Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

The 5′ UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5′ untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The “winner” viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication

Damaging the Integrated HIV Proviral DNA with TALENs.

HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs) to target a highly conserved sequence in the transactivation response element (TAR) of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication

HIV-1 genome conservation analysis to select TALEN sites.

<p>A. Schematic diagram of HIV-1 genome adapted from the Los Alamos National Laboratory HIV website [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125652#pone.0125652.ref044" target="_blank">44</a>]. Bolded boxes are regions with HT-TALEN DNA targets, one of which is shown in B. B. 5’ LTR DNA TALEN target sequence. The TALE binding targets are indicated by black lines. The endonuclease target site sequence is in lower case font and indicated by grey lines. C. TAR RNA with partial 5’ TALE binding site in upper-case font and endonuclease target site in lower-case font. D. HIV-1 DNA sequences (274,874 total) from the Los Alamos HIV Sequence Database were aligned with ClustalΩ to determine conservation which is presented in a position specific-scoring matrix [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125652#pone.0125652.ref033" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125652#pone.0125652.ref044" target="_blank">44</a>]. The nucleotide frequency for the most conserved regions were chosen as TALEN target sequences found in the TAR coding region (B) of the LTRs (226 sequences) (A).</p

TALEN targeting of integrated complete HIV-1 proviral DNA in cell culture.

<p>A. Schematic diagram of the complete HIV-1 proviral DNA to be targeted by the HT-TALEN pair or the NS-TALEN pair. The target region is found in both the 5’ and 3’LTRs. The host genome is indicated in grey. B. Western blot analysis of HeLa/LAV cells transfected with a HT-TALEN plasmid pair or the NS-TALEN pair. The blot was probed with anti-Flag and anti-Actin as a loading control. C. Bar graph showing quantitation of flow cytometry analysis of cytotoxicity. Transiently transfected HeLa/LAV cells were analyzed by flow cytometry (n = 3) to identify Annexin V positive cells. Standard deviations are indicated by error bars with no statistical significance (NS) p>0.05 in cytotoxicity between the control and the TALEN pairs. D. Sequences of clones containing mutated target regions represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125652#pone.0125652.g003" target="_blank">Fig 3</a>. E. A schematic of the 5’ target region of wild type plai.2 HIV-1 proviral DNA and the mutated plai.2 HIV-1 proviral DNA. The mutated proviral DNA was designed based on the sequence from HeLa/LAV clone HL-16 (Fig 4D). The Gag coding region (containing capsid) is indicated. Western blot analysis of cell lysates harvested from pEAK Rapid cells transfected with mutant or wild type plai.2 proviral DNA. The blot was probed with anti-Capsid qingto detect Gag production and anti-Actin as a loading control.</p

HT-TALENs and NS-TALENs cleave an HIV-1 DNA fragment <i>in vitro</i>.

<p>A. Schematic diagram representing HT-TALENs and NS-TALENs bound to their cognate DNA target sequence (thick lines). Relative locations of the Fok1 endonuclease, Flag epitope tag, and nuclear localization sequence (NLS) are indicated. Asterisks and grey boxes designate where a “NS” coding TALE repeat was used in the 5’ NS-TALEN construction. B. Western blot of <i>in vitro</i> transcription/translation reactions containing no expression plasmids, each TALEN alone, the HT-TALEN pair, or the NS-TALEN pair. C. Gel electrophoresis analysis of <i>in vitro</i> cleavage reactions containing no TALEN plasmids, the HT-TALEN pair, or the NS-TALEN pair. The HIV-1 target DNA fragment size is 747 bp, with expected on-target cleavage products of approximately 430 bp and 317 bp. Quantification of cleavage was performed using ImageJ software and is shown below the gel image. D. The HIV-1 target DNA fragment from (C) was mutated in the 5’ TALE binding site to create a set of triple mutant templates (Mut1-Mut4). The sequences of Mut1-Mut4 are depicted in bold, lowercase font and mutated positions are indicated by asterisks. Cleavage reactions containing either the HT-TALEN or NS-TALEN pairs incubated with the HIV-1 target templates were size fractionated by electrophoresis and quantified by densitometry with ImageJ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125652#pone.0125652.ref039" target="_blank">39</a>].</p

The Role of Social Capital in the Success of Fair Trade

Fair Trade companies have pulled off an astonishingtour de force. Despite their relatively small size and lack of resources, they have managed to achieve considerable commercial success and, in so doing, have put the fair trade issue firmly onto industry agendas. We analyse the critical role played by social capital in this success and demonstrate the importance of values as an exploitable competitive asset. Our research raises some uncomfortable questions about whether fair trade has ‘sold out' to the mainstream and whether these companies have any independent future or whether their ultimate success lies in the impact they have had on day-to-day trading behavio

Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases