Description: Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (〉200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3 , located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 ( Tnfaip3 ) gene, is an early-primary response gene controlled by nuclear factor-B (NF-B) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-B-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-B/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-B/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-B-induced lincRNA-Tnfaip3 to act as a coactivator of NF-B for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-B to modulate inflammatory gene transcription in mouse macrophages.

Description: This study was designed to evaluate and characterize the molecular basis of antitumor activity of naturally occurring resveratrol (RES; 3,5,4'-trihydroxy- trans -stilbene) derivatives. The compounds were isolated from plants in previous studies and characterized spectroscopically. The antitumor activities of 31 RES derivatives, including dimers, trimers, and tetramers of RES, were evaluated using cell-based assays and validated on a murine model. Several trimeric and a tetrameric stilbenoids induced tumor cell apoptosis or growth arrest of several tumor cell lines with IC 50 values (2.8–19.7 μM), significantly lower than that of RES (IC 50 〉70 μM). Using pauciflorol B (PauB) as an example, we showed that the compound induced apoptosis p53 dependently, inducing p53 accumulation and p53-modulated gene expression in cells with wild-type p53, but not in those with nonfunctional p53. Reexpression of p53 in p53-null cells rescued cell death response. In parallel, the MAPK/p38 was activated and critical for PauB-induced killing. Interestingly, activation of p38 in p53 deficient cells was sufficient to drive cells into senescence via the p16–pRb pathway. Finally, PauB dose-dependently inhibited tumor growth on nude mice. Naturally occurring trimeric and tetrameric stilbenoids are potent antitumor agents. Those compounds exert antitumor effect through p53-dependent induction of apoptosis or senescence.—Qiao, H., Chen, X., Xu, L., Wang, J., Zhao, G., Hou, Y., Ge, H. M., Tan, R-X., Li, E. Antitumor effects of naturally occurring oligomeric resveratrol derivatives.

Description: The purpose of this study was to develop a novel in vivo albumin-labeling method to allow PET of cardiac function after myocardial infarction and vascular leakage and increased permeability in inflammatory diseases and malignant tumors. Methods: To label albumin in vivo, we synthesized a NOTA (1,4,7-triazacyclononane- N,N',N'' -triacetic acid)-conjugated truncated form of Evans blue (NEB). 18 F labeling was achieved by the formation of an 18 F-aluminum fluoride ( 18 F-AlF) complex, and 64 Cu labeling was obtained by a standard chelation method. Sixty-minute dynamic PET imaging was performed on normal mice to evaluate the distribution of 18 F-AlF-NEB, which was compared with in vitro–labeled mouse serum albumin ( 18 F-fluorobenzyl-MSA). Electrocardiography-gated PET imaging was performed in a mouse model of myocardial infarction. Both dynamic and static PET scans were obtained in a mouse inflammation model induced by local injection of turpentine to evaluate vascular leakage. Tumor permeability was studied by dynamic and late-point static PET using 64 Cu-NEB in a UM-22B xenograft model. Results: NEB was successfully synthesized, and 18 F labeling including work-up took about 20–30 min, with a radiochemical purity greater than 95% without the need for high-performance liquid chromatography purification. Most of the radioactivity was retained in the circulation system at 60 min after injection (26.35 ± 1.52 percentage injected dose per gram [%ID/g]). With electrocardiography-gated PET, ventricles of the heart and major arteries were clearly visualized. The myocardial infarction mice showed much lower left ventricular ejection fraction than the control mice. Inflammatory muscles showed significantly higher tracer accumulation than the contralateral healthy ones. UM-22B tumor uptake of 64 Cu-NEB gradually increased with time (5.73 ± 1.11 %ID/g at 1 h and 8.03 ± 0.77 %ID/g at 2 h after injection). Conclusion: The distribution and local accumulation of serum albumin can be noninvasively visualized and quantified by 18 F-AlF-NEB and 64 Cu-NEB PET. The simple labeling and broad applications make these imaging probes attractive for clinical translation.

Description: Glucagonlike peptide (GLP-1) and its receptor (GLP-1R) exhibit cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in both animal studies and clinical trials. However, the kinetics of GLP-1R expression in the infarcted/ischemic myocardium has not yet been explored. The purpose of this study was to monitor the presence and time course of regional myocardial GLP-1R expression after MI/R with noninvasive PET. Methods: Male Sprague–Dawley rats underwent a 45-min transient left coronary artery occlusion, followed by reperfusion. The myocardial infarction was confirmed by electrocardiogram and cardiac ultrasound. In vivo PET was performed to determine myocardial uptake of 18 F-FBEM-Cys 40 -exendin-4 at different time points after reperfusion. The localization of 18 F-FBEM-Cys 40 -exendin-4 accumulation was determined by coregistering 18 F-FDG PET and CT images. Ex vivo autoradiography, GLP-1R immunohistochemical staining, and Western blot analysis were performed to confirm the PET results. Results: Myocardial origin and infarcted/ischemic area localization of 18 F-FBEM-Cys 40 -exendin-4 accumulation was confirmed by coregistration of small-animal CT and 18 F-FDG images. At 8 h after MI/R, tracer uptake in the infarcted/ischemic region was 0.37 ± 0.05 percentage injected dose per gram, significantly higher than that in the control group ( P 〈 0.01). The localized tracer uptake decreased, relative to the 8-h time point, but was still significantly higher than the control group on days 1 and 3 after MI/R. At 2 wk after MI/R, the tracer uptake in the affected area showed no significant difference, compared with that in the healthy myocardium. Autoradiography showed the same trend of 18 F-FBEM-Cys 40 -exendin-4 uptake in the myocardial infarcted/ischemic area. The specificity of tracer uptake into ischemic myocardium was supported by decreased tracer uptake after the rats were pretreated with an excess amount of unlabeled exendin-4. Immunohistochemical staining and Western blotting of GLP-1R protein of excised cardiac sections confirmed that the change in uptake observed by PET corresponded to a change in GLP-1R expression. Conclusion: Noninvasive PET using 18 F-FBEM-Cys 40 -exendin-4 revealed a dynamic pattern of GLP-1R upregulation in the infarcted/ischemic area after MI/R. The imaging results will deepen our understanding of the mechanism of the cardioprotective effect of GLP-1 and its analogs and potentially provide guidance for optimization of the time frame of therapeutic intervention.

Description: 18 F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide ( 18 F-AlF-NOTA-PRGD2, denoted as 18 F-alfatide) using a fluoride–aluminum complex that significantly simplified the labeling procedure. Methods: Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18 F-alfatide, and 1 tuberculosis patient was investigated using both 18 F-alfatide and 18 F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohistochemistry and staining intensity quantification were performed to confirm the expression of integrin α v β 3 . Results: Under the optimal conditions, the whole radiosynthesis including purification was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18 F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. Conclusion: 18 F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18 F-alfatide allows specific imaging of α v β 3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogenesis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.

Description: Isoflurane and propofol are known to depress cardiac contraction, but the molecular mechanisms involved are not known. In this study, we determined whether decreasing myofilament Ca 2+ responsiveness underlies anesthesia-induced depression of contraction and uncovered the molecular targets of isoflurane and propofol. Force and intracellular Ca 2+ ([Ca 2+ ] i ) were measured in rat trabeculae superfused with Krebs-Henseleit solution, with or without propofol or isoflurane. Photoaffinity labeling of myofilament proteins with meta-Azi-propofol (AziP m ) and Azi-isoflurane (Azi-iso) and molecular docking were also used. Both propofol and isoflurane dose dependently depressed force from low doses (propofol, 27 ± 6 μM; isoflurane, 1.0 ± 0.1%) to moderate doses (propofol, 87 ± 4 μM; isoflurane, 3.0 ± 0.25%), without significant alteration [Ca 2+ ] i . During steady-state activations in both intact and skinned preparations, propofol and isoflurane depressed maximum Ca 2+ -activated force and increased the [Ca 2+ ] i required for 50% of activation. Myofibrils photolabeled with AziP m and Azi-iso identified myosin, actin, and myosin light chain as targets of the anesthetics. Several adducted residues in those proteins were located in conformationally sensitive regions that underlie contractile function. Thus, propofol and isoflurane decrease force development by directly depressing myofilament Ca 2+ responsiveness and have binding sites in key regions for contraction in both actin and myosin.—Meng, T., Bu, W., Ren, X., Chen, X., Yu, J., Eckenhoff, R. G., Gao, W. D. Molecular mechanism of anesthetic-induced depression of myocardial contraction.

Description: Separation of germ cells from somatic cells is a widespread feature of animal sexual reproduction, with a core set of germ cell factors conserved among diverse animals. It is not known what controls their conserved gonad-specific expression. Core components of epigenetic machinery are ancient, but its role in conserved tissue expression regulation remains unexplored. We found that promoters of the reproductive genes BOULE and DAZL exhibit differential DNA methylation, consistent with their gonad-specific expression in humans and mice. Low or little promoter methylation from the testicular tissue is attributed to spermatogenic cells of various stages in the testis. Such differential DNA methylation is present in the orthologous promoters not only of other mammalian species, but also of chickens and fish, supporting a highly conserved epigenetic mechanism. Furthermore, hypermethylation of DAZL and BOULE promoters in human sperm is associated with human infertility. Our data strongly suggest that epigenetic regulation may underlie conserved germ-cell–specific expression, and such a mechanism may play an important role in human fertility.—Zhang, C., Xue, P., Gao, L., Chen, X., Lin, K., Yang, X., Dai, Y., Xu, E. Y. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

Description: A novel stress-inducible protein, Sestrin2 (Sesn2), declines in the heart with aging. AMPK has emerged as a pertinent stress-activated kinase that has been shown to have cardioprotective capabilities against myocardial ischemic injury. We identified the interaction between Sesn2 and AMPK in the ischemic heart. To determine whether ischemic AMPK activation—modulated by the Sesn2-AMPK complex in the heart—is impaired in aging that sensitizes the heart to ischemic insults, young C57BL/6 mice (age 3–4 mo), middle-aged mice (age 10–12 mo), and aged mice (age 24–26 mo) were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia. The ex vivo working heart system was used for measuring substrate metabolism. The protein level of Sesn2 in hearts was gradually decreased with aging. Of interest, ischemic AMPK activation was blunted in aged hearts compared with young hearts ( P 〈 0.05); the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in aged hearts during ischemia and reperfusion ( P 〈 0.05 vs. young hearts). Myocardial infarction size was larger in aged hearts ( P 〈 0.05 vs. young hearts). Immunoprecipitation with Sesn2 Ab revealed that cardiac Sesn2 forms a complex with AMPK and upstream liver kinase B1 (LKB1) during ischemia. Of interest, the binding affinity between Sesn2 and AMPK upstream LKB1 is impaired in aged hearts during ischemia ( P 〈 0.05 vs. young hearts). Furthermore, Sesn2-knockout hearts demonstrate a cardiac phenotype and response to ischemic stress that is similar to wild-type aged hearts ( i.e., impaired ischemic AMPK activation and higher sensitivity to ischemia- and reperfusion- induced injury). Adeno-associated virus–Sesn2 was delivered to aged hearts via a coronary delivery approach and significantly rescued the protein level of Sesn2 and the ischemic tolerance of aged hearts; therefore, Sesn2 is a scaffold protein that mediates AMPK activation in the ischemic myocardium via an interaction with AMPK upstream LKB1. Decreased Sesn2 levels in aging lead to a blunted ischemic AMPK activation, alterations in substrate metabolism, and an increased sensitivity to ischemic insults—Quan, N., Sun, W., Wang, L., Chen, X., Bogan, J. S., Zhou, X., Cates, C., Liu, Q., Zheng, Y., Li J. Sestrin2 prevents age-related intolerance to ischemia and reperfusion injury by modulating substrate metabolism.

Description: Exposure to microgravity leads to alterations in multiple systems, but microgravity-related changes in the gastrointestinal tract and its clinical significance have not been well studied. We used the hindlimb unloading (HU) mouse model to simulate a microgravity condition and investigated the changes in intestinal microbiota and colonic epithelial cells. Compared with ground-based controls (Ctrls), HU affected fecal microbiota composition with a profile that was characterized by the expansion of Firmicutes and decrease of Bacteroidetes. The colon epithelium of HU mice showed decreased goblet cell numbers, reduced epithelial cell turnover, and decreased expression of genes that are involved in defense and inflammatory responses. As a result, increased susceptibility to dextran sulfate sodium–induced epithelial injury was observed in HU mice. Cohousing of Ctrl mice with HU mice resulted in HU-like epithelial changes in Ctrl mice. Transplantation of feces from Ctrl to HU mice alleviated these epithelial changes in HU mice. Results indicate that HU changes intestinal microbiota, which leads to altered colonic epithelial cell homeostasis, impaired barrier function, and increased susceptibility to colitis. We further demonstrate that alteration in gastrointestinal motility may contribute to HU-associated dysbiosis. These animal results emphasize the necessity of evaluating astronauts’ intestinal homeostasis during distant space travel.—Shi, J., Wang, Y., He, J., Li, P., Jin, R., Wang, K., Xu, X., Hao, J., Zhang, Y., Liu, H., Chen, X., Wu, H., Ge, Q. Intestinal microbiota contributes to colonic epithelial changes in simulated microgravity mouse model.