Printing Three‐Dimensional Refractory Metal Patterns in Ambient Air: Toward High Temperature Sensors

Abstract Refractory metals offer exceptional benefits for high temperature electronics including high‐temperature resistance, corrosion resistance and excellent mechanical strength, while their high melting temperature and poor processibility poses challenges to manufacturing. Here this work reports a direct ink writing and tar‐mediated laser sintering (DIW‐TMLS) technique to fabricate three‐dimensional (3D) refractory metal devices for high temperature applications. Metallic inks with high viscosity and enhanced light absorbance are designed by utilizing coal tar as binder. The printed patterns are sintered into oxidation‐free porous metallic structures using a low‐power (<10 W) laser in ambient environment, and 3D freestanding architectures can be rapidly fabricated by one step. Several applications are presented, including a fractal pattern‐based strain gauge, an electrically small antenna (ESA) patterned on a hemisphere, and a wireless temperature sensor that can work up to 350 °C and withstand burning flames. The DIW‐TMLS technique paves a viable route for rapid patterning of various metal materials with wide applicability, high flexibility, and 3D conformability, expanding the possibilities of harsh environment sensors

Requirement of PEA3 for transcriptional activation of FAK gene in tumor metastasis.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides -170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis

Data underlying the publication: Cytotoxic activities against MCF-7 and MDA-MB-231, antioxidant and α-glucosidase inhibitory activities of Trachelospermum jasminoides extracts in vitro

cytotoxic activities against MCF-7 and MDA-MB-231 and the antioxidant and α-glucosidase inhibitory activities of the extracts of Trachelospermum jasminoides in vitr

E1AF involved in the mouse FAK gene transcription activation in B16F1 cells.

<p>(A) Activation of FAK promoter by ETS transcription factor E1AF. The B16F1 cells were transfected with 1 µg plasmids expressing ETS-1, ETS-2, E1AF or mock plasmid along with 1 µg p-842/+43-luc and Renilla expressing plasmid. The luciferase activity of each sample was normalized to the internal Renilla control. Then the normalized luciferase activity was standardized to that of p-842/+43-luc with vector alone. Each value is the mean ±S.D. of at least three independent experiments. (B) Mapping the regions of the FAK promoter necessary for E1AF responsiveness. The B16F1 cells were transfected with p-842/+43-luc construct or the truncated FAK promoter constructs shown above and with or without E1AF expression vector. Luciferase activity was normalized to Renilla luciferase activity and standardized to the normalized activity from p-842/+43-luc with control vector alone. Data shown are the means ±S.D. of at least three independent experiments. (C) Site-directed mutation analysis of the FAK promoter. Cells were transfected with 2 µg p-170/+43-luc, p-55/+43-luc, or p-170/+43-luc devoid of E1AF binding site (p-170/+43 m) along with Renilla expressing plasmid. FAK promoter activity in B16F1 cells was decreased in the absence of E1AF binding site. (D) Stimulatory effect of E1AF overexpression on FAK promoter activation was prevented in the absence of E1AF binding site. B16F1 cells were co-transfected with 1 µg E1AF expressing plasmid, or empty vector along with 1 µg wide type p-170/+43-luc or mutated p-170/+43 m-luc and Renilla expressing plasmid. Luciferase activity of B16F1 cells co-transfected with empty vector and wide type p-170-luc was arbitrarily set at 1. E. EMSA was performed using nuclear proteins of B16F10 cells and the ³²P-labeled mouse FAK promoter E1AF element probe. F. EMSA of the same amounts nuclear extracts from B16F10 cells and B16F1 cells incubated with ³²P -labeled E1AF element probe and supershift by PEA3 antibody. The arrows indicate the supershifted bands.</p

Schematic diagram of signal pathways underlying PEA3-mediated FAK gene transcription activation in melanoma tumor cells.

<p>Schematic diagram of signal pathways underlying PEA3-mediated FAK gene transcription activation in melanoma tumor cells.</p

Progression deletion analysis of mouse FAK promoter.

<p>(A) Deletion constructs were cloned into the promoter-less pGL3-Basic vector. Different constructs with different deletions of the promoter fragments are shown. Numbers indicate 5′ and 3′ ends relative to the first transcription initiation site in each construct. (B) Identification of the minimal promoter for the FAK gene basal transcription. Luciferase plasmids harboring various lengths of FAK promoter regions were transiently transfected into B16F10 cells along with Renilla expressing plasmid. The luciferase activity was normalized to the internal Renilla control and standardized to the normalized activity from pGL3-Basic. Each value is the mean ±S.D. of at least three independent experiments.</p

PEA3 and FAK were detected by the immunohistochemistry (IHC).

