Detection of <i>EML4-ALK</i> in Lung Adenocarcinoma Using Pleural Effusion with FISH, IHC, and RT-PCR Methods

<div><p>Anaplastic lymphoma kinase (<i>ALK</i>) and echinoderm microtubule-associated protein-like 4 (<i>EML4</i>) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the <i>EML4-ALK</i> fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The <i>EML4-ALK</i> fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription—polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). <i>EML4-ALK</i> was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the <i>EML4-ALK</i> fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting <i>EML4-ALK</i> in blocks of pleural effusion cells from lung cancer patients.</p></div

Detection of the <i>EML4-ALK</i> gene rearrangement with FISH.

<p>Dual-color, break-apart fluorescent <i>in situ</i> hybridization was performed on cell block slides. The centromeric (green) and telomeric (red) flanks of the <i>ALK</i> locus. Splitting the red and green signals indicates <i>ALK</i> rearrangement.</p

Representative examples of Ventana immunohistochemical staining of ALK.

<p>A: H&E staining of a cell block shows lung carcinoma cells. B: Strong staining of ALK protein in a cell block slide (corresponding to sample A). C: H&E staining of a cell block shows lung carcinoma. D: Weak staining of ALK protein in a cell block (corresponding to sample C).</p

Au/Polypyrrole@Fe₃O₄ Nanocomposites for MR/CT Dual-Modal Imaging Guided-Photothermal Therapy: An <i>in Vitro</i> Study

Construction of multifunctional nanocomposites as theranostic platforms has received considerable biomedical attention. In this study, a triple-functional theranostic agent based on the cointegration of gold nanorods (Au NRs) and superparamagnetic iron oxide (Fe₃O₄) into polypyrrole was developed. Such a theranostic agent (referred to as Au/PPY@Fe₃O₄) not only exhibits strong magnetic property and high near-infrared (NIR) optical absorbance but also produces high contrast for magnetic resonance (MR) and X-ray computed tomography (CT) imaging. Importantly, under the irradiation of the NIR 808 nm laser at the power density of 2 W/cm² for 10 min, the temperature of the solution containing Au/PPY@Fe₃O₄ (1.4 mg/mL) increased by about 35 °C. Cell viability assay showed that these nanocomposites had low cytotoxicity. Furthermore, an <i>in vitro</i> photothermal treatment test demonstrates that the cancer cells can be efficiently killed by the photothermal effects of the Au/PPY@Fe₃O₄ nanocomposites. In summary, this study demonstrates that the highly versatile multifunctional Au/PPY@Fe₃O₄ nanocomposites have great potential in simultaneous multimodal imaging-guided cancer theranostic applications

BMP‑2 Derived Peptide and Dexamethasone Incorporated Mesoporous Silica Nanoparticles for Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells

Bone morphogenetic protein-2 (BMP-2), a growth factor that induces osteoblast differentiation and promotes bone regeneration, has been extensively investigated in bone tissue engineering. The peptides of bioactive domains, corresponding to residues 73–92 of BMP-2 become an alternative to reduce adverse side effects caused by the use of high doses of BMP-2 protein. In this study, BMP-2 peptide functionalized mesoporous silica nanoparticles (MSNs-pep) were synthesized by covalently grafting BMP-2 peptide on the surface of nanoparticles via an aminosilane linker, and dexamethasone (DEX) was then loaded into the channel of MSNs to construct nanoparticulate osteogenic delivery systems (DEX@MSNs-pep). The in vitro cell viability of MSNs-pep was tested with bone mesenchymal stem cells (BMSCs) exposure to different particle concentrations, revealing that the functionalized MSNs had better cytocompatibility than their bare counterparts, and the cellular uptake efficiency of MSNs-pep was remarkably larger than that of bare MSNs. The in vitro results also show that the MSNs-pep promoted osteogenic differentiation of BMSCs in terms of the levels of alkaline phosphatase (ALP) activity, calcium deposition, and expression of bone-related protein. Moreover, the osteogenic differentiation of BMSCs can be further enhanced by incorporating of DEX into MSNs-pep. After intramuscular implantation in rats for 3 weeks, the computed tomography (CT) images and histological examination indicate that this nanoparticulate osteogenic delivery system induces effective osteoblast differentiation and bone regeneration in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs can act synergistically to enhance osteogenic differentiation of BMSCs, which have potential applications in bone tissue engineering

Construction of protein fragments containing HA-13–263 with or without Fd or Fc and analysis of protein expression.

<p>(A) Proteins containing residues of HA-13–263 with Fc (HA-13–263-Fc), with Fd (HA-13–263-Fd-His), or without fusion with Fd and Fc (HA-13–263-His) were constructed. Purified proteins were either boiled at 95°C for 5 min, or not boiled, followed by detection of the expression by SDS-PAGE and Coomassie Blue staining (B), and Western blot (C) by using a HA1-specific mAb. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.</p

One-Pot Synthesis of MoS₂ Nanoflakes with Desirable Degradability for Photothermal Cancer Therapy

Developing biodegradable photothermal agent holds great significance for potential clinical translation of photothermal therapy. In the current study, one-pot hydrothermal synthesis of MoS₂ nanoflakes with desirable degradation capability was presented. The participation of poly­(acrylic acid) (PAA) in hydrothermal process could not only facilitate the modification of polyethylene glycol (PEG), but also bestow degradability to the prepared MoS₂ nanoflakes. Moreover, the PEGylated hybrid nanoflakes (MoS₂–PPEG) also exhibited excellent stability in various medium and outstanding photothermal properties. Interestingly, MoS₂–PPEG behaved distinctly different degradation rate in diverse condition. The rapid degradation of MoS₂–PPEG was observed in neutral pH solution, whereas much slower degradation occurred in an acidic tumor microenvironment. Furthermore, data indicated that the major degradation product of MoS₂–PPEG was water-soluble Mo-based ion. Meanwhile, the good in vitro biocompatibility of MoS₂–PPEG was also confirmed in terms of cytotoxicity and hemolysis. With favorable photothermal performance, MoS₂–PPEG can efficiently killing cancer cells in vitro and suppress the tumor growth in vivo. More importantly, the gradual decreasing content of MoS₂–PPEG in organs and detectable Mo element in urine of mice suggested that the degradability of MoS₂–PPEG might facilitate its excretion to some degree. Hence, the degradable MoS₂ nanoflakes prepared by one-pot hydrothermal routine may provide insight for further biomedical applications of inorganic photothermal agent

SDS-PAGE and Western blot analysis of expressed proteins containing truncated HA1 fragments.

<p>Protein samples were either boiled at 95°C for 5 min, or not boiled, followed by addition of 6× SDS-PAGE sample buffer. SDS-PAGE with Coomassie Blue staining was then run (A), as well as Western blot (B) by using a HA1-specific mAb. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left.</p

Comparison of neutralizing antibodies in sera of mice vaccinated with HA-13–263 proteins.

<p>Sera were collected at 10 days post-last vaccination. PBS was used as the negative control. (A) Neutralizing antibody titers against HA of homologous AH-HA strain (clade 2.3.4) and heterologous A/Hong Kong/156/97 (HK-HA, clade 0) and A/VietNam/1194/2004 (1194-HA, clade 1) strains of H5N1 pseudovirus. The data are presented as mean NT₅₀± SD from five mice per group. * indicates significant difference (<i>P</i><0.05), respectively, between HA-13–263-Fdc, HA-13–263-Fc, or HA-13–263-Fd-His vaccination groups and HA-13–263-His group or PBS control for the three H5N1 pseudoviruses. (B) Neutralizing antibody titers against heterologous strains of A/VietNam/1194/2004 (VN/1194, clade 1) and A/Shenzhen/406H/06 (SZ/406H, clade 2.3.4) H5N1 live viruses. The titers of neutralizing antibodies are presented as mean ± SD of five mice per group. * indicates significant difference (<i>P</i><0.05), respectively, between HA-13–263-Fdc, HA-13–263-Fc, or HA-13–263-Fd-His vaccination groups and HA-13–263-His group or PBS control for both VN/1194 and SZ/406H viruses. The dotted line shows the limit of detection.</p

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Spectral evolution of the coexistence patterns as a function of pump powe