Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

<p>Abstract</p> <p>Background</p> <p>The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.</p> <p>Results</p> <p>We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that <it>Agrobacterium </it>VirE2 protein interacts with the <it>Arabidopsis </it>nuclear transport adapter protein importin α-1 in the cytoplasm, whereas interaction of VirE2 with a different importin α isoform, importin α-4, occurs predominantly in the nucleus.</p> <p>Conclusion</p> <p>Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.</p

Observational connection of non-thermal X-ray emission from pulsars with their timing properties and thermal emission

The origin and radiation mechanisms of high energy emissions from pulsars have remained mysterious since their discovery. Here we report, based on a sample of 68 pulsars, observational connection of non-thermal X-ray emissions from pulsars with their timing properties and thermal emissions, which may provide some constraints on theoretical modeling. Besides strong correlations with the spin-down power $\dot{E}$ and the magnetic field strength at the light cylinder $B_{\rm lc}$, the non-thermal X-ray luminosity in 0.5 - 8 keV, $L_{\rm p}$, represented by the power-law component in the spectral model, is found to be strongly correlated with the highest possible electric field strength in the polar gap, $E_{\rm pc}$, of the pulsar. The spectral power index $\Gamma_{\rm p}$ of that power-law component is also found, for the first time in the literature, to strongly correlate with $\dot{E}$, $B_{\rm lc}$ and $E_{\rm pc}$, thanks to the large sample. In addition, we found that $L_{\rm p}$ can be well described by $L_{\rm p}\propto T^{5.96\pm 0.64}R^{2.24\pm 0.18}$, where $T$ and $R$ are the surface temperature and the emitting-region radius of the surface thermal emission, represented by the black-body component in the spectral model. $\Gamma_{\rm p}$, on the other hand, can be well described only when timing variables are included, and the relation is $\Gamma_{\rm p} = \log(T^{-5.8\pm 1.93}R^{-2.29\pm 0.85}P^{-1.19\pm 0.88}\dot{P}^{0.94\pm 0.44})$ plus a constant. These relations strongly suggest the existence of connections between surface thermal emission and electron-positron pair production in pulsar magnetospheres.Comment: 13 pages, 11 figures, accepted by MNRA

Improving Success Rates of Percutaneous Coronary Intervention for Chronic Total Occlusion at a Rural Hospital in East Taiwan

SummaryBackgroundWe aimed to report the results of percutaneous coronary intervention for chronic total occlusion (CTO) in a remote hospital of southeast Taiwan that does not have on-site coronary artery bypass graft support and has insufficient medical resources.MethodsFrom 2006 to 2009, we identified 96 patients who underwent percutaneous coronary intervention and whose coronary angiogram showed CTO lesions. On-site cardiovascular surgeons were unavailable from 2006 to 2009.ResultsThe success rate (test for trend, p = 0.02) and numbers of guidewires used (test for trend, p = 0.59) significantly increased from 2006 to 2009, and the procedural time reduced significantly (test for trend, p = 0.001). The volume of contrast media injected decreased, although this result was not statistically significant (p = 0.70).ConclusionOur experience in managing CTO lesions substantially improved and the procedural time reduced over 4 years, even when constrained by a relative shortage of medical resources

Adaptor protein Shc acts as an immune-regulator for the LPS-stimulated maturation of bone marrow-derived dendritic cells

<p>Abstract</p> <p>Background</p> <p>The Shc isoforms is known to mediate immune responses and has been indicated as a negative regulator of autoimmunity and lymphocyte activation. We aimed to evaluate the immune-regulatory role of Shc in rat bone marrow-derived DCs in the maturation process triggered by LPS.</p> <p>Results</p> <p>We found that, in response to LPS, expression of Shc proteins was induced and that neutralization of Shc inhibited the LPS-induced transient phosphorylation of p52Shc on pTyr239/240 in DCs of Lewis (LEW; RT1^l) rats. Moreover, the significantly enhanced expression of IL-10 and the surface level of costimulatory molecule CD80, as well as suppressed expression of IL-6 and IL-12 in the Shc-silenced DCs were also observed. Similar IκB phosphorylation occurred in Shc-silenced DCs primed by LPS, indicating Shc is not associated with NF-κB pathway. We further demonstrate that Shc blockade on LPS-treated DCs results in significant increase of the overall STAT3 phosphorylation and the relative levels of phospho-STAT3 in the nuclear fraction. STAT3 activation by LPS with or without Shc blockade was totally abolished by SU6656, a selective Src family kinases inhibitor, underscoring the critical role of Src-mediated activation.</p> <p>Conclusions</p> <p>We conclude that Shc blockade in LPS-primed DC leads to the development of tolerogenic DC via Src-dependent STAT3 activation and that adaptor protein Shc might play a pivotal role in mediating immunogenic and tolerogenic properties of DCs.</p

