Multiple shRNA combinations for near-complete coverage of all HIV-1 strains

<p>Abstract</p> <p>Background</p> <p>Combinatorial RNA interference (co-RNAi) approaches are needed to account for viral variability in treating HIV-1 with RNAi, as single short hairpin RNAs (shRNA) are rapidly rendered ineffective by resistant strains. Current work suggests that 4 simultaneously expressed shRNAs may prevent the emergence of resistant strains.</p> <p>Results</p> <p>In this study we assembled combinations of highly-conserved shRNAs to target as many HIV-1 strains as possible. We analyzed intersecting conservations of 10 shRNAs to find combinations with 4+ matching the maximum number of strains using 1220+ HIV-1 sequences from the Los Alamos National Laboratory (LANL). We built 26 combinations of 2 to 7 shRNAs with up to 87% coverage for all known strains and 100% coverage of clade B subtypes, and characterized their intrinsic suppressive activities in transient expression assays. We found that all combinations had high combined suppressive activities, though there were also large changes in the individual activities of the component shRNAs in our multiple expression cassette configurations.</p> <p>Conclusion</p> <p>By considering the intersecting conservations of shRNA combinations we have shown that it is possible to assemble combinations of 6 and 7 highly active, highly conserved shRNAs such that there is always at least 4 shRNAs within each combination covering all currently known variants of entire HIV-1 subtypes. By extension, it may be possible to combine several combinations for complete global coverage of HIV-1 variants.</p

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER- /PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase-independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease

A conceptual enquiry into communities of practice as praxis in international doctoral education

Undertaking a PhD entails diverse and multi-faceted challenges as doctoral researchers enter a distinct academic culture that requires transition to a new level and threshold of learning – with both knowledge acquisition and production at the core. While doctoral researchers are expected to secure different dimensions of knowledge, which necessitates meaningful ‘dialogue’ with experts, the colossal task is still ironically associated with isolated doctoral experience and somewhat limited postgraduate supervision provision. With the extra concerns typically confronting the international doctoral cohort, the pressure tends be intensified, and may lead to psychological well-being concerns. Nevertheless, there is evidence from the literature that highlights the often unacknowledged forms of learning opportunities and support mechanisms via community participation. By employing communities of practice as the main framework, this conceptual paper exemplifies the crucial role played by these communities – how these communities serve to scaffold doctoral researchers’ academic progress, support their psychological adjustments, and reinforce the crucial, but perhaps limited, formal doctoral support provision. By featuring effective examples of educational praxis via these communities, our paper offers a holistic understanding of formal and informal infrastructures as part of the wider doctoral ecology with a view to achieving a more holistic and meaningful doctoral experience

A conceptual enquiry into communities of practice as praxis in international doctoral education

Undertaking a PhD entails diverse and multi-faceted challenges as doctoral researchers enter a distinct academic culture that requires transition to a new level and threshold of learning – with both knowledge acquisition and production at the core. While doctoral researchers are expected to secure different dimensions of knowledge, which necessitates meaningful ‘dialogue’ with experts, the colossal task is still ironically associated with isolated doctoral experience and somewhat limited postgraduate supervision provision. With the extra concerns typically confronting the international doctoral cohort, the pressure tends be intensified, and may lead to psychological well-being concerns. Nevertheless, there is evidence from the literature that highlights the often unacknowledged forms of learning opportunities and support mechanisms via community participation. By employing communities of practice as the main framework, this conceptual paper exemplifies the crucial role played by these communities – how these communities serve to scaffold doctoral researchers’ academic progress, support their psychological adjustments, and reinforce the crucial, but perhaps limited, formal doctoral support provision. By featuring effective examples of educational praxis via these communities, our paper offers a holistic understanding of formal and informal infrastructures as part of the wider doctoral ecology with a view to achieving a more holistic and meaningful doctoral experience

Training community healthcare workers on the use of information and communication technologies: a randomised controlled trial of traditional versus blended learning in Malawi, Africa

Background: Despite the increasing uptake of information and communication technologies (ICT) within healthcare services across developing countries, community healthcare workers (CHWs) have limited knowledge to fully utilise computerised clinical systems and mobile apps. The ‘Introduction to Information and Communication Technology and eHealth’ course was developed with the aim to provide CHWs in Malawi, Africa, with basic knowledge and computer skills to use digital solutions in healthcare delivery. The course was delivered using a traditional and a blended learning approach. Methods: Two questionnaires were developed and tested for face validity and reliability in a pilot course with 20 CHWs. Those were designed to measure CHWs’ knowledge of and attitudes towards the use of ICT, before and after each course, as well as their satisfaction with each learning approach. Following validation, a randomised controlled trial was conducted to assess the effectiveness of the two learning approaches. A total of 40 CHWs were recruited, stratified by position, gender and computer experience, and allocated to the traditional or blended learning group using block randomisation. Participants completed the baseline and follow-up questionnaires before and after each course to assess the impact of each learning approach on their knowledge, attitudes, and satisfaction. Per-item, pre-post and between-group, mean differences for each approach were calculated using paired and unpaired t-tests, respectively. Per-item, between-group, satisfaction scores were compared using unpaired t-tests. Results: Scores across all scales improved after attending the traditional and blended learning courses. Self-rated ICT knowledge was significantly improved in both groups with significant differences between groups in seven domains. However, actual ICT knowledge scores were similar across groups. There were no significant differences between groups in attitudinal gains. Satisfaction with the course was generally high in both groups. However, participants in the blended learning group found it more difficult to follow the content of the course. Conclusions: This study shows that there is no difference between blended and traditional learning in the acquisition of actual ICT knowledge among community healthcare workers in developing countries. Given the human resource constraints in remote resource-poor areas, the blended learning approach may present an advantageous alternative to traditional learning