Ensemble-Empirical-Mode-Decomposition based micro-Doppler signal separation and classification

The target echo signals obtained by Synthetic Aperture Radar (SAR) and Ground Moving Target Indicator (GMTI platforms are mainly composed of two parts, the micro-Doppler signal and the target body part signal. The wheeled vehicle and the track vehicle are classified according to the different character of their micro-Doppler signal. In order to overcome the mode mixing problem in Empirical Mode Decomposition (EMD), Ensemble Empirical Mode Decomposition (EEMD) is employed to decompose the original signal into a number of Intrinsic Mode Functions (IMF). The correlation analysis is then carried out to select IMFs which have a relatively high correlation with the micro-Doppler signal. Thereafter, four discriminative features are extracted and Support Vector Machine (SVM) classifier is applied for classification. The experimental results show that the features extracted after EEMD decomposition are effective, with up 90% success rate for classification using one feature. In addition, these four features are complementary in different target velocity and azimuth angles

G protein, adenylate cyclase, and cAMP generation in orbital fibroblasts and fibrocytes.

<p>(A) Confluent cultures were treated with bTSH (5 mIU/mL), PGE₂ (1 µM), forskolin (20 µM) or nothing (control) for 16 h. Cell layers were analyzed for cAMP content and protein determination. (B) Adenylate cyclase mRNA levels were determined in orbital fibroblasts and fibrocytes from healthy donors (n = 5) and those with GD (n = 5). (C) RNA from orbital fibroblasts, fibrocytes, and thyroid tissue was subjected to RT-PCR for Gq and Gs using the C_T method. Data are expressed as mean±SD of triplicate determinations from three different strains of each. (D) Cultures indicated were treated with nothing, bTSH, PKA inhibitors H89 (10 µM), KT5720 (10 µM) or Rp-cAMP (1 mM) alone or in the combinations indicated for 6 h. RNA was extracted and subjected to RT-PCR for IL-6. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. In 3 separate experiments, H89 failed to inhibit TSH-dependent IL-6 expression (1.62±0.23-fold and 1.06±0.3-fold increase in fibroblasts and fibrocytes, respectively vs TSH alone).</p

Divergent PKCµ and PKCβII mRNA expression in pure CD34⁺ and CD34⁻ orbital fibroblast subsets.

<p>A parental strain of TAO orbital fibroblasts (containing mixed CD34⁺ and CD34⁻ cells) was sorted into pure CD34⁺ and CD34⁻ subsets by FACS as described in Experimental Procedures. These were then cultured for 48 h., RNA was isolated, andRT-PCR performed for (left panel) PKCµ and (right panel) PKCβII using the C_T method. Ct values were normalized to their respective GAPDH levels. Data are expressed as the mean ± SD of three independent replicates from a single experiment, representative of three performed. PKCµ mRNA was undetectable in CD34⁺ subsets in all 3 studies while PKCβII mRNA levels were 44±7-fold above their respective parental fibroblast cultures.</p

Involvement of NF-κB in the induction of IL-6 by bTSH.

<p>(A) Confluent orbital fibroblast and fibrocyte cultures were treated without or with bTSH for 60 min. Cytosolic and nuclear protein fractions were prepared as described in Experimental Procedures. Nuclear protein extract was probed with anti-NF-κB-p65 Abs by Western blot analysis. Densitometry: nuclear p65, control vs bTSH-treated fibroblasts, 11.72 AU vs 58.5 AU; fibrocytes, 10.2 AU vs 75.3 AU, respectively. Cytosolic extracts were subjected to Western blot analysis of IκBα, pIKK, and IKKβ. (B) Orbital fibroblasts, in this case from a patient with TAO, and fibrocytes were treated with nothing or bTSH (5 mIU/mL) in the absence or presence of PDTC (100 µM) for 16 h. Media were analyzed for IL-6 content by ELISA. Data are presented as the mean ± SD of 3 independent determinations. (##, P<0.01 vs untreated controls; **, P<0.01 vs TSH alone). (C) Sub-confluent orbital fibroblast and fibrocyte cultures were transfected with control siRNA or that targeting Rel A, incubated for 48 h. and then treated with nothing or bTSH (5 mIU/mL) for 16 h. Media were analyzed for IL-6. Inset: Western blot confirming knockdown of Rel A. Data are expressed as the mean ± SD of three independent determinations. ***, p<0.001 vs control siRNA. (D) Orbital fibroblasts and fibrocytes were treated with nothing or bTSH (5 mIU/mL) in the absence or presence of GFX (10 µM) or Akti (1 