ABCC6 is a basolateral plasma membrane protein

RATIONALE:: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. OBJECTIVE:: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. METHODS AND RESULTS:: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. CONCLUSIONS:: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation. © 2013 American Heart Association, Inc

Radiation-Induced Galectin-1 by Endothelial Cells: A Promising Molecular Target for Preferential Drug Delivery to the Tumor Vasculature

The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature-targeting ligand, anginex, to improve the therapeutic ratio for strategies developed against solid tumors. Our findings indicate that galectin-1 is (a) one of the major receptors for anginex (b) overexpressed by tumor neovasculature and (c) further specifically upregulated in endothelial cells in response to radiation exposure as low as 0.5 Gy. An investigation of [18]-F-labeled anginex biodistribution in SCK tumors indicates that anginex is an effective targeting molecule for image and radiation-guided therapy of solid tumors. An anginex-conjugated liposome capable of being loaded with drug was shown to selectively target endothelial cells post-radiation. The presence of endothelial cells in a three-dimensional co-culture system with tumor cells developed to study tumor/endothelial cell interactions in vitro led to higher levels of galectin-1 and showed a further increase in expression upon radiation exposure when compared to tumor cell spheroids alone. Similar increase in galectin-1 was observed in tumor tissue originating from the tumor‐endothelial cell spheroids in vivo and radiation exposure further induced galectin-1 in these tumors. The overall results suggest feasibility of using a clinical or subclinical radiation dose to increase expression of the galectin-1 receptor on the tumor microvasculature to promote delivery of therapeutics via the anginex peptide. This approach may reduce systemic toxicity, overcome drug resistance, and improve the therapeutic efficacy of conventional chemo/radiation strategies

FILIPINO TIKTOK INFLUENCERS AND PURCHASING BEHAVIOR OF YOUNG PROFESSIONALS

The traditional use of conventional media by businesses for audience targeting has shifted with the rise of influencer marketing, notably on platforms like TikTok, posing challenges in content adaptation and technological adaptation. Albert Bandura's Social Cognitive Theory examines factors shaping purchasing behavior, particularly relevant for young professionals. A quantitative correlational study focused on young professionals engaging with TikTok and influenced by Filipino TikTok creators, revealing education level as a key determinant of purchasing behavior. Extended TikTok engagement positively correlates with increased purchasing likelihood. Filipino TikTok influencers significantly impact young professionals' purchasing decisions, emphasizing the need for tailored marketing strategies targeting this demographic. Recommendations were provided for effective influencer and digital marketing strategies targeting young professionals

Regulation of Nuclear Receptor Nur77 by miR-124

<div><p>The nuclear receptor Nur77 is commonly upregulated in adult cancers and has oncogenic functions. Nur77 is an immediate-early response gene that acts as a transcription factor to promote proliferation and protect cells from apoptosis. Conversely, Nur77 can translocate to the mitochondria and induce apoptosis upon treatment with various cytotoxic agents. Because Nur77 is upregulated in cancer and may have a role in cancer progression, it is of interest to understand the mechanism controlling its expression. MicroRNAs (miRNAs) are responsible for inhibiting translation of their target genes by binding to the 3ʹUTR and either degrading the mRNA or preventing it from being translated into protein, thereby making these non-coding endogenous RNAs vital regulators of every cellular process. Several miRNAs have been predicted to target Nur77; however, strong evidence showing the regulation of Nur77 by any miRNA is lacking. In this study, we used a luciferase reporter assay containing the 3ʹUTR of Nur77 to screen 296 miRNAs and found that miR-124, which is the most abundant miRNA in the brain and has a role in promoting neuronal differentiation, caused the greatest reduction in luciferase activity. Interestingly, we discovered an inverse relationship in Daoy medulloblastoma cells and undifferentiated granule neuron precursors in which Nur77 is upregulated and miR-124 is downregulated. Exogenous expression to further elevate Nur77 levels in Daoy cells increased proliferation and viability, but knocking down Nur77 via siRNA resulted in the opposite phenotype. Importantly, exogenous expression of miR-124 reduced Nur77 expression, cell viability, proliferation, and tumor spheroid size in 3D culture. In all, we have discovered miR-124 to be downregulated in instances of medulloblastoma in which Nur77 is upregulated, resulting in a proliferative state that abets cancer progression. This study provides evidence for increasing miR-124 expression as a potential therapy for cancers with elevated levels of Nur77.</p></div

Overview of Nur77 regulation by miR-124.

<p>Nur77 can be directly targeted by miR-124, as revealed by our studies reported here (indicated by the red line), or indirectly affected by miR-124 via Sp1. Nur77 may act through several downstream target genes to promote cell proliferation and survival.</p

Nur77 knockdown decreases cell viability and proliferation.

<p>(A) Daoy cells were transfected with 20 nM siNur77 or non-targeting control (NT), and cell viability was measured via the CellTiter-Glo assay every day for 3 days. Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day; *<i>p</i> < 0.0001. (B) Cells were stained with crystal violet every day for 3 days to measure proliferation over time. The absorbance was measured and normalized to that of Day 0 (0 hours). The statistical significance was calculated for each day; *<i>p</i> < 0.01. (C) Proliferation was monitored via the IncuCyte live-cell imager. Cell confluence was averaged, with 4 replicates of each condition; *<i>p</i> < 0.0001. (D) Nur77 mRNA was significantly (<i>p</i> < 0.0001) decreased after transfecting Daoy cells with siNur77. (E) Images shown for each NT and siNur77 panel over 5 days are the same image view within the same well and are representative of 3 independent experiments with 4 wells for each condition. These images correspond to the data in C. Data shown in A are representative of 5 independent experiments; data in B are representative of 4 independent experiments, and data in C and E are representative of 2 independent experiments. Data shown in D is the average of 4 independent experiments.siNur77, SMARTpool siNur77 (Catalog # M-003426-04) from GE Healthcare.</p

Long-Term Exposure and Safety of a Novel Topical Rapamycin Cream for the Treatment of Facial Angiofibromas in Tuberous Sclerosis Complex: Results From a Single-Center, Open-Label Trial

Facial angiofibromas, composed of fibrous tissue and blood vessels appearing on the face, are closely associated with tuberous sclerosis complex. Historically, oral rapamycin, a mammalian target of the rapamycin inhibitor of cell proliferation, has been used to treat visceral tuberous sclerosis–related tumors; however, the side effect profile of this medicine generally precludes its use in patients lacking significant internal involvement. The authors developed a novel topical formulation of rapamycin cream to treat the facial angiofibroma without exposing patients to possible systemic side effects. We followed 11 patients in a long-term, open-label, prospective study to evaluate the safety and effectiveness of rapamycin cream when used chronically. All of the patients showed an improvement in the appearance of their facial angiofibroma which was maintained in long-term follow-up without safety concerns or systemic absorption. The novel rapamycin cream was tolerated well by all patients and represents a way to address the cutaneous manifestation of tuberous sclerosis complex

Nur77 promotes cell viability and proliferation.

<p>(A) Daoy cells were transduced with pSIN-Nur77 (Nur77) or pSIN vector (EV), and cell viability was measured via the CellTiter-Glo assay every day for 4 days. Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day. (B) Cells were stained with crystal violet every day for 4 days to measure proliferation over time. The absorbance was measured and normalized to that of Day 0 (0 hours). The statistical significance was calculated for each day. (C) Cell proliferation was monitored by using an IncuCyte live-cell imager for real-time imaging. The resulting cell confluence was recorded every 12 hours for 4 days. (D) Nur77 mRNA level was measured after transduction with Nur77. All experiments were performed by using Daoy cells transduced with EV or Nur77 lentivirus. All data shown are representative of 3 independent experiments; *<i>p</i>≤ 0.0001.</p

miR-124 decreases cell proliferation in 2D and 3D cultures.

<p>(A) Expression of miR-124 was significantly (<i>p</i> < 0.0001) increased after antibiotic selection of Daoy cells transduced with pEZX-MR03-miR-124. As a result, Nur77 mRNA levels were significantly decreased (<i>p</i> < 0.0001). Data shown are the average of 6 independent experiments. (B) The CellTiter-Glo assay was used to analyze the cell viability of Daoy cells stably expressing exogenous miR-124 or vector control (MR03). Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day; *<i>p</i> < 0.0001. (C) Stable cells were imaged by using the IncuCyte live-cell imager to determine cell proliferation over the course of 3.5 days, and statistical significance was determined for each day; *<i>p</i> < 0.0001. (D) Parental Daoy cells (Daoy) and Daoy cells stably expressing exogenous miR-124 (miR-124) or its control vector (MR03) were seeded at 3 densities (288, 800, and 2500 cells/well) and grown using 3D culture techniques. After 23 days (left panel), the cells’ spheroid areas (*<i>p</i> < 0.01) were measured by using the IN Cell Analyzer (middle panel). Viability (*<i>p</i> < 0.05) was determined by performing CellTiter-Glo 3D Cell Viability Assays and is shown as raw luminescence units (RLU) (right panel). The spheroid area data shown are for cells seeded at an initial density of 800 cells per well. Data from B are representative of 5 independent experiments; data from C are representative of 4 independent experiments, and data from D are representative of 2 independent experiments.</p