miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.     

Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes

Molecular Biology - When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be...     

A novel domain-duplicated SlitFAR3 gene involved in sex pheromone biosynthesis in Spodoptera litura

Fatty acyl reductases (FARs) are key enzymes that participate in sex pheromone biosynthesis by reducing fatty acids to fatty alcohols. Lepidoptera typically harbor numerous FAR gene family members. Although FAR genes are involved in the biosynthesis of sex pheromones in moths, the key FAR gene of Spodoptera litura remains unclear. In this work, we predicted 30 FAR genes from the S. litura genome and identified a domain duplication within gene SlitFAR3, which exhibited high and preferential expression in the sexually mature female pheromone glands (PGs) and a rhythmic expression pattern during the scotophase of sex pheromone production. The molecular docking of SlitFAR3, as predicted using a 3D model, revealed a co-factor NADPH binding cavity and 2 substrate binding cavities. Functional expression in yeast cells combined with comprehensive gas chromatography indicated that the SlitFAR3 gene could produce fatty alcohol products. This study is the first to focus on the special phenomenon of FAR domain duplication, which will advance our understanding of biosynthesis-related genes from the perspective of evolutionary biology.     

Tau pathogenesis is promoted by Aβ1-42 but not Aβ1-40

Background

The relationship between the pathogenic amyloid β-peptide species Aβ1–42 and tau pathology has been well studied and suggests that Aβ1–42 can accelerate tau pathology in vitro and in vivo. The manners if any in which Aβ1–40 interacts with tau remains poorly understood. In order to answer this question, we used cell-based system, transgenic fly and transgenic mice as models to study the interaction between Aβ1–42 and Aβ1–40.

Results

In our established cellular model, live cell imaging (using confocal microscopy) combined with biochemical data showed that exposure to Aβ1–42 induced cleavage, phosphorylation and aggregation of wild-type/full length tau while exposure to Aβ1–40 didn’t. Functional studies with Aβ1–40 were carried out in tau-GFP transgenic flies and showed that Aβ1–42, as previously reported, disrupted cytoskeletal structure while Aβ1–40 had no effect at same dose. To further explore how Aβ1–40 affects tau pathology in vivo, P301S mice (tau transgenic mice) were injected intracerebrally with either Aβ1–42 or Aβ1–40. We found that treatment with Aβ1–42 induced tau phosphorylation, cleavage and aggregation of tau in P301S mice. By contrast, Aβ1–40 injection didn’t alter total tau, phospho-tau (recognized by PHF-1) or cleavage of tau, but interestingly, phosphorylation at Ser²⁶² was shown to be significantly decreased after direct inject of Aβ1–40 into the entorhinal cortex of P301S mice.

Conclusions

These results demonstrate that Aβ1–40 plays different role in tau pathogenesis compared to Aβ1–42. Aβ1–40 may have a protective role in tau pathogenesis by reducing phosphorylation at Ser²⁶², which has been shown to be neurotoxic.

     

Low-Dose Prospectively Electrocardiogram-Gated Axial Dual-Source CT Angiography in Patients with Pulsatile Bilateral Bidirectional Glenn Shunt: An Alternative Noninvasive Method for Postoperative Morphological Estimation

Objective

To explore the clinical value of low-dose prospectively electrocardiogram-gated axial dual-source CT angiography (low-dose PGA scanning, CTA) in patients with pulsatile bilateral bidirectional Glenn shunt (bBDG) as an alternative noninvasive method for postoperative morphological estimation.

Methods

Twenty patients with pulsatile bBDG (mean age 4.2±1.6 years) underwent both low-dose PGA scanning and conventional cardiac angiography (CCA) for the morphological changes. The morphological evaluation included the anatomy of superior vena cava (SVC) and pulmonary artery (PA), the anastomotic location, thrombosis, aorto-pulmonary collateral circulation, pulmonary arteriovenous malformations, etc. Objective and subjective image quality was assessed. Bland–Altman analysis and linear regression analyses were used to evaluate the correlation on measurements between CTA and CCA. Effective radiation dose of both modalities was calculated.

Results

The CT attenuation value of bilateral SVC and PA was higher than 300 HU. The average subjective image quality score was 4.05±0.69. The morphology of bilateral SVC and PA was displayed completely and intuitively by CTA images. There were 24 SVC above PA and 15 SVC beside PA. Thrombosis was found in 1 patient. Collateral vessels were detected in 13 patients. No pulmonary arteriovenous malformation was found in our study. A strong correlation (R²>0.8, P<0.001) was observed between the measurements on CTA images and on CCA images. Bland–Altman analysis demonstrated a systematic overestimation of the measurements by CTA (the mean value of bias>0).The mean effective dose of CTA and CCA was 0.50±0.17 mSv and 4.85±1.34 mSv respectively.

Conclusion

CT angiography with a low-dose PGA scanning is an accurate and reliable noninvasive examination in the assessment of morphological changes in patients with pulsatile bBDG.     

Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.     

新疆地区小麦白粉病菌寄主范围的研究

用小麦白粉病菌11个生理小种的混合菌种,对新疆地区的小麦近缘植物的7个属22个种的47份材料进行接种,除6份免疫外,其余均接种成功.用其中6个属19个种的29份小麦近缘植物产生的白粉病菌,对小麦回接,参试的29份材料全部回接成功.小麦白粉病菌对小麦近缘植物的寄生像在小麦上一样,有明显的寄生专化性.感病的小麦近缘植物的78.0%对小麦白粉病菌的感病性,随生育期增长而急剧下降.文中并对小麦白粉病中间寄主的作用进行了讨论.