SLUG is activated by nuclear factor kappa B and confers human alveolar epithelial A549 cells resistance to tumor necrosis factor-alpha-induced apoptosis

BACKGROUND: The role of tumor necrosis factor alpha (TNF-α) in cancer is complex with both apoptotic and anti-apoptotic roles proposed. However the mechanism is not clear. In the study, we designed to investigate the effect of TNF-α on the activation and expression of nuclear factor kappa B (NF-κB)/p65/SLUG/PUMA/Bcl-2 levels in human lung cancer A549 cell line, and in conditions of TNF-α-induced apoptosis. METHODS: We have engineered three A549 cell lines that were transiently transfected with PUMA siRNA, SLUG siRNA and Bcl-2 siRNA, respectively. We have measured the in vitro effects of siRNA on apoptosis, and sensitivity to 20 ng/ml of TNF-α treatment for 24–48 h. RESULTS: We found the NF-κB activity and PUMA mRNA/protein was significantly increased after treatment of TNF-α for 24 h in untreated A549 cells, and led to a significant increase in TNF-α-induced apoptosis, no significant increase of SLUG and Bcl-2 level was shown. However, after treatment of TNF-α for 48 h in untreated A549 cells, SLUG and Bcl-2 level was significant increased, and PUMA level was significant decreased, and TNF-α-induced apoptosis was significantly decreased compared to the apoptosis level after treatment of TNF-α for 24 h. Inhibition of the NF-κB activity could effectively decrease the PUMA level and increase the SLUG and Bcl-2 level. PUMA silencing by siRNA led to a significant decrease in TNF-α-induced apoptosis after treatment of TNF-α for 24 h. Bcl-2 and SLUG silencing by siRNA led to a significant increase in TNF-α-induced apoptosis for 48 h. Furthermore, SLUG silencing increased PUMA level and decreased Bcl-2 level. CONCLUSIONS: The findings suggested that TNF-α treatment promoted apoptosis via the NF-κB-dependent PUMA pathway. The anti-apoptotic role of TNF-α was via NF-κB-dependent SLUG and Bcl-2 pathway at a later time

Comparison of [11C]TZ1964B and [18F]MNI659 for PET imaging brain PDE10A in nonhuman primates

Phosphodiesterase 10A (PDE10A) inhibitors show therapeutic effects for diseases with striatal pathology. PET radiotracers have been developed to quantify in vivo PDE10A levels and target engagement for therapeutic interventions. The aim of this study was to compare two potent and selective PDE10A radiotracers, [(11)C]TZ1964B and [(18)F]MNI659 in the nonhuman primate (NHP) brain. Double scans in the same cynomolgus monkey on the same day were performed after injection of [(11)C]TZ1964B and [(18)F]MNI659. Specific uptake was determined in two ways: nondisplaceable binding potential (BP(ND)) was calculated using cerebellum as the reference region and the PDE‐10A enriched striatum as the target region of interest (ROI); the area under the time–activity curve (AUC) for the striatum to cerebellum ratio was also calculated. High‐performance liquid chromatography (HPLC) analysis of solvent‐extracted NHP plasma identified the percentage of intact tracer versus radiolabeled metabolites samples post injection of each radiotracer. Both radiotracers showed high specific accumulation in NHP striatum. [(11)C]TZ1964B has higher striatal retention and lower specific striatal uptake than [(18)F]MNI659. The BP(ND) estimates of [(11)C]TZ1964B were 3.72 by Logan Reference model (LoganREF) and 4.39 by simplified reference tissue model (SRTM); the BP(ND) estimates for [(18)F]MNI659 were 5.08 (LoganREF) and 5.33 (SRTM). AUC ratios were 5.87 for [(11)C]TZ1964B and 7.60 for [(18)F]MNI659. Based on BP(ND) values in NHP striatum, coefficients of variation were ~10% for [(11)C]TZ1964B and ~30% for [(18)F]MNI659. Moreover, the metabolism study showed the percentage of parent compounds were ~70% for [(11)C]TZ1964B and ~50% for [(18)F]MNI659 60 min post injection. These data indicate that either [(11)C]TZ1964B or [(18)F]MNI659 could serve as suitable PDE10A PET radiotracers with distinguishing features for particular clinical application

Radiation dosimetry of [ 18 F]VAT in nonhuman primates

BACKGROUND: The objective of this study is to determine the radiation dosimetry of a novel radiotracer for vesicular acetylcholine transporter (−)-(1-((2R,3R)-8-(2-[(18)F]fluoro-ethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone ([(18)F]VAT) based on PET imaging in nonhuman primates. [(18)F]VAT has potential for investigation of neurological disorders including Alzheimer’s disease, Parkinson’s disease, and dystonia. METHODS: Three macaque fascicularis (two males, one female) received 185.4–198.3 MBq [(18)F]VAT prior to whole-body imaging in a MicroPET-F220 scanner. Time activity curves (TACs) were created from regions of interest (ROIs) that encompassed the entire small organs or samples with the highest activity within large organs. Organ residence times were calculated based on the TACs. We then used OLINDA/EXM 1.1 to calculate human radiation dose estimates based on scaled organ residence times. RESULTS: Measurements from directly sampled arterial blood yielded a residence time of 0.30 h in agreement with the residence time of 0.39 h calculated from a PET-generated time activity curve measured in the left ventricle. Organ dosimetry revealed the liver as the critical organ (51.1 and 65.4 μGy/MBq) and an effective dose of 16 and 19 μSv/MBq for male and female, respectively. CONCLUSIONS: The macaque biodistribution data showed high retention of [(18)F]VAT in the liver consistent with hepatobiliary clearance. These dosimetry data support that relatively safe doses of [(18)F]VAT can be administered to obtain imaging in humans

Lack of neuroinflammation in the HIV-1 transgenic rat: An [18 F]-DPA714 PET imaging study

BACKGROUND: HIV-associated neuroinflammation is believed to be a major contributing factor in the development of HIV-associated neurocognitive disorders (HAND). In this study, we used micropositron emission tomography (PET) imaging to quantify neuroinflammation in HIV-1 transgenic rat (Tg), a small animal model of HIV, known to develop neurological and behavioral problems. METHODS: Dynamic [(18)F]DPA-714 PET imaging was performed in Tg and age-matched wild-type (WT) rats in three age groups: 3-, 9-, and 16-month-old animals. As a positive control for neuroinflammation, we performed unilateral intrastriatal injection of quinolinic acid (QA) in a separate group of WT rats. To confirm our findings, we performed multiplex immunofluorescent staining for Iba1 and we measured cytokine/chemokine levels in brain lysates of Tg and WT rats at different ages. RESULTS: [(18)F]DPA-714 uptake in HIV-1 Tg rat brains was generally higher than in age-matched WT rats but this was not statistically significant in any age group. [(18)F]DPA-714 uptake in the QA-lesioned rats was significantly higher ipsilateral to the lesion compared to contralateral side indicating neuroinflammatory changes. Iba1 immunofluorescence showed no significant differences in microglial activation between the Tg and WT rats, while the QA-lesioned rats showed significant activation. Finally, cytokine/chemokine levels in brain lysates of the Tg rats and WT rats were not significantly different. CONCLUSION: Microglial activation might not be the primary mechanism for neuropathology in the HIV-1 Tg rats. Although [(18)F]DPA-714 is a good biomarker of neuroinflammation, it cannot be reliably used as an in vivo biomarker of neurodegeneration in the HIV-1 Tg rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-015-0390-9) contains supplementary material, which is available to authorized users

Avoiding Steric Congestion in Dendrimer Growth through Proportionate Branching: A Twist on da Vinci’s Rule of Tree Branching

Making defect-free macromolecules is a challenging issue in chemical synthesis. This challenge is especially pronounced in dendrimer synthesis where exponential growth quickly leads to steric congestion. To overcome this difficulty, proportionate branching in dendrimer growth is proposed. In proportionate branching, both the number and the length of branches increase exponentially but in opposite directions to mimic tree growth. The effectiveness of this strategy is demonstrated through the synthesis of a fluorocarbon dendron containing 243 chemically identical fluorine atoms with a MW of 9082 Da. Monodispersity is confirmed by nuclear magnetic resonance spectroscopy, mass spectrometry, and small-angle X-ray scattering. Growing different parts proportionately, as nature does, could be a general strategy to achieve defect-free synthesis of macromolecules

pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?

National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health; NIBIB; National Institute of Standards and TechnologyChemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pK(a) = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pK(a)s of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as similar to 10 in no-wash live-cell imaging. The dye 1-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye 1 make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators

Advanced Neuroimaging Approaches to Pediatric Brain Tumors

Central nervous system tumors are the most common pediatric solid tumors; they are also the most lethal. Unlike adults, childhood brain tumors are mostly primary in origin and differ in type, location and molecular signature. Tumor characteristics (incidence, location, and type) vary with age. Children present with a variety of symptoms, making early accurate diagnosis challenging. Neuroimaging is key in the initial diagnosis and monitoring of pediatric brain tumors. Conventional anatomic imaging approaches (computed tomography (CT) and magnetic resonance imaging (MRI)) are useful for tumor detection but have limited utility differentiating tumor types and grades. Advanced MRI techniques (diffusion-weighed imaging, diffusion tensor imaging, functional MRI, arterial spin labeling perfusion imaging, MR spectroscopy, and MR elastography) provide additional and improved structural and functional information. Combined with positron emission tomography (PET) and single-photon emission CT (SPECT), advanced techniques provide functional information on tumor metabolism and physiology through the use of radiotracer probes. Radiomics and radiogenomics offer promising insight into the prediction of tumor subtype, post-treatment response to treatment, and prognostication. In this paper, a brief review of pediatric brain cancers, by type, is provided with a comprehensive description of advanced imaging techniques including clinical applications that are currently utilized for the assessment and evaluation of pediatric brain tumors

pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?

Chemically synthesized near-infrared aza-BODIPY dyes displayed off–on fluorescence at acidic pH (p<i>K</i>_a = 6.2–6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent p<i>K</i>_as of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to “turned-on” fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as ∼10 in no-wash live-cell imaging. The dye <b>1</b>-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye <b>1</b> make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators