Three dimensional drift control at nano-scale in single molecule localization microscopy.

Super-resolution imaging based on single molecule localization of cellular structures on nanometer scale requires to record a series of wide-field or TIRF images resulting in a considerable recording time (typically of minutes). Therefore, sample drift becomes a critical problem and will lower the imaging precision. Herein we utilized morphological features of the specimen (mammalian cells) itself as reference markers replacing the traditionally used markers (e.g., artificial fiduciary markers, fluorescent beads, or metal nanoparticles) for sample drift compensation. We achieved sub-nanometer localization precision <1.0 nm in lateral direction and <6.0 nm in axial direction, which is well comparable with the precision achieved with the established methods using artificial position markers added to the specimen. Our method does not require complex hardware setup, extra labelling or markers, and has the additional advantage of the absence of photobleaching, which caused precision decrease during the course of super-resolution measurement. The achieved improvement of quality and resolution in reconstructed super-resolution images by application of our drift-correction method is demonstrated by single molecule localization-based super-resolution imaging of F-actin in fixed A549 cells.

miR-18a-5p Promotes Proliferation and Migration of Vascular Smooth Muscle Cells by Activating the AKT/Extracellular Regulated Protein Kinases (ERK) Signaling Pathway.

BACKGROUND microRNAs (miRNAs) play important roles in abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), which lead to restenosis in coronary artery disease. Nevertheless, the role of miR-18a-5p and how it works in VSMCs remain unknown. MATERIAL AND METHODS miR-18a-5p expression was determined by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR) analysis of tissues from 20 patients with stent restenosis, and rats with carotid artery injury, as well as VSMCs. A cell viability assay was used to measure cell proliferation. Cell migration abilities were assessed by transwell migration assay and wound healing assays. To identify miR-18a-5p targets, a dual-luciferase reporter assay was performed. Western blot analysis and immunofluorescence techniques were used to assess the protein expression levels of AKT and ERK. The rescue effects of miR-18a-5p on the proliferation or migration of VSMCs were evaluated after exposure to the AKT inhibitor MK-2206 and ERK inhibitor PD98059. RESULTS The expression level of miR-18a-5p was significantly higher in the blood serum of patients with stent restenosis and in rats with carotid artery injury, and the expression of AKT and ERK was higher after carotid artery injury. The proliferation and migration abilities of VSMCs were accelerated by the overexpression of miR-18a-5p. It was found that miR-18a-5p directly modulates AKT/ERK signaling. Upregulated miR-18a-5p increased the protein expression levels of AKT and ERK and we found a positive correlation between miR-18a-5p expression level and expression of AKT and ERK. Additionally, the promoting effect of miR-18a-5p on VSMCs proliferation, migration, and invasion was reversed by ERK inhibitor or AKT inhibitor. CONCLUSIONS miR-18a-5p can promote proliferation of VSMCs by activating the AKT/ERK signaling pathway.

Inhibition of G9a by a small molecule inhibitor, UNC0642, induces apoptosis of human bladder cancer cells.

Urinary bladder cancer (UBC) is characterized by frequent recurrence and metastasis despite the standard chemotherapy with gemcitabine and cisplatin combination. Histone modifiers are often dysregulated in cancer development, thus they can serve as an excellent drug targets for cancer therapy. Here, we investigated whether G9a, one of the histone H3 methyltransferases, was associated with UBC development. We first analyzed clinical data from public databases and found that G9a was significantly overexpressed in UBC patients. The TCGA Provisional dataset showed that the average expression level of G9a in primary UBC samples (n = 408) was 1.6-fold as much as that in normal bladder samples (n = 19; P < 0.001). Then we used small interfering RNA to knockdown G9a in human UBC T24 and J82 cell lines in vitro, and observed that the cell viability was significantly decreased and cell apoptosis induced. Next, we choosed UNC0642, a small molecule inhibitor targeting G9a, with low cytotoxicity, and excellent in vivo pharmacokinetic properties, to test its anticancer effects against UBC cells in vitro and in vivo. Treatment with UNC0642 dose-dependently decreased the viability of T24, J82, and 5637 cells with the IC50 values of 9.85 ± 0.41, 13.15 ± 1.72, and 9.57 ± 0.37 µM, respectively. Furthermore, treatment with UNC0642 (1-20 µM) dose-dependently decreased the levels of histone H3K9me2, the downstream target of G9a, and increased apoptosis in T24 and J82 cells. In nude mice bearing J82 engrafts, administration of UNC0642 (5 mg/kg, every other day, i.p., for 6 times) exerted significant suppressive effect on tumor growth without loss of mouse body weight. Moreover, administration of UNC0642 significantly reduced Ki67 expression and increased the level of cleaved Caspase 3 and BIM protein in J82 xenografts evidenced by immunohistochemistry and western blot analysis, respectively. Taken together, our data demonstrated that G9a may be a promising therapeutic target for UBC, and an epigenetics-based therapy by UNC0642 is suggested.

Design, synthesis and biological evaluation of novel benzo-α-pyrone containing piperazine derivatives as potential BRAFV600E inhibitors.

The increasingly acquired resistance to vemurafenib and side effects of known inhibitors motivate the search for new and more effective anti-melanoma drugs. In this Letter, virtual screening and scaffold growth were combined together to achieve new molecules as BRAFV600E inhibitors. Along with docking simulation, a primary screen in vitro was performed to filter the modifications for the lead compound, which was then substituted, synthesized and evaluated for their inhibitory activity against BRAFV600E and several melanoma cell lines. Out of the obtained compounds, derivative 3l was identified as a potent BRAFV600E inhibitor and exerted an anticancer effect through BRAFV600E inhibition. The following biological evaluation assays confirmed that 3l could induce cell apoptosis and marked DNA fragmentation. Furthermore, 3l could arrest the cell cycle at the G0/G1 phase in melanoma cells. The docking simulation displayed that 3l could tightly bind with the crystal structure of BRAFV600E at the active site. Overall, the biological profile of 3l suggests that this compound may be developed as a potential anticancer agent.

Discovery of Chromeno[4,3-c]pyrazol-4(2H)-one Containing Carbonyl or Oxime Derivatives as Potential, Selective Inhibitors PI3Kα.

A series of novel chromeno[4,3-c]pyrazol-4(2H)-one containing carbonyl or oxime derivatives (4a-n, 5a-n) have been synthesized and evaluated their biological activities as phosphatidyl inositol 3-kinase (PI3K) inhibitors. Out of them, compound 5l showed the most potent antiproliferative activities against HCT-116 with IC50 of 0.10 µM in vitro, and exhibited the most potent activity for PI3Kα with the value of 0.012 µM. Docking simulation of 5l into PI3Kα active site were performed to determine the probable binding model, and it indicated that compound 5l could be optimized as a potential inhibitor of PI3Kα in the further study.

Vaspin contributes to autophagy and endothelial-to-mesenchymal transition via PI3K-/AKT-mTOR pathway.

BACKGROUND: Visceral adipose tissue-derived serine protease inhibitor (Vaspin) was found to have anti-inflammatory, anti-apoptosis, and pro-autophagy activities. Our investigation is aimed to ascertain the effect of Vaspin on hypoxia-evoked endothelial-mesenchymal transition (EndMT) in human cardiac microvascular endothelial cells (HCMECs). METHODS: In vitro assays including CCK8, TUNEL, western blots, RT-qPCR to assess the effect of Vaspin on hypoxia-induced cell injuries, endothelial-mesenchymal transition (EndMT) and the inflammatory state in HCMECs. Transmission electron microscopy (TEM) was used to monitor autophagosome formation in HCMECs. The autophagy related proteins coupled with the critical effectors of PI3K/AKT-mTOR signaling pathway were also detected by western blots. In vivo assays including HE and ELISA to assess the effects of Vaspin on myocardial fibrosis pathology and type I and type III collagen in rats. RESULTS: Vaspin pretreatment dramatically dose-dependently restored the proliferative impairment and the induced EndMT in HCMECs by hypoxia. The Vaspin-pretreated HCMECs also presented with attenuated expression of increased IL-1ß, TNF-α and IL-6 by hypoxia a dose-dependent manner. Vaspin alleviated rat MF. The impact of Vaspin is also related to the increased autophagy and activated PI3K/AKT-mTOR signaling pathway. The protective and pro-autophagy activity of Vaspin was antagonized by the PI3K/AKT-mTOR inhibitor LY294002. CONCLUSION: Vaspin ameliorated the hypoxia-stimulated cell injuries and EndMT by activating autophagy via PI3K/AKT-mTOR signaling pathway.

Nano-Structural Effects on Gene Transfection: Large, Botryoid-Shaped Nanoparticles Enhance DNA Delivery via Macropinocytosis and Effective Dissociation.

Effective delivery is the primary barrier against the clinical translation of gene therapy. Yet there remains too much unknown in the gene delivery mechanisms, even for the most investigated polymeric carrier (i.e., PEI). As a consequence, the conflicting results have been often seen in the literature due to the large variability in the experimental conditions and operations. Therefore, some key parameters should be identified and thus strictly controlled in the formulation process. Methods: The effect of the formulation processing parameters (e.g., concentration or mixture volume) and the resulting nanostructure properties on gene transfection have been rarely investigated. Two types of the PEI/DNA nanoparticles (NPs) were prepared in the same manner with the same dose but at different concentrations. The microstructure of the NPs and the transfection mechanisms were investigated through various microscopic methods. The therapeutic efficacy of the NPs was demonstrated in the cervical subcutaneous xenograft and peritoneal metastasis mouse models. Results: The high-concentration process (i.e., small reaction-volume) for mixture resulted in the large-sized PEI/DNA NPs that had a higher efficiency of gene transfection, compared to the small counterpart that was prepared at a low concentration. The microstructural experiments showed that the prepared small NPs were firmly condensed, whereas the large NPs were bulky and botryoid-shaped. The large NPs entered the tumor cells via the macropinocytosis pathway, and then efficiently dissociated in the cytoplasm and released DNA, thus promoting the intranuclear delivery. The enhanced in vivo therapeutic efficacy of the large NPs was demonstrated, indicating the promise for local-regional administration. Conclusion: This work provides better understanding of the effect of formulation process on nano-structural properties and gene transfection, laying a theoretical basis for rational design of the experimental process.

Clinicopathological, immunohistochemical, and ultrastructural study of 13 cases of melanotic schwannoma.

BACKGROUND: Melanotic schwannoma is a rare variant of schwannoma composed of melanin-producing cells with ultrastructural features of schwann cells. The description of the course of the tumors differs somewhat, but it is generally considered as a benign lesion. We investigated the clinicopathologic features, immunophenotypes, and ultrastructural features of 13 patients with nonpsammomatous melanotic schwannoma (NPMS). METHODS: Tumor specimens of each patient were sectioned and stained with hematoxylin-eosin, Fontana-Masson, Prussian blue, and periodic acid-Schiff (PAS). Immunohistochemical markers such as S-100, Leu-7, HMB-45, Melan-A, CK, EMA, vimentin, GFAP, laminin, collagen IV and MIB-1 were detected with the Envision immunohistochemical staining method. Four of the cases were observed by electron microscopy. RESULTS: Of the 13 patients, 8 were male and 5 female, aged from 11 to 92 years (mean, 38.6 years). The tumor sites included the spinal nerve root (5 patients), cranial nerve (1), greater omentum (1), subcutaneous tissue (3), mesentery (1), bone (1) and mediastinum (1). Eleven patients were followed up for over 2 years, with a mean of 5.9 years. One patient (9.1%) with a primary tumor in the greater omentum developed another primary tumor of the same type in the subcutaneous tissue of the abdominal wall after the first operation. Local recurrence of the tumor was seen in 2 patients (18.2%). One patient (9.1%) showed the local recurrence and metastasis. Seven patients (63.6%) showed no evidence of the recurrence or metastasis. Grossly, all tumors were well-circumscribed and the gross findings were suggestive of melanin-containing tumors. The tumor was composed of spindled and epithelioid cells with abundant intracytoplasmic melanin pigments. Nuclei were round and contained delicate, evenly distributed chromatins as well as small, distinct nucleoli. In some areas, the nucleoli were large and prominent. Rare mitoses were seen in most lesions except the larger omentum lesion. The pigment was shown to be positive for the Fontana-Masson and negative for Prussian blue and PAS. Immunohistochemical staining for S-100, Leu-7, HMB-45, Melan-A, and vimentin were strongly positive. Linear immunoreactions of both laminin and collagen IV was detected in all patients. Ultrastructurally, numerous elongated tumor-cell processes, duplicated basement membrane and melanosomes were observed in all developmental stages. CONCLUSIONS: Histologically, melanotic schwannoma is a rare variant of schwannoma composed of melanin-producing cells with ultrastructural features of schwann cells. Distinguishing between this tumor and malignant melanoma is of paramount importance in planning of management. Immunohistochemically, combined use of laminin and collagen IV is valuable in distinguishing melanotic schwannoma from malignant melanoma. Wide local resection and additional radiotherapy should be advocated. Further studies including cytogenetic or molecular biology are still required to better delineate melanotic schwannoma from malignant melanoma. Appropriate long-term follow-up is needed for all melanotic schwannomas.

[Diagnostic value of A103 and inhibin-alpha in adrenocortical tumors: an immunohistochemical study using tissue microarray techniques].

OBJECTIVE: To investigate the potential diagnostic value of A103 and inhibin-alpha in adrenocortical tumors and to evaluate the applicability of tissue microarray/tissue chip in pathological studies using immunohistochemistry. METHODS: A tissue microarray/tissue chip was constructed to contain 179 formalin-fixed, paraffin-embedded adrenal tissue samples which include 3 normal adrenal cortex, 2 fetal adrenal cortex, 2 nodular adrenocortical hyperplasia samples, 72 adrenocortical adenomas, 39 adrenocortical carcinomas, 3 adrenal medulla, 13 metastatic carcinomas, 4 metastatic malignant melanomas and 44 pheochromocytomas. Additional 20 cases of normal adult adrenal gland were used as controls. Immunohistochemical markers, including A103, inhibin-alpha, calretinin and Ki-67 were used on the tissue array sections by EnVision immunohistochemical staining methods. RESULTS: Positive staining of A103 was seen in all of the 23 (100%) adrenal cortex, 2 fetal adrenal cortex, 2 nodular adrenocortical hyperplasia samples, 60 of 66 (90.9%) adrenocortical adenomas samples, 35 of 37 (94.6%) adrenocortical carcinomas samples, 3 of 3 malignant melanomas, but in none of the adrenal medulla, pheochromocytomas or adrenal metastatic carcinoma samples. In all of the adrenal cortex, fetal adrenal cortex and nodular adrenocortical hyperplasia cases, inhibin-alpha immunoreactivity was limited to the zona reticularis and the innermost zona fasciculata. Fifty of the 66 (75.8%) adrenocortical adenomas, 28 of the 37 (75.7%) adrenocortical carcinomas were positive for inhibin-alpha. None of the adrenal medulla, pheochromocytoma, metastatic malignant melanoma or carcinoma samples showed a positive inhibin-alpha immunostain. CONCLUSIONS: The tissue microarray/tissue chip technique provides a reliable method to investigate marker expression by offering a rapid, economic and accurate screening of tissue specimens on a large scale. The combined use of A103 and inhibin-alpha is valuable in distinguishing adrenocortical tumor from pheochromocytoma and other metastatic neoplasms.

Transplanted bone marrow mesenchymal stem cells protects myocardium by regulating 14-3-3 protein in a rat model of diabetic cardiomyopathy.

OBJECTIVE: This study examined the mechanism of bone marrow mesenchymal stem cells (BMSCs) up-regulating the expression of 14-3-3 protein, blocking the myocardial apoptosis in diabetic cardiomyopathy and thereby improving cardiac function. METHODS AND RESULTS: (1) Rat model of diabetic cardiomyopathy was made by feeding rats with high fat/high sugar diet and intraperitoneal injection of small dose of streptozocin (STZ). The model was successfully established as confirmed by the detection of blood sugar, lipid profile, ultrasonographic and hemodynamic examinations. (2) Bone marrow (BM) liquid was taken from the rat femur and tibia bones. The BMSCs were obtained by culture and were confirmed by phase-contrast microscopy and flow cytometry. The BMSCs were transplanted into the rats and fluorescent microscopy showed that transplantation was successful. (3) TUNNEL, Western blotting revealed that in rats of DCM group, myocardial apoptosis was more severe and expression of capase-3 was significantly up-regulated while in rats receiving transplantation of BMSCs showed opposite changes, with the differences being statistically significant (P < 0.05). (4) Western blotting exhibited that, compared with DCM group, 14-3-3 and p-Ask1 protein was significantly increased while Ask1 was obviously decreased. CONCLUSION: Our findings suggested that transplantation of bone marrow mesenchymal stem cells could inhibit the myocardial apoptosis in diabetic cardiomyopathy, possibly by up-regulating the expression of 14-3-3 protein and inhibiting the phosphorylation of Ask1.