Functional Characterization of <i>Hevea brasiliensis CRT/DRE Binding Factor 1</i> Gene Revealed Regulation Potential in the CBF Pathway of Tropical Perennial Tree

<div><p>Rubber trees (<i>Hevea brasiliensis</i>) are susceptible to low temperature and therefore are only planted in the tropical regions. In the past few decades, although rubber trees have been successfully planted in the northern margin of tropical area in China, they suffered from cold injury during the winter. To understand the physiological response under cold stress, we isolated a C-repeat binding factor 1 (<i>CBF1</i>) gene from the rubber tree. This gene (<i>HbCBF1)</i> was found to respond to cold stress but not drought or ABA stress. The corresponding HbCBF1 protein showed CRT/DRE binding activity in gel shift experiment. To further characterize its molecular function, the <i>HbCBF1</i> gene was overexpressed in <i>Arabidopsis</i>. The <i>HbCBF1</i> over expression (OE) line showed enhanced cold resistance and relatively slow dehydration, and the expression of <i>Arabidopsis</i> CBF pathway downstream target genes, e.g. <i>AtCOR15a</i> and <i>AtRD29a</i>, were significantly activated under non-acclimation condition. These data suggest <i>HbCBF1</i> gene is a functional member of the CBF gene family, and may play important regulation function in rubber tree.</p></div

Automated Synthesis and Preclinical Evaluation of Optimized Integrin α6-Targeted Positron Emission Tomography Imaging of Pancreatic Cancer

Integrin α6 has been considered a promising biomarker, is overexpressed in many tumors, and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we identified a novel high-affinity integrin α6-targeted peptide named RD2 (Arg-Trp-Tyr-Asp-PEG4)2-Lys-Lys and developed a 18F-radiolabeled peptide tracer ([18F]-AlF-NOTA-RD2) and evaluated its potential application in positron emission tomography (PET) imaging of pancreatic cancer. [18F]-AlF-NOTA-RD2 was produced using GMP (Good Manufacturing Practice of Medical Products)-compliant automatic radiosynthesis on a single GE FASTLab2 cassette-type synthesis module. The stability of [18F]-AlF-NOTA-RD2 was analyzed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). The cell uptake assay of the tracer was assessed using PANC-1 cells. In addition, small-animal PET imaging and biodistribution studies of [18F]-AlF-NOTA-RD2 were performed in pancreatic cancer subcutaneous tumor-bearing mice. The PET tracer [18F]-AlF-NOTA-RD2 was obtained with a radiochemical yield of 23.7 ± 4.7%, radiochemical purity of >99%, and molar activity of 165.7 ± 59.1 GBq/μmol. [18F]-AlF-NOTA-RD2 exhibited good in vitro stability in PBS and FBS. LogP octanol water value for the tracer was −2.28 ± 0.05 (n = 3). The binding affinity of RD2 to the integrin α6 protein (Kd = 0.13 ± 3.65 μM, n = 3) was significantly higher than that of the RWY (CRWYDENAC) (Kd = 6.97 ± 1.44 μM, n = 3). Small-animal PET imaging and biodistribution also revealed that [18F]-AlF-NOTA-RD2 displayed rapid and good tumor uptake and lower liver background uptake in PANC-1 tumor-bearing mice. [18F]-AlF-NOTA-RD2 showed significant radioactivity accumulation in tumors and was successfully blocked by NOTA-RD2. Compared with [18F]-FDG, [18F]-AlF-NOTA-RD2 PET imaging and biodistribution studies in PANC-1 xenograft tumor-bearing mice confirmed a good tumor-to-muscle ratio (8.69 ± 2.03 vs 1.41 ± 0.23, respectively) at 0.5 h and (2.99 ± 3.02 vs 1.43 ± 0.17, respectively) at 1 h post injection. Autoradiography of human pancreatic cancer tumor tissues further confirmed high accumulation of [18F]-AlF-NOTA-RD2. In summary, we developed an optimized integrin α6-targeted imaging tracer and obtained high radioactivity products with a cassette-type synthesis module; moreover, the tracer exhibited good binding affinity with integrin α6 and good target specificity for PANC-1 cells in xenograft pancreatic tumor-bearing mice, demonstrating its promising application as a noninvasive PET radiotracer of integrin α6 expression in pancreatic cancer

<i>HbCBF1</i> overexpression in <i>Arabidopsis</i>.

<p>(A) Southern blotting of <i>HbCBF1</i> in transgenic plants. (B) <i>HbCBF1</i> expression in OE-3, OE-6 and OE-13 lines. The gene expression was detected by Northern blotting analysis using the <i>HbCBF1</i> probe. (C) Thirty days old seedlings of <i>col-0</i>, OE-3, OE-6 and OE-13 lines showed decreased plant height.</p

Characterization of <i>HbCBF1</i> gene.

<p>(A) Southern blotting analysis of <i>HbCBF1</i> gene in <i>Hevea brasiliensis</i> genome. (B) <i>HbCBF1</i> expression in response to cold, drought stress or ABA treatment. Twenty microgram total RNA from the leaves of the seedlings treated with cold, drought or ABA for the indicated time were used for Northern blotting analysis. <i>HbCBF1</i> full length cDNA was used as probe. EtBr-stained rRNA was used as an internal standard to monitor equal loading of total RNA.</p

Gel mobility shift assay of recombinant HbCBF1 protein indicated CRT/DRE binding activity.

<p><b>(A)</b> Probes sequences used in this analysis. Bolded nucleotides are CRT/DRE motif, while underlined nucleotides are the mutation sites. <b>(B)</b> HbCBF1 bound with CRT/DRE elements specifically <i>in vitro</i>. Four microgram total proteins of GST (lane 1 from the left) or GST::HbCBF1 (lane 2–9 from the left) were used for the binding with labeled hot COR15a probe. One hundred and twenty-five folds molar unlabelled cold competitor probes were added as indicated (labe 3–9 from the left) in each reactions. The retarded binding bands and free non-binding probe were indicated as "B" and "F" respectively.</p

<i>HbCBF1</i> overexpression conferred cold resistance and relatively slow dehydration in <i>Arabidopsis</i>.

<p>(A) The electrolyte leakage rates of <i>col-0</i>, OE-3, OE-6 and OE-13 lines under nonacclimated (warm) or 5-days cold—acclimated conditions (cold). Electrolyte leakage was measured using leaves from two-week-old seedlings frozen to -7°C at a refrigerating rate of 1°C per h from -1°C. The star symbol (*) indicates significant difference between each individual OE line and wild type plants (WT). (B) Representative seedlings of <i>col-0</i>, OE-3, OE-6 and OE-13 lines recovered from 18 hours frozen at -6°C. The survival rates of each line were listed. (C) The dehydration rates of the detached leaves from <i>col-0</i>, OE-3, OE-6 and OE-13 seedlings. The star (*) symbol indicates significant difference between WT and each individual OE line, while plus symbols (+) shows significant difference between WT and individual OE-6 and OE-13 line. Data in A and C were presented as mean ± standard error of mean of five biological replicates. Significance was determined by Student’s <i>t</i> test at the probability levels of <i>P</i> < 0.05. Comparisons were made between the wild type plant and each individual overexpressing line. For B, totally thirty to fifty plants were tested for each line and about ten seedlings were grown in each pot.</p