Autophagy is an evolutionarily conserved lysosomal self-digestion process involved in degradation of long-lived proteins and damaged organelles. In recent years, increasing evidence indicates that autophagy is associated with a number of pathological processes, including cancer. In this review, we focus on the recent studies of the evolutionarily conserved autophagy-related genes (ATGs) that are implicated in autophagosome formation and the pathways involved. We discuss several key autophagic mediators (eg, Beclin-1, UVRAG, Bcl-2, Class III and I PI3K, mTOR, and p53) that play pivotal roles in autophagic signaling networks in cancer. We discuss the Janus roles of autophagy in cancer and highlighted their relationship to tumor suppression and tumor progression. We also present some examples of targeting ATGs and several protein kinases as anticancer strategy, and discuss some autophagy-modulating agents as antitumor agents. A better understanding of the relationship between autophagy and cancer would ultimately allow us to harness autophagic pathways as new targets for drug discovery in cancer therapeutics.

Background

Circulating endothelial cells (CECs) are markers of vascular damage that have clinical relevance in many diseases, including acute myocardial infarction (AMI), and may be predictors of treatment responses. Herein, we investigated the diagnostic and prognostic value of CEC monitoring in AMI patients and a murine model.

Methodology/Principal Findings

CECs were defined as Hoechst 33342+/CD45−/CD31+/CD146+/CD133− in human blood samples and Hoechst 33342+/CD45−/CD31+/KDR+/CD117− in murine samples. To evaluate the validity and variability of our CEC detection system, peripheral blood samples of vascular endothelial growth factor-treated athymic nude mice and AMI patients were collected and subjected to intra-assay analysis. CEC detection by flow cytometry and real-time PCR were compared. Blood samples were obtained from 61 AMI patients, 45 healthy volunteers and 19 samples of the original AMI patients accepted one month treatment, via flow cytometry and expressed as a percentage of peripheral blood mononuclear cells.

Results

Our CEC detection method was validated and had limited variability. CEC concentrations were higher in AMI patients compared to healthy controls. One month post-treatment, CECs levels decreased significantly.

Conclusions/Significance

CEC levels may be useful as a diagnostic and prognostic biomarker in AMI patients.

Purpose

The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.

Methods

Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO4. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.

Results

FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO4, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.

Conclusions

AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.

Background

Nanoparticles are potentially used for early cancer detection, accurate diagnosis, and cancer treatment.

Results

In this paper, the breast cancer cells treated with gold colloidal suspension were carefully studied by surface-enhanced Raman scattering (SERS) spectra. Raman spectroscopy combining with high-resolution electron microscope is employed to investigate the interaction of gold nanoparticles (GNPs) with the intracellular components. The TEM images show that the GNPs are taken into the living cells and enveloped into some vesicles named ‘lick up vesicles’ in the cytosol.

Conclusions

The SERS spectra and SERS mapping of cells indicate that the major Raman bands are mostly assigned to the vibration characteristics of proteins, and the C-H in-plane bending mode of the substituted benzene in Phenylalanine is remarkably enhanced. Finally, the interaction mechanisms of the GNPs with the intracellular components are further discussed in detail.

Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress–responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

Author Summary

Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling arthropod susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. Although it was postulated that apoptosis (a genetically controlled form of cellular suicide) may play a very important role in insect innate immunity against viral infection, direct evidence has been lacking due to the lack of knowledge on the regulatory pathways responsible for the induction of apoptosis following viral infection. In this study, we found that there is a rapid induction of pro-apoptotic genes within 1–3 hours of exposure to virus. This rapid pro-apoptotic response was only observed in live animals but not in cultured cells. Genetic analysis indicated that animals lacking this rapid pro-apoptotic response were hypersensitive to viral infection. Thus our work provides unequivocal evidence indicating that rapid induction of apoptosis plays a very important role in mediating insect resistance to viral infection.

Recent studies have shown that multivariate pattern analysis (MVPA) can be useful for distinguishing brain disorders into categories. Such analyses can substantially enrich and facilitate clinical diagnoses. Using MPVA methods, whole brain functional networks, especially those derived using different frequency windows, can be applied to detect brain states. We constructed whole brain functional networks for groups of vascular dementia (VaD) patients and controls using resting state BOLD-fMRI (rsfMRI) data from three frequency bands - slow-5 (0.01∼0.027 Hz), slow-4 (0.027∼0.073 Hz), and whole-band (0.01∼0.073 Hz). Then we used the support vector machine (SVM), a type of MVPA classifier, to determine the patterns of functional connectivity. Our results showed that the brain functional networks derived from rsfMRI data (19 VaD patients and 20 controls) in these three frequency bands appear to reflect neurobiological changes in VaD patients. Such differences could be used to differentiate the brain states of VaD patients from those of healthy individuals. We also found that the functional connectivity patterns of the human brain in the three frequency bands differed, as did their ability to differentiate brain states. Specifically, the ability of the functional connectivity pattern to differentiate VaD brains from healthy ones was more efficient in the slow-5 (0.01∼0.027 Hz) band than in the other two frequency bands. Our findings suggest that the MVPA approach could be used to detect abnormalities in the functional connectivity of VaD patients in distinct frequency bands. Identifying such abnormalities may contribute to our understanding of the pathogenesis of VaD.

It is generally accepted that the de-differentiation of smooth muscle cells (SMCs) from contractile to proliferative/synthetic phenotype has an important role during vascular remodeling and diseases. Here we provide evidence that challenges this theory. We identify a new type of multipotent vascular stem cell (MVSC) in blood vessel wall. MVSCs express markers including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell (MSC)-like cells that subsequently differentiate into SMCs. On the other hand, we use lineage tracing with smooth muscle myosin heavy chain as a marker to show that MVSCs and proliferative or synthetic SMCs do not arise from the de-differentiation of mature SMCs. Upon vascular injuries, MVSCs, instead of SMCs, become proliferative, and MVSCs can differentiate into SMCs and chondrogenic cells, thus contributing to vascular remodeling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of MVSCs, rather than the de-differentiation of SMCs, contributes to vascular remodeling and diseases.

Astrocyte elevated gene-1 (AEG-1) and endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling have been demonstrated to be important in osteosarcoma (OS) progression. In the present study, we explored the interaction between AEG-1 and ET-1/ETAR signaling in OS cells, and investigated the mechanism(s) through which the functional interaction may impact OS cell invasion and chemoresistance. Overexpression and knockdown of AEG-1 were performed in Saos-2 and MG-63 OS cells, respectively. Overexpression of AEG-1 in Saos-2 cells significantly increased ET-1 expression (at both the mRNA and protein levels), cell invasion, MMP-2 expression and cell survival against cisplatin. These effects were eradicated using a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or a selective ETAR inhibitor, BQ123. Knockdown of AEG-1 in MG-63 cells significantly decreased ET-1 expression (at both the mRNA and protein levels), cell invasion, MMP-2 expression and cell survival against cisplatin. Exogenous ET-1 restored cell invasion and MMP-2 expression levels in MG-63 cells, in which AEG-1 had been knocked down, in the presence of LY294002, but not in the presence of BQ123. However, exogenous ET-1 only partially rescued cell survival against cisplatin-induced apoptosis in the presence of LY294002, in cells in which AEG-1 had been knocked down. In conclusion, we have demonstrated that AEG-1 regulates ET-1 expression at the transcriptional level in a PI3K-dependent manner in OS cells. Downstream of PI3K, ET-1/ETAR signaling primarily mediates the promoting effect of AEG-1 on OS cell invasion, likely through the upregulation of MMP-2 expression, thus, ET-1/ETAR signaling partially, but significantly, mediates the AEG-1-induced chemoresistance in OS cells. To the best of our knowledge, this study has provided the first evidence of a functional association between AEG-1 and ET-1/ETAR signaling in OS cells, which adds novel insights into the molecular mechanism of OS metastasis and chemoresistance.

Purpose. To determine whether administration of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) and whether placement of the outer volume saturation bands significantly affect shimming and water suppression on hepatic MR spectroscopic prescanning. Method. Region of interest (ROI) of 2 cm × 2 cm × 2 cm was carefully positioned in the region of the middle portion of the right hepatic lobe. 32 patients were examined before and after administration of Gd-DTPA with and without outer-volume saturation bands. Linewidths (Full-Width Half-Maximum (FWHM)) and water suppression were obtained. A paired t-test for comparison of means was used. Results. (1) The group with the outer volume saturation bands demonstrated slightly better water suppression effect than the group without outer volume saturation bands before administration. (2) The group with the outer volume saturation bands demonstrated better water suppression effect than the group without outer volume saturation bands after administration. (3) Both shimming and water suppression effectswere decreased on enhanced MR spectroscopic prescanning (all P < 0.05). Conclusions. Placement of the outer volume saturation bands is helpful to improve water suppression both before and after contrast agent administration. Gd-DTPA exerts a slightly adverse effect (a statistically significant but clinically unimportant) on magnetic resonance spectroscopic prescanning at 3T.

In the title compound, [Zn2(C8H4O6)(C12H8N2)2(H2O)6](C8H4O6), the complete ions of both the binuclear dication and the dianion are generated by crystallographic inversion symmetry. The Zn atom is bonded to an N,N′-bidentate phenanthroline ligand, three water moleules and an O-mono­denate 2,5-dihy­droxy­terephthalate dianion. In the resulting distorted octa­hedral ZnN2O4 coordination poly­hedron, the water O atoms are in a mer orientation. Two intra­molecular O—H⋯O hydrogen bonds occur in the bridging 2,5-dihy­droxy­terephthalate dianion within the complex cation and also in the free dianion. An intra­molecular Ow—H⋯O (w = water) hydrogen bond also occurs within the dication. In the crystal, O—H⋯O hydrogen bonds link the component ions into a three-dimensional network.

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis.

Progressive reduction in β-cell mass is responsible for the development of type 2 diabetes mellitus, and alteration in insulin receptor substrate 2 (IRS-2) abundance plays a critical role in this process. IRS-2 expression is stimulated by the transcription factor cAMP response element-binding protein (CREB) and we recently demonstrated that Ca2+/calmodulin dependent kinase 4 (CaMK4) is upstream of CREB activation in β-cells. This study investigated whether CaMK4 is also a potential target to increase β-cell mass through CREB-mediated IRS-2 expression, by quantifying mouse MIN6 β-cell proliferation and apoptosis following IRS-2 knockdown, CaMKs inhibition and alterations in CaMK4 and CREB expression. Expression of constitutively active CaMK4 (ΔCaMK4) and CREB (CREBDIEDLM) significantly stimulated β-cell proliferation and survival. In contrast, expression of their corresponding dominant negative forms (ΔK75ECaMK4 and CREBM1) and silencing of IRS-2 increased apoptosis and reduced β-cell division. Moreover, CREBDIEDLM and CREBM1 expression completely abolished the effects of ΔK75ECaMK4 and of ΔCaMK4, respectively. Our results indicate that CaMK4 regulates β-cell proliferation and apoptosis in a CREB-dependent manner and that CaMK4-induced IRS-2 expression is important in these processes.

Fat tissue is viewed as an active endocrine organ that secretes a variety of bioactive substances. Resistin, an adipocyte-secreted factor, is thought to be closely related to obesity, insulin resistance and inflammation, the three most significant risk factors for the progression of pancreatic cancer. However, the association between resistin and pancreatic cancer is still unknown. In this study, pancreatic tumor samples from 45 patients with pancreatic ductal adenocarcinoma were analyzed with immunohistochemistry for the expression of resistin. The correlation between resistin expression and clinicopathological features and prognosis were evaluated. Resistin staining was observed in 48.9% (22 of 45) of the cases. Resistin expression was more frequent in poorly differentiated tumors (9 of 9, 100%) compared to moderately differentiated tumors (11 of 28, 39.3%) and well-differentiated tumors (2 of 8, 25%) (p<0.01). The incidence of resistin expression in patients with Japan Pancreas Society stages III–IV (18 of 27, 66.7%) was significantly higher than in subjects with stages I–II (4 of 18, 22.2%) (p<0.01). Patients with resistin-stained tumors had significantly shorter relapse-free survival times (median, 9 months) than patients with negative tumors (median, 18 months) (p<0.05). In addition, multivariate analysis showed that resistin expression was an independent prognostic factor for relapse-free survival of patients with pancreatic ductal adenocarcinoma (p<0.05). These results demonstrate that resistin may influence the progression of pancreatic tumors and may be a useful predictor of relapse-free survival in patients with pancreatic ductal adenocarcinoma.

In this study, we synthesized monodispersed polystyrene (PS)-silica core-shell spheres with various shell thicknesses for the fabrication of photonic crystals. The shell thickness of the spheres was controlled by various additions of tetraethyl orthosilicate during the shell growth process. The shrinkage ratio of the inverse opal photonic crystals prepared from the core-shell spheres was significantly reduced from 14.7% to within 3%. We suspected that the improvement resulted from the confinement of silica shell to the contraction of PS space during calcination. Due to the shell effect, the inverse opals prepared from the core-shell spheres have higher filling fraction and larger wavelength of stop band maximum.

Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.

Objective

The Calcium Chloride (CaCl2) model is a widely accepted rodent model for abdominal aortic aneurysm (AAA). Calcium deposition, mainly consisting of calcium phosphate (CaPO4) crystals, has been reported to exist in both human and experimental aneurysms. CaPO4 crystal has been utilized for in vitro DNA transfection by mixing CaCl2 and Phosphate Buffered Saline (PBS). Here, we describe accelerated aneurysm formation resulting from a modification of the CaCl2 model.

Methods

The modified CaCl2, the CaPO4 model, was created by applying PBS onto the mouse infrarenal aorta after CaCl2 treatment. Morphological, histological and immunohistochemical analyses were performed on arteries treated with both the CaPO4 model and the conventional CaCl2 model as control. In vitro methods were carried out using a mixture of CaCl2 and PBS to create CaPO4 crystals. CaPO4 induced apoptosis of primary cultured mouse vascular smooth muscle cells (VSMCs) was measured by DNA fragmentation ELISA.

Results

First, we showed that the CaPO4 model produces AAA, defined as an increase of 50% or greater in the diameter of the aorta; faster than in the CaCl2 model. CaPO4 model showed significantly larger aneurysmal dilation at 7, 28, and 42 days as reflected by a maximum diameter fold change (measured in mm) of 1.69 ± 0.07, 1.99 ± 0.14 and 2.13 ± 0.09 as opposed to 1.22 ± 0.04, 1.48 ± 0.07 and 1.68±0.06 as seen in CaCl2 model, respectively (n=6; P<0.05). A semi-quantitative grading analysis of elastin fiber integrity at 7 days revealed a significant increase in elastin degradation in the CaPO4 model as compared to CaCl2 model (2.7±0.2 vs 1.5±0.2, p<0.05, n=6). Significantly higher level of apoptosis occurred in the CaPO4 model (apoptosis index at 1, 2, and 3 days post-surgery: 0.26 ± 0.14, 0.37± 0.14, and 0.33 ± 0.08 for CaPO4 model and 0.012 ± 0.10, 0.15± 0.02, and 0.12 ± 0.05 for conventional CaCl2 model) (n=3; p<0.05). An enhancement of macrophage infiltration and calcification was also observed at 3 and 7 days in CaPO4. CaPO4 induced approximately 3.7 times more apoptosis in VSMCs when compared to a mixture of CaCl2 (n=4; p<0.0001) in vitro.

Conclusion

Our data shows that the CaPO4 model accelerates aneurysm formation with the enhancement of apoptosis, macrophage infiltration and calcium deposition. This modified model, with its rapid and robust dilation, can be utilized as a new model for AAA.

Introduction

We have previously demonstrated that TGF-β in the presence of elevated levels of its primary signaling protein, Smad3, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. Moreover, we have shown that the mechanism in part, is through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluate the role of the ERK mitogen-activated protein kinase (ERK MAPK) in TGF-β-induced VSMC proliferation.

Methods

Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with ansiRNA. For in vivo experiments, Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK and Proliferating Cell Nuclear Antigen (PCNA).

Results

In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time and concentration-dependent manner. Overexpression of the signaling protein, Smad3 enhanced TGF-β-induced activation of ERK MAPK whereas inhibition of Smad3 with ansiRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by PCNA.

Conclusion

Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that through a Smad3/ERK MAPK signaling pathway, TGF-β enhances VSMC proliferation. These findings explain at least in part, the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Both gene knockout and chemical inhibition show that PKCδ is critical for efficient secretion of type I collagen by arterial smooth muscle cells. The data suggest that PKCδ regulates trafficking of collagen I by controlling its exit from the trans-Golgi network through a mechanism involving Cdc42.

Collagen type I is the most abundant component of extracellular matrix in the arterial wall. Mice knocked out for the protein kinase C δ gene (PKCδ KO) show a marked reduction of collagen I in the arterial wall. The lack of PKCδ diminished the ability of arterial smooth muscle cells (SMCs) to secrete collagen I without significantly altering the intracellular collagen content. Moreover, the unsecreted collagen I molecules accumulate in large perinuclear puncta. These perinuclear structures colocalize with the trans-Golgi network (TGN) marker TGN38 and to a lesser degree with cis-Golgi marker (GM130) but not with early endosomal marker (EEA1). Associated with diminished collagen I secretion, PKCδ KO SMCs exhibit a significant reduction in levels of cell division cycle 42 (Cdc42) protein and mRNA. Restoring PKCδ expression partially rescues Cdc42 expression and collagen I secretion in PKCδ KO SMCs. Inhibition of Cdc42 expression or activity with small interfering RNA or secramine A in PKCδ WT SMCs eliminates collagen I secretion. Conversely, restoring Cdc42 expression in PKCδ KO SMCs enables collagen I secretion. Taken together, our data demonstrate that PKCδ mediates collagen I secretion from SMCs, likely through a Cdc42-dependent mechanism.

The asymmetric unit of the title structure, C28H19NO·0.5C3H6O, comprises one 2-(anthracen-9-yl)-10-meth­oxy­benzo[h]­quinoline mol­ecule and an acteone mol­ecule with an occupany of 0.5. The solvent mol­ecule is disordered around a centre of symmetry. Its occupancy was determined from NMR data and kept fixed during the refinement. The two conjugated ring systems of the mol­ecule are almost perpendicular to each other; the inter­planar angle between the anthracene and quinoline ring systems is 84.9 (2)°.

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K+- and Mg2+-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca2+ and Mg2+, and the current increase could be inhibited by Ni2+ or Ba2+. The TRPM7-like current was potentiated by acidic pH and inhibited by La3+ and 2-aminoethoxydiphenyl borate. In addition, Ca2+-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca2+ ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.

Spontaneous spinal epidural hematoma (SSEH) during pregnancy is rare and may result in permanent damage if not promptly treated. There were few studies discussing the etiology, presentation and treatment of SSEH during pregnancy. The authors describe a case of spontaneous cervical epidural hematoma during pregnancy, which was diagnosed by magnetic resonance imaging (MRI) and managed with surgical evacuation. A retrospective review of a case of spontaneous epidural hematoma of spine during pregnancy was performed. The clinical features, diagnoses, treatments and outcomes of all cases were analyzed. Precise diagnosis without delay and rapid surgical treatment are essential for the management of SSEH during pregnancy.

Aims

High shear stress (HSS) can have significant impact on angiogenesis and atherosclerosis in collateral arteries near the bifurcation and curvature regions. Here, we investigate the spatiotemporal pattern of HSS-induced intracellular calcium alteration.

Methods and results

Genetically encoded biosensors based on fluorescence resonance energy transfer were targeted in the cytoplasm and the endoplasmic reticulum (ER) to visualize the subcellular calcium dynamics in bovine aortic endothelial cells under HSS (65 dyn/cm2). Upon HSS application, the intracellular Ca2+ concentration ([Ca2+]i) increased immediately and maintained a sustained high level, while the ER-stored calcium had a significant decrease only after 300 s. The perturbation of calcium influx across the plasma membrane (PM) by the removal of extracellular calcium or the blockage of membrane channels inhibited the early phase of [Ca2+]i increase upon HSS application, which was further shown to be sensitive to the magnitudes of shear stress and the integrity of cytoskeletal support. In contrast, Src, phospholipase C(PLC), and the inositol 1,4,5-trisphosphate receptor (IP3R) can regulate the late phase of HSS-induced [Ca2+]i increase via the promotion of the ER calcium efflux.

Conclusion

The HSS-induced [Ca2+]i increase consists of two well-co-ordinated phases with different sources and mechanisms: (i) an early phase due to the calcium influx across the PM which is dependent on the mechanical impact and cytoskeletal support and (ii) a late phase originated from the ER-calcium efflux which is regulated by the Src, PLC, and IP3R signalling pathway. Therefore, our work presented new molecular-level insights into systematic understanding of mechanotransduction in cardiovascular systems.

Background

Acupuncture in humans can produce clinical effects via the central nervous system. However, the neural substrates of acupuncture’s effects remain largely unknown.

Results

We utilized functional MRI to investigate the topological efficiency of brain functional networks in eighteen healthy young adults who were scanned before and after acupuncture at the ST36 acupoints (ACUP) and its sham point (SHAM). Whole-brain functional networks were constructed by thresholding temporal correlations matrices of ninety brain regions, followed by a graph theory-based analysis. We showed that brain functional networks exhibited small-world attributes (high local and global efficiency) regardless of the order of acupuncture and stimulus points, a finding compatible with previous studies of brain functional networks. Furthermore, the brain networks had increased local efficiency after ACUP stimulation but there were no significant differences after SHAM, indicating a specificity of acupuncture point in coordinating local information flow over the whole brain. Moreover, significant (P<0.05, corrected by false discovery rate approach) effects of only acupuncture point were detected on nodal degree of the left hippocampus (higher nodal degree at ACUP as compared to SHAM). Using an uncorrected P<0.05, point-related effects were also observed in the anterior cingulate cortex, frontal and occipital regions while stimulation-related effects in various brain regions of frontal, parietal and occipital cortex regions. In addition, we found that several limbic and subcortical brain regions exhibited point- and stimulation-related alterations in their regional homogeneity (P<0.05, uncorrected).

Conclusions

Our results suggest that acupuncture modulates topological organization of whole-brain functional brain networks and the modulation has point specificity. These findings provide new insights into neuronal mechanism of acupuncture from the perspective of functional integration. Further studies would be interesting to apply network analysis approaches to study the effects of acupuncture treatments on brain disorders.

The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

Background

Obesity has reached epidemic proportions, affecting more than one tenth of the world’s population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.

Methodology/Principal Findings

Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a Kd value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.

Conclusions/Significance

Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes.