtdIns(3,4)P2 through its PX domain, was recruited to regions on the plasma membrane containing EGF receptor or positive for PtdIns(3,4)P2 as detected with the PH domain of TAPP1. Treatment with AG1478, an EGF receptor specific tyrosine kinase inhibitor, prevented the recruitment of SNX5 to the cytosolic face of the plasma membrane and inhibited the formation of macropinosomes in response to EGF treatment. CONCLUSION: Based on these data, we propose that SNX5 requires the generation of phosphoinositides for recruitment to the plasma membrane and, moreover, influences the level of macropinocytic activity.

The beer and biofuels laboratory: A report on implementing and supporting a large, interdisciplinary, yeast-focused course-based undergraduate research experience.

Course-integrated Undergraduate Research Experiences (CUREs) involve large numbers of students in real research. We describe a late-year microbiology CURE in which students use yeast to address a research question around beer brewing or synthesizing biofuel; the interdisciplinary student-designed project incorporates genetics, bioinformatics, biochemistry, analytical chemistry, and microbiology. Students perceived significant learning gains around multiple technical and "becoming a scientist" aspects of the project. The project is demanding for both the students and the academic implementers. We examine the rich landscape of support and interaction that this CURE both encourages and requires while also considering how we can support the exercise better and more sustainably. The findings from this study provide a picture of a CURE implementation that has begun to reach the limits of both the students' and the academics' capacities to complete it. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):213-222, 2018.

A laboratory competency examination in microbiology.

The American Society for Microbiology's curricular guidelines for Introductory Microbiology highlighted key laboratory skills in the isolation, visualization and identification of microorganisms as core learning objectives in the discipline. Since the publication of these guidelines in 2012, there has been a paucity of diagnostic assessment tools in the literature that can be used to assess competencies in the microbiology laboratory. This project aimed to establish a laboratory competency examination for introductory microbiology, with tasks specifically aligned to laboratory skills and learning outcomes outlined in curricular guidelines for microbiology. A Laboratory Competency Examination assessing student skills in light microscopy, Gram-staining, pure culture, aseptic technique, serial dilution, dilution calculations and pipetting was developed at The University of Queensland, Australia. The Laboratory Competency Examination was field-tested in a large introductory microbiology subject (∼400 students), and student performance and learning gains data were collected from 2016 to 2017 to evaluate the validity of the assessment. The resulting laboratory assessment is presented as an endpoint diagnostic tool for assessing laboratory competency that can be readily adapted towards different educational contexts.

Communication Ambassadors-an Australian Social Media Initiative to Develop Communication Skills in Early Career Scientists.

Science communication is a skill set to be developed through ongoing interactions with different stakeholders across a variety of platforms. Opportunities to engage the general public are typically reserved for senior scientists, but the use of social media in science communication allows all scientists to instantaneously disseminate their findings and interact with online users. The Communication Ambassador program is a social media initiative launched by the Australian Society for Microbiology to expand the online presence and science communication portfolios of early-career scientists. Through their participation in the program, a rotating roster of Australian microbiologists have broadened the online reach of the Society's social media channels as well as their own professional networks by attending and live-tweeting microbiology events throughout the year. We present the Communication Ambassador program as a case study of coordinated social media activity in science communication to the general public, and describe the potential for its applications in science education and training.

Course-based undergraduate research experiences in molecular biosciences-patterns, trends, and faculty support.

Inquiry-driven learning, research internships and course-based undergraduate research experiences all represent mechanisms through which educators can engage undergraduate students in scientific research. In life sciences education, the benefits of undergraduate research have been thoroughly evaluated, but limitations in infrastructure and training can prevent widespread uptake of these practices. It is not clear how faculty members can integrate complex laboratory techniques and equipment into their unique context, while finding the time and resources to implement undergraduate research according to best practice guidelines. This review will go through the trends and patterns in inquiry-based undergraduate life science projects with particular emphasis on molecular biosciences-the research-aligned disciplines of biochemistry, molecular cell biology, microbiology, and genomics and bioinformatics. This will provide instructors with an overview of the model organisms, laboratory techniques and research questions that are adaptable for semester-long projects, and serve as starting guidelines for course-based undergraduate research.

An oligodendrocyte lineage-specific semaphorin, Sema5A, inhibits axon growth by retinal ganglion cells.

In the mammalian CNS, glial cells repel axons during development and inhibit axon regeneration after injury. It is unknown whether the same repulsive axon guidance molecules expressed by glia and their precursors during development also play a role in inhibiting regeneration in the injured CNS. Here we investigate whether optic nerve glial cells express semaphorin family members and, if so, whether these semaphorins inhibit axon growth by retinal ganglion cells (RGCs). We show that each optic nerve glial cell type, astrocytes, oligodendrocytes, and their precursor cells, expressed a distinct complement of semaphorins. One of these, sema5A, was expressed only by purified oligodendrocytes and their precursors, but not by astrocytes, and was present in both normal and axotomized optic nerve but not in peripheral nerves. Sema5A induced collapse of RGC growth cones and inhibited RGC axon growth when presented as a substrate in vitro. To determine whether sema5A might contribute to inhibition of axon growth after injury, we studied the ability of RGCs to extend axons when cultured on postnatal day (P) 4, P8, and adult optic nerve explants and found that axon growth was strongly inhibited. Blocking sema5A using a neutralizing antibody significantly increased RGC axon growth on these optic nerve explants. These data support the hypothesis that sema5A expression by oligodendrocyte lineage cells contributes to the glial cues that inhibit CNS regeneration.

Do you kiss your mother with that mouth? An authentic large-scale undergraduate research experience in mapping the human oral microbiome.

Clinical microbiology testing is crucial for the diagnosis and treatment of community and hospital-acquired infections. Laboratory scientists need to utilize technical and problem-solving skills to select from a wide array of microbial identification techniques. The inquiry-driven laboratory training required to prepare microbiology graduates for this professional environment can be difficult to replicate within undergraduate curricula, especially in courses that accommodate large student cohorts. We aimed to improve undergraduate scientific training by engaging hundreds of introductory microbiology students in an Authentic Large-Scale Undergraduate Research Experience (ALURE). The ALURE aimed to characterize the microorganisms that reside in the healthy human oral cavity-the oral microbiome-by analyzing hundreds of samples obtained from student volunteers within the course. Students were able to choose from selective and differential culture media, Gram-staining, microscopy, as well as polymerase chain reaction (PCR) and 16S rRNA gene sequencing techniques, in order to collect, analyze, and interpret novel data to determine the collective oral microbiome of the student cohort. Pre- and postsurvey analysis of student learning gains across two iterations of the course (2012-2013) revealed significantly higher student confidence in laboratory skills following the completion of the ALURE (p < 0.05 using the Mann-Whitney U-test). Learning objectives on effective scientific communication were also met through effective student performance in laboratory reports describing the research outcomes of the project. The integration of undergraduate research in clinical microbiology has the capacity to deliver authentic research experiences and improve scientific training for large cohorts of undergraduate students.

Macropinosome quantitation assay.

In contrast to phagocytosis, macropinocytosis is not directly initiated by interactions between cell surface receptors and cargo ligands, but is a result of constitutive membrane ruffling driven by dynamic remodelling of cortical actin cytoskeleton in response to stimulation of growth factor receptors. Wang et al. (2010) [13] developed a reliable assay that allows quantitative assessment of the efficiency and kinetics of macropinosome biogenesis and/or maturation in cells where the function of a targeted protein has been perturbed by pharmacological inhibitors or by knock-down or knock-out approaches. In this manuscript we describe a modified quantitative protocol to measure the rate and volume of fluid phase uptake in adherent cells. This assay:•uses fluorescent dextran, microscopy and semi-automated image analysis;•allows quantitation of macropinosomes within large numbers of individual cells;•can be applied also to non-homogenous cell populations including transiently transfected cell monolayers. We present the background necessary to consider when customising this protocol for application to new cell types or experimental variations.

Purification and culture of retinal ganglion cells.

Retinal ganglion cells (RGCs) are the neurons that extend axons through the optic nerve, connecting and transmitting information from the retina to the brain. In mammals, RGCs receive information from bipolar and amacrine cells and synapse onto target cells in the lateral geniculate nucleus (LGN) as well as the superior colliculus. Methods for acute purification of RGCs from rodent retina by immunopanning followed by culture in a serum-free medium have facilitated the study of neuronal biology and function in a defined environment. These methods are introduced here, and modifications for achieving optimal RGC purity and culture are described.

Purification and culture of retinal ganglion cells from rodents.

Here we describe methods for acute purification of retinal ganglion cells (RGCs) from rodent retina by immunopanning, followed by culture in serum-free medium. Though the method was initially established and verified with rats, we have included modifications for the purification of mouse RGCs. This protocol is written for isolation of cells from one litter of pups. All of the volumes and numbers of panning plates should be scaled according to the number of litters used, particularly for rat RGCs.

Culturing hybridoma cell lines for monoclonal antibody production.

This protocol describes how to culture hybridoma cell lines (e.g., Thy1.1) for monoclonal antibody production. Supernatants harvested from such cultures can be used to purify various rodent neural cell types by immunopanning.

How much is too much assessment? Insight into assessment-driven student learning gains in large-scale undergraduate microbiology courses.

Designing and implementing assessment tasks in large-scale undergraduate science courses is a labor-intensive process subject to increasing scrutiny from students and quality assurance authorities alike. Recent pedagogical research has provided conceptual frameworks for teaching introductory undergraduate microbiology, but has yet to define best-practice assessment guidelines. This study assessed the applicability of Biggs' theory of constructive alignment in designing consistent learning objectives, activities, and assessment items that aligned with the American Society for Microbiology's concept-based microbiology curriculum in MICR2000, an introductory microbiology course offered at the University of Queensland, Australia. By improving the internal consistency in assessment criteria and increasing the number of assessment items explicitly aligned to the course learning objectives, the teaching team was able to efficiently provide adequate feedback on numerous assessment tasks throughout the semester, which contributed to improved student performance and learning gains. When comparing the constructively aligned 2011 offering of MICR2000 with its 2010 counterpart, students obtained higher marks in both coursework assignments and examinations as the semester progressed. Students also valued the additional feedback provided, as student rankings for course feedback provision increased in 2011 and assessment and feedback was identified as a key strength of MICR2000. By designing MICR2000 using constructive alignment and iterative assessment tasks that followed a common set of learning outcomes, the teaching team was able to effectively deliver detailed and timely feedback in a large introductory microbiology course. This study serves as a case study for how constructive alignment can be integrated into modern teaching practices for large-scale courses.

Gap junctions are essential for generating the correlated spike activity of neighboring retinal ganglion cells.

Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets.

Axon degeneration: where the Wlds things are.

Expression of the Wld(s) protein significantly delays axon degeneration in injuries and diseases, but the mechanism for this protection is unknown. Two recent reports present evidence that axonal mitochondria are required for Wld(S)-mediated axon protection.

Axon degeneration: molecular mechanisms of a self-destruction pathway.

Axon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. However, the molecular pathways that regulate this process remain unclear. Axon loss in chronic neurodegenerative diseases share many morphological features with those in acute injuries, and expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in both events, indicating a common mechanism of axonal self-destruction in traumatic injuries and degenerative diseases. A proposed model of axon degeneration is that nerve insults lead to impaired delivery or expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown.