The effects of stem length and core placement on shRNA activity

<p>Abstract</p> <p>Background</p> <p>Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined.</p> <p>Results</p> <p>Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity.</p> <p>Conclusion</p> <p>We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.</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

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells

<p>Abstract</p> <p>Background</p> <p>Tumors expressing a transforming growth factor-beta type I receptor (TβRI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFβ signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of TβRI.</p> <p>Results</p> <p>We cloned and identified four new in-frame deletion variants of TβRI, designated DM1 to DM4, in pleural effusion-derived tumor cells. Intriguingly, DM1 and DM2, with a small region truncated in the putative signal peptide of TβRI, had a serious defect in their protein expression compared with that of the wild-type receptor. Using serial deletion mutagenesis, we characterized a region encoded by nucleotides 16–51 as a key element controlling TβRI protein expression. Consistently, both DM1 and DM2 have this peptide deleted. Experiments using cycloheximde and MG132 further confirmed its indispensable role for the protein stability of TβRI. In contrast, truncation of the 9A-stretch itself or a region downstream to the stretch barely affected TβRI expression. However, variants lacking a region C-terminal to the stretch completely lost their capability to conduct TGFβ-induced transcriptional activation. Intriguingly, expression of DM3 in a cell sensitive to TGFβ made it significantly refractory to TGFβ-mediated growth inhibition. The effect of DM3 was to ablate the apoptotic event induced by TGFβ.</p> <p>Conclusion</p> <p>We identified four new transcript variants of TβRI in malignant effusion tumor cells and characterized two key elements controlling its protein stability and transcriptional activation. Expression of one of variants bestowed cancer cells with a growth advantage in the presence of TGFβ. These results highlight the potential roles of some naturally occurring TβRI variants on the promotion of tumor malignancy.</p

Energy hole mitigation through cooperative transmission in wireless sensor networks

The energy balancing capability of cooperative communication is utilized to solve the energy hole problem in wireless sensor networks. We first propose a cooperative transmission strategy, where intermediate nodes participate in two cooperative multi-input single-output (MISO) transmissions with the node at the previous hop and a selected node at the next hop, respectively. Then, we study the optimization problems for power allocation of the cooperative transmission strategy by examining two different approaches: network lifetime maximization (NLM) and energy consumption minimization (ECM). For NLM, the numerical optimal solution is derived and a searching algorithm for suboptimal solution is provided when the optimal solution does not exist. For ECM, a closed-form solution is obtained. Numerical and simulation results show that both the approaches have much longer network lifetime than SISO transmission strategies and other cooperative communication schemes. Moreover, NLM which features energy balancing outperforms ECM which focuses on energy efficiency, in the network lifetime sense

SCintillation and IONosphere eXtended (SCION-X): A 12U CubeSat for Ionospheric and Atmospheric Science

SCION-X (SCintillation and IONosphere eXtended) is a 12U CubeSat that is being designed and developed by Upper Air Dynamics Laboratory, National Central University (NCU). SCION-X is the second funded CubeSat project being developed by NCU and is the largest self-developed spacecraft to date. This mission will help to further understand the variation of ionospheric irregularities, remote sensing methods for PM2.5 pollution distribution, and thermospheric photochemistry while serving as a relay station for amateur radio. Furthermore, it will help increase the communication and cooperation between universities in developing spaceflight capacity

Dam (Canis familiaris)Welfare throughout the Peri-Parturient Period in Commercial Breeding Kennels

Publication history: Accepted - 10 October 2022; Published - 18 October 2022.Poor dam welfare throughout the peri-parturient period can also negatively affect that of their offspring. This study aimed to identify changes in physical, physiological, and behavioral metrics indicative of dam welfare throughout the peri-parturient period. Dams (n = 74) from eight U.S. Midwest commercial breeding (CB) kennels were tested at 6 and 1 week prepartum, and 4 and 8 weeks postpartum. At each time point dams underwent a stranger approach test, physical health assessment, hair collection for hair cortisol concentration (HCC) and fecal collection for fecal glucocorticoid metabolites (FGM), fecal secretory immunoglobulin A (sIgA) and parasite detection. Linear mixed-effects models indicated dams exhibited more affiliative behaviors towards the stranger at 4 weeks postpartum than 6 weeks prepartum (p = 0.03), increased HCC from 4-weeks to 8 weeks postpartum (p = 0.02), and increased FGM from 1 week prepartum to 8 weeks postpartum (p = 0.04). At each respective time point, the percentage of dams with intestinal parasites was 11%, 4%, 23%, and 15%. Most changes are likely due to increased energy requirements and hormonal variations. However, deviations from expected changes may have resulted from changes in environment and/ or management, which should be explored in future studies.This research was funded by The Stanton Foundation, grant number 41000444

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

Present-Day Lake Level Variation from Envisat Altimetry over the Northeastern Qinghai-Tibetan Plateau: Links with Precipitation and Temperature

Lakes in permafrost regions are highly sensitive to changes in air temperature, snowmelt, and soil frost. In particular, the Qinghai-Tibetan Plateau (QTP) is one of the most sensitive regions in the world influenced by global climate change. In this study, we use retracked Enivsat radar altimeter measurements to generate water level change time series over Lake Qinghai and Lake Ngoring in the northeastern QTP and examine their relationships with precipitation and temperature changes. The response of water levels in Lake Qinghai and Lake Ngoring is positive with regards to precipitation amount. There is a negative relationship between water level and temperature change. These findings further the idea that the arid and high-elevation lakes in the northeastern QTP are highly sensitive to climate variations. Water level increases in Lake Qinghai in winter may indicate inputs of subsurface water associated with freeze-thaw cycles in the seasonally frozen ground and the active layer