Therapeutic Effect of Repurposed Temsirolimus in Lung Adenocarcinoma Model

Lung cancer is one of the major cause of cancer-related deaths worldwide. The poor prognosis and resistance to both radiation and chemotherapy urged the development of potential targets for lung cancer treatment. In this study, using a network-based cellular signature bioinformatics approach, we repurposed a clinically approved mTOR inhibitor for renal cell carcinomans, temsirolimus, as the potential therapeutic candidate for lung adenocarcinoma. The PI3K-AKT-mTOR pathway is known as one of the most frequently dysregulated pathway in cancers, including non-small-cell lung cancer. By using a well-documented lung adenocarcinoma mouse model of human pathophysiology, we examined the effect of temsirolimus on the growth of lung adenocarcinoma in vitro and in vivo. In addition, temsirolimus combined with reduced doses of cisplatin and gemcitabine significantly inhibited the lung tumor growth in the lung adenocarcinoma mouse model compared with the temsirolimus alone or the conventional cisplatin–gemcitabine combination. Functional imaging techniques and microscopic analyses were used to reveal the response mechanisms. Extensive immunohistochemical analyses were used to demonstrate the apparent effects of combined treatments on tumor architecture, vasculature, apoptosis, and the mTOR-pathway. The present findings urge the further exploration of temsirolimus in combination with chemotherapy for treating lung adenocarcinoma

Inhibitory Effect of Anoectochilus formosanus Extract on Hyperglycemia-Related PD-L1 Expression and Cancer Proliferation

Traditional herb medicine, golden thread (Anoectochilus formosanus Hayata) has been used to treat various diseases. Hyperglycemia induces generation of reactive oxygen species (ROS) and enhancement of oxidative stress which are risk factors for cancer progression and metastasis. In this study, we evaluated hypoglycemic effect of A. formosanus extracts (AFEs) in an inducible hyperglycemia animal model and its capacity of free-radical scavenging to establish hyperglycemia-related carcinogenesis. AFE reduced blood glucose in hyperglycemic mice while there was no change in control group. The incremental area under blood glucose response curve was decreased significantly in hyperglycemic mice treated with AFE in a dose-dependent manner. AFE and metformin at the same administrated dose of 50 mg/kg showed similar effect on intraperitoneal glucose tolerance test in hyperglycemic mice. Free-radical scavenger capacity of AFE was concentration dependent and 200 μg/ml of AFE was able to reduce more than 41% of the free radical. Treatment of cancer cells with AFE inhibited constitutive PD-L1 expression and its protein accumulation. It also induced expression of pro-apoptotic genes but inhibited proliferative and metastatic genes. In addition, it induced anti-proliferation in cancer cells. The results suggested that AFE not only reduced blood glucose concentration as metformin but also showed its potential use in cancer immune chemoprevention/therapy via hypoglycemic effect, ROS scavenging and PD-L1 suppression

Potential of Cellular Therapy for ALS: Current Strategies and Future Prospects

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive upper and lower motor neuron (MN) degeneration with unclear pathology. The worldwide prevalence of ALS is approximately 4.42 per 100,000 populations, and death occurs within 3–5 years after diagnosis. However, no effective therapeutic modality for ALS is currently available. In recent years, cellular therapy has shown considerable therapeutic potential because it exerts immunomodulatory effects and protects the MN circuit. However, the safety and efficacy of cellular therapy in ALS are still under debate. In this review, we summarize the current progress in cellular therapy for ALS. The underlying mechanism, current clinical trials, and the pros and cons of cellular therapy using different types of cell are discussed. In addition, clinical studies of mesenchymal stem cells (MSCs) in ALS are highlighted. The summarized findings of this review can facilitate the future clinical application of precision medicine using cellular therapy in ALS