Identification and Characterization of Small Regulatory RNA in Streptococcus

Background: The human gut is a rich habitat for a diverse population of bacteria. These bacteria play a vital role in a multitude of functions. They affect immune responses, metabolism, and even neurological activities. Some inhabitants of the gut biome include the lactic acid bacteria: Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. These bacteria are essential members of the flora and are non-pathogenic, however, some of their relatives like Streptococcus pyogenes cause severe diseases such as flesh-eating disease. The metabolic functions of these bacteria are mediated by small regulatory RNAs (sRNAs), which are noncoding RNA sequences that fold into complex stem-loop structures. Goal of Study: The goal of this project was to identify and characterize sRNAs that mediate bacterial metabolism and host interactions in non-pathogenic, lactic acid bacteria with homologous RNAs in pathogenic strains. Methods and Results: A small regulatory RNA that is conserved among different streptococcal species was identified. In S.pyogenes, this conserved sRNA, named MarS, is associated with virulence, however, the function of its homolog found in non-pathogenic bacteria, AsdS sRNA, has not been characterized. Computational methods were used to elucidate the function of this sRNA and predict its 2D and 3D structures. The gene containing the target sRNA was isolated and RNA constructs were designed to characterize regions that are part of it. Conclusions: A conserved sRNA species was successfully identified in S.thermophilus and cloned for synthesis by in-vitro transcription. Future work will be focused on structure determination and characterizing interactions to target sRNA through biophysical methods

An Analysis of the Reflection Component in the EPICS Model of Service Learning

Service learning is a pedagogy providing a structured environment for students to link service with course learning objectives. Key to the service learning experience is critical reflection. This gives students the opportunity to examine their coursework in the context of the service they provide to their community and, in a broader sense, the impact they can have on the world. Research has shown that students participating in service learning have a higher comprehension of the course material and also develop an awareness of their local community and the issues it faces. In engineering, there are many examples of service-learning programs ranging from freshman introductory courses to senior capstone courses. Despite their successes, an area that the engineering education community has yet to fully develop is the reflection component of service learning. This paper addresses the development of reflection activities and materials in the Engineering Projects in Community Service (EPICS) program at Purdue University. EPICS engages students in long-term design projects that provide technical solutions to problems faced by local community service organizations. It is a multidisciplinary (composed of students from 20 majors), vertically integrated (freshman-senior), engineering-based design course. Students design, build, test, and deploy projects meeting the specific needs of their community partners. Reflection has been integrated in the EPICS program through curricular activities and key milestones of the course. These activities guide students through the reflection process on a variety of topics. Critical reflection on the design process and teaming complement those on more traditional areas of ethics and social context to enhance a student\u27s service learning experience. This paper presents an overview of the reflection activities that have been developed, interpretations of student reflections from these activities, and plans to evolve the reflection component in EPICS

Tensions of Integration in Professional Formation: Investigating Development of Engineering Students\u27 Social and Technical Perceptions

Tensions of Integration in Professional Formation: Investigating Development of Engineering Students\u27 Social and Technical PerceptionsTwenty-first century engineers face incredible challenges and opportunities, many of which aresocially complex, transcending the traditional “technical” boundaries of engineering. Thetechnology produced by engineers must not only function as predicted by mathematical andtheoretical models but must also operate beneficially and seamlessly in complex social contexts.In this sense, engineers must embody an integrated social and technical – or sociotechnical –identity rather than a dualistic social/technical one.A growing body of scholarship has discussed how dominant cultures of engineering shapestudents’ and professionals’ understandings of social and technical dimensions of their work.Further, engineering education research has advanced understanding of how engineering identityis formed by external, structural forces. Yet, from a psychological perspective, we know littleabout how engineering students come to perceive and embody their identities as engineers,especially in relation to social and technical dimensions of these identities. Thus, we organizedthis study around the following research questions.RQ0: How do students psychologically experience identity trajectories of becoming engineers?RQ1: How do students perceive the social and technical features of engineering identity?RQ2: How do students internally experience their identities as engineers, particularly with regard to social and technical dimensions of these identities?RQ3: How do social and technical perceptions of their engineering identity develop and change in the course of the engineering curriculum or in the transition to the workplace?To respond to these research questions, we have conducted two longitudinal studies usinginterpretative phenomenological analysis (IPA). One study focused on graduating seniors as theytransitioned into the workplace, and the second study focused on first-year students transitioningto engineering degree coursework. These investigations produced robust and nuancedunderstanding of students’ engineering identity trajectories throughout and beyond thecurriculum. These findings are being leveraged in order to provide our initial understanding in athematic analysis on sophomore engineering students.Thus far, the findings of the investigation highlight the complexity of becoming both engineers,specifically by demonstrating a somewhat contradictory relationship between what participantsperceived to be engineering and how they actually embodied an engineering-self. They furtherdemonstrate the manifold ways that participants realized and prioritized identities outside ofengineering and how these multiple selves interacted in ways that affected their engineeringidentities. Additionally, findings for both male and female groups suggest that somepsychological patterns might be related to gender. In sum, the findings depict a complex pictureof engineering-students-turned-engineers as whole persons. By focusing on how engineeringidentity development is embodied, the findings generate multiple theoretical insights that bearrelevance for engineering education research and provocative implications that bear significancefor engineering educators, students, and employers

Deweyan tools for inquiry and the epistemological context of critical pedagogy

This article develops the notion of resistance as articulated in the literature of critical pedagogy as being both culturally sponsored and cognitively manifested. To do so, the authors draw upon John Dewey\u27s conception of tools for inquiry. Dewey provides a way to conceptualize student resistance not as a form of willful disputation, but instead as a function of socialization into cultural models of thought that actively truncate inquiry. In other words, resistance can be construed as the cognitive and emotive dimensions of the ongoing failure of institutions to provide ideas that help individuals both recognize social problems and imagine possible solutions. Focusing on Dewey\u27s epistemological framework, specifically tools for inquiry, provides a way to grasp this problem. It also affords some innovative solutions; for instance, it helps conceive of possible links between the regular curriculum and the study of specific social justice issues, a relationship that is often under-examined. The aims of critical pedagogy depend upon students developing dexterity with the conceptual tools they use to make meaning of the evidence they confront; these are background skills that the regular curriculum can be made to serve even outside social justice-focused curricula. Furthermore, the article concludes that because such inquiry involves the exploration and potential revision of students\u27 world-ordering beliefs, developing flexibility in how one thinks may be better achieved within academic subjects and topics that are not so intimately connected to students\u27 current social lives, especially where students may be directly implicated

Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation

DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

Entry Assessment in Community Colleges: Tracking or Facilitating?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66989/2/10.1177_009155219302100302.pd

Serologic and PCR testing of persons with chronic fatigue syndrome in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses

In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases

Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs

The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces false-positive MLV PCR results. We report here the finding of a large number of PCR reagents that have low levels of MLV sequences. We found that recombinant reverse-transcriptase (RT) enzymes from six companies derived from either MLV or avian myeloblastosis virus contained MLV pol DNA sequences but not gag or mouse DNA sequences. Sequence and phylogenetic analysis showed high relatedness to Moloney MLV, suggesting residual contamination with an RT-containing plasmid. In addition, we identified contamination with mouse DNA and a variety of MLV sequences in commercially available human DNAs from leukocytes, brain tissues, and cell lines. These results identify new sources of MLV contamination and highlight the importance of careful pre-screening of commercial specimens and diagnostic reagents to avoid false-positive MLV PCR results