Nondestructive and Quantitative Analysis of Cell Wall Regeneration in the Medicinal Macrofungus Ganoderma lingzhi by a Membrane-Fusing Fluorescent Probe.

Ganoderma is a prize medicinal macrofungus with a broad range of pharmaceutical values. To date, various attempts have been made to cultivate Ganoderma to improve the production of secondary metabolites with pharmacological activity. Among the adopted techniques, protoplast preparation and regeneration are indispensable. However, the evaluation of protoplasts and regenerated cell walls usually relies on electron microscopy assays, which require time-consuming and destructive sample preparation and merely provide localized information in the selected area. In contrast, fluorescence assays enable sensitive real-time detection and imaging in vivo. They can also be applied to flow cytometry, providing a collective overview of every cell in a sample. However, for macrofungi such as Ganoderma, the fluorescence analysis of protoplasts and regenerated cell walls is difficult owing to the hindrance of the homologous fluorescent protein expression and the lack of an appropriate fluorescence marker. Herein, a specific plasma membrane probe, TAMRA perfluorocarbon nucleic acid probe (TPFN), is proposed for the nondestructive and quantitative fluorescence analysis of cell wall regeneration. Exploiting the perfluorocarbon membrane-anchoring chains, hydrophilic nucleic acid linker, and fluorescent dye TAMRA, the probe is proven to be selective, soluble, and stable, enabling rapid fluorescence detection of a protoplast sample free of transgenic expression or immune staining. Based on the TPFN and flow cytometry techniques, a quantitative approach is constructed to monitor the process of cell wall growth in a fast, quantitative, and high-throughout manner, and the obtained results are consistent with those of conventional electron microscopy. In principle, with slight modifications or integration, the proposed probe and approach can be adapted to the preparation of cell protoplasts, inspection of cell wall integrity under environmental stress, and programmable membrane engineering for cytobiology and physiology research.

Detection of Incomplete Bladder Duplication by SPECT/CT.

Bladder duplication is an extremely rare congenital urinary tract malformation that is often discovered incidentally. Here, we report a case of incomplete bladder duplication diagnosed by SPECT/CT in a 65-y-old man with lung cancer. Compared with the results of whole-body planar bone scintigraphy, this SPECT/CT finding caused the diagnosis to be revised in this patient with suspected bone metastases. To our knowledge, this is the first documented case of incomplete bladder duplication discovered by SPECT/CT.

Structural Insight into the Mechanism of 4-Aminoquinolines Selectivity for the alpha2A-Adrenoceptor.

BACKGROUND: α2A-adrenoceptor (AR) is a potential target for the treatment of degenerative diseases of the central nervous system, and α2A-AR agonists are effective drugs for this condition. However, the lack of high selectivity for α2A-AR subtype of traditional drugs greatly limits their clinic usage. METHODS: A series of homobivalent 4-aminoquinolines conjugated by two 4-aminoquinoline moieties via varying alkane linker length (C2-C12) were characterized for their affinities for each α2-AR subtype. Subsequently, docking, molecular dynamics and mutagenesis were applied to uncover the molecular mechanism. RESULTS: Most 4-aminoquinolines (4-aminoquinoline monomer, C2-C6, C8-C10) were selective for α2A-AR over α2B- and α2C-ARs. Besides, the affinities are of similar linker length-dependence for each α2-AR subtype. Among all the compounds tested, C10 has the highest affinity for α2A-AR (pKi=-7.45±0.62), which is 12-fold and 60-fold selective over α2B-AR and α2C-AR, respectively. Docking and molecular dynamics suggest that C10 simultaneously interacts with orthosteric and "allosteric" sites of the α2A-AR. The mutation of F205 decreases the affinity by 2-fold. The potential allosteric residues include S90, N93, E94 and W99. CONCLUSION: The specificity of C10 for the α2A-AR and the potential orthosteric and allosteric binding sites proposed in this study provide valuable guidance for the development of novel α2A-AR subtype selective compounds.

Vanillin enhances the passive transport rate and absorption of drugs with moderate oral bioavailability in vitro and in vivo by affecting the membrane structure.

Vanillin is a popular flavoring agent in the food, tobacco, and perfume industries. In this paper, we investigated the effect of vanillin on the transport rates of drugs with different levels of permeability (acyclovir, hydrochlorothiazide, propranolol and carbamazepine) through a Caco-2 cell bidirectional transport experiment. We also explored the underlying mechanism using an in silico technique and fluorescence anisotropy measurements. The influence of vanillin on the pharmacokinetics of drugs whose transport rates were affected by vanillin in vitro was then studied in vivo. Results showed that vanillin (100 µM) increased the cumulative amount of passively transported drugs (2.1-fold of hydrochlorothiazide, 1.49-fold of propranolol, 1.35-fold of acyclovir, and 1.34-fold of carbamazepine) in vitro. Molecular dynamics simulations revealed that vanillin disordered the structure of the lipid bilayer and reduced the energy barrier of drugs across the center of the membrane. The anisotropy of TMA-DPH also decreased in Caco-2 cells after treatment with vanillin (25 and 100 µM) and indicated an increase in membrane fluidity, which was dose-dependent. An oral bioavailability study indicated that vanillin (100 mg kg-1) significantly enhanced the Cmax and AUC0-6 of hydrochlorothiazide by 1.42-fold and 1.28-fold, respectively, and slightly elevated the Cmax of propranolol. In conclusion, vanillin can significantly increase the absorption of drugs with moderate oral bioavailability in vitro and in vivo by loosening the membrane. Thus, the concurrent consumption of drugs with food containing vanillin may result in increased drug plasma concentration and pose potential health risks.

RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, especially in developing countries. Although the chronic infections of hepatitis B and C viruses have been established as the etiological factors of HCC, the mechanism for the tumorigenesis and development of HCC is still unclear. The liver-specific microRNA-122 (miR-122), an established tumor-suppressor miRNA, is often down-regulated in HCC, while the underlying mechanism is not well understood. Here we report that the AU-rich element-binding factor AUF1 suppresses the expression of Dicer1, the type III RNase that is required for microRNA maturation, leading to the inhibited biogenesis of miR-122. Overexpression of AUF1 led to the decreased expression of Dicer1 and miR-122, while the level of the miR-122 precursor (pre-miR-122) was increased. On the other hand, siRNA of AUF1 (siAUF1) increased the levels of Dicer1 mRNA and miR-122, but it reduced the abundance of pre-miR-122. Consistent with the reported data, this study demonstrated that AUF1 and Dicer1 showed opposite expression pattern in both human HCC tissues and cell lines. In addition, AUF1 inhibited the expression of Dicer1 by interacting with the 3' untranslated region (3'UTR) and coding region of DICER1 mRNA. Moreover, the knockdown of AUF1 by siRNA altered the expression of other miRNAs and promoted HCC cell death. In conclusion, AUF1 down-regulates the expression miR-122 by interacting with the 3'UTR and coding region of DICER1 mRNA and suppressing Dicer1 expression. The AUF1/Dicer1/miR-122 pathway might play a critical role in the development of HCC.

Astrocyte elevated gene-1 (AEG-1) shRNA sensitizes Huaier polysaccharide (HP)-induced anti-metastatic potency via inactivating downstream P13K/Akt pathway as well as augmenting cell-mediated immune response.

Astrocyte elevated gene-1 (AEG-1) is an important force in the development and progression of hepatocellular carcinoma (HCC). To extend our study, we examined here the role of AEG-1 in anti-metastatic effects of Huaier polysaccharide (HP) on the human HCC MHCC97-H cell line. AEG-1 shRNA contributed to the anti-proliferation effect of HP on MHCC97-H cells. Furthermore, results of Transwell insert chambers showed that low expression of AEG-1 could effectively facilitate HP to suppress MHCC97-H cell migration and invasion. We achieved this by reducing phosphoinositide 3-kinases (P13K) and phosphorylated Akt (pAkt) expression as well as enhancing natural killer (NK) cell activity. Taken together, our data strongly suggested that AEG-1 shRNA could block the carcinogenesis and progression of MHCC97-H cells and highlight the therapeutic potential of HP in HCC treatment, at least by part, by inhibiting the activation of the PI3K/Akt pathway and enhancing the NK cell-mediated immune response. These findings may provide a new strategy for HCC treatment.

Astrocyte elevated gene-1 is a novel biomarker of epithelial-mesenchymal transition and progression of hepatocellular carcinoma in two China regions.

Astrocyte elevated gene-1 (AEG-1) is involved in important biological processes including cell invasion, metastasis, and carcinogenesis. However, its clinical significance has remained largely unknown in hepatocellular carcinoma. Here, specimens from 144 patients with hepatocellular carcinomas in Beijing and Heilongjiang regions were investigated by immunohistochemical staining for AEG-1, vimentin, and E-cadherin expressions. A clinicopathological study revealed that AEG-1 expression level in tumor cells was significantly correlated with TNM stage (P = 0.001) and Edmonson grade (P < 0.0001). In addition, AEG-1, vimentin, and E-cadherin (epithelial-mesenchymal transition (EMT) biomarker) expressions were correlated with each other. These findings suggest that AEG-1 may be an epithelial-mesenchymal transition-associated biomarker in human hepatocellular carcinoma and play important roles in the progression of hepatocellular carcinoma. In addition, the AEG-1 gene is a potential target for elimination of hepatocellular carcinoma in the future.

AU-binding factor 1 expression was correlated with metadherin expression and progression of hepatocellular carcinoma.

RNA-binding factor 1 (AUF1) was found to be up-regulated in numerous tumors compared with untransformed tissues. Furthermore, it has been identified to regulate mRNAs en masse in hepatocellular carcinoma (HCC). Metadherin (MTDH) as a novel oncogene also promotes tumor progression and metastasis in HCC. Our study aimed to investigate the correlation between AUF1 and MTDH expressions by immunochemistry in 146 HCC patients from Heilongjiang region. AUF1 expression in HCC tumors was higher than that in the matched normal liver tissues. Particularly, AUF1 overexpression was closely associated with tumor size (P < 0.022), TNM stage (P < 0.003), hepatitis B surface antigen status, and AFP serum levels (P < 0.05). Furthermore, AUF1 overexpression led to poor outcome during 5-year follow-up (P < 0.001). Additionally, AUF1 and MTDH expressions were correlated with each other. Our findings suggest that the AUF1 gene may play an important role in HCC progression and be a novel biomarker in the future.

Huaier polysaccharides suppresses hepatocarcinoma MHCC97-H cell metastasis via inactivation of EMT and AEG-1 pathway.

We have recently reported that astrocyte elevated gene-1 (AEG-1) might be an epithelial-mesenchymal transition (EMT) associated biomarker in human hepatocellular carcinoma (HCC), and play an important role in the progression of hepatocellular carcinoma. To extend our study, we examined here the anti-invasive and metastatic effects of Huaier polysaccharide (HP) on human HCC cell line MHCC97-H and explored its possible mechanism of action. Treatment with HP dose-dependently inhibited the proliferation, adhesion, migration and invasion of MHCC97-H cells in vitro. This was achieved not only by reducing the expression of AEG-1 and N-cadherin, but also by enhancing E-cadherin expression. Therefore, these data suggested that HP can inhibit the growth and metastatic potential of MHCC97-H cells through modulation of the AEG-1/EMT pathway.