<p>Immunohistochemical analysis for PEA3 and FAK protein expression in paraffin wax embedded human primary oral squamous cell carcinoma specimen. Nuclear expression of PEA3 and cytoplasmic expression of FAK can be seen in representative cases of metastatic cancer tissue and are absent in some nonmetastasis carcinoma tissues. (original magnification, ×200). Scale bar = 100 µm.</p

Increased FAK expression in highly metastatic B16F10 melanoma cells.

<p>(A–B) Comparison of the <i>in vivo</i> and <i>in vitro</i> metastatic potential of B16F10 and B16F1 melanoma cells. B16F10 and B16F1 cells (1×10⁶) were injected intravenously into C57BL/6J mice via tail vein. After 20 days, lungs from the mice were resected and analyzed for metastasis. The representative lung in mice injected with F1 or F10 cells are shown (A-1). After fixation in Bouin’s solution, metastatic nodules found on lung surface are counted and averaged (A-2). Data are presented as mean ± SEM of five mice. ***P<0.01. (B-1) Migration of F10 and F1 in vitro. F1 and F10 cells were incubated in the transwell insert with 10% FBS in the lower chamber for 12 h. Photo show representative membrane attached migrated cells. Scale bar = 100 µm. Quantitative analysis of the number of the cells migrated to the down side of the membrane (B-2). Data are mean ±SEM of three independent experiments. ***P<0.01. (C–D) Northern blot and RT-PCR analyses of FAK mRNA in B16F10 and B16F1 cells. β-actin was amplified in RT-PCR and used as an internal control. (E-1) Western blot analysis of FAK expression level and p-FAK level in B16F10 and B16F1 cells. Whole cell lysates from B16F10 and B16F1 cells were blotted with antibodies to FAK, phosphorylated FAK (Y397 p-FAK) or tubulin as a control. (E-2) Densitometric quantification revealed a significant increase in expression of FAK protein in F10 cell line. Densitometric units were normalized to Tubulin and then divided by F1 group results. (n = 3; ***P<0.01, **p<0.05 ).</p

Involvement of PEA3 in B16F10 cells migration and invasion.

<p>(A) Elevated expression of PEA3 protein in nuclear extracts from B16F10 cells. PEA3 protein of each cell line nuclear extract was determined by western blotting using the anti-PEA3 antibody. Knockdown of PEA3 mRNA in B16F10 cells led to a reduction of FAK mRNA (B) or FAK protein (C). The B16F10 cells were transfected with 2 µg psi-PEA3 or mock plasmid for 48 h, RT-PCR analysis was performed to measure the PEA3 and FAK mRNAs (B), western blot analysis was performed to determine the PEA3,FAK and phosphorylated FAK (Y397 p-FAK) protein levels in the whole lysates of the B16F10 cells (C-1), tubulin or actin was used as a control. (C-2) Densitometric quantification analysis of western blot results in C-1. Densitometric units were normalized to tubulin and then divided by mock group results. (<i>n</i> = 3; ***, <i>p</i><0.01) (D-E) Knockdown of PEA3 suppressed B16F10 cell invasiveness. After transfected B16F10 cells with mock or psi-PEA3 for 24 h, cells were collected and re-suspended in culture medium at a density of 1 × 10⁶ cells/ml. 100 µl of the cell suspension was plated into the upper wells of Transwell inserts containing 8 µm pore polycarbonate membranes pre-coated with fibronectin (10 µg/ml) on the under surface. The cells were allowed to migrate for 24 h at 37°C, then cells on upper wells were removed gently and those migrated to the under surface were fixed and stained. Representative membranes stained with crystal violet are shown (D-1), the arrows indicated the migrant cells. Scale bar = 200 µm. Quantitative analysis of the number of the cells migrated to the down side of the membrane (D-2). Data are mean ±SEM of three independent experiments. ***P<0.01, vs. mock control. (E) Knockdown of PEA3 in B16F10 cells suppressed the tumor cells migration. The B16F10 cells were transfected with si-PEA3 or mock vector. Then wound-healing scratch motility assays were performed in the fibronectin-coated plates in the presence of serum. Cell migration was assessed at 0 and 48 h. Representative images are shown (E1). Migration of cells into the wound was quantified (E-2). (<i>n</i> = 3; ***, <i>p</i><0.01).</p

Schematic diagram of mouse FAK promoter region as well as putative transacting factor binding sites.

<p>This figure shows the genomic sequence (FAK genomic sequence from mouse RefSeq: NT_039621) which includes exon-5 and exon-4 of the FAK gene (in grey shadow). The sequence contains −861 bp genomic region immediately upstream of the FAK gene transcription start site (+1) and downstream of the +290 bp sequence. The position of the transcription initiation site was defined as +1 determined by us with 5′ RACE in the B16F10 cells and indicated with black bent arrow. This putative TATA box-less promoter contains GC-rich sequence. The putative binding sites for transcription factors Sp1, E1AF, AP-2 and AML-1 predicted by TRANSFAC program are underlined and labeled.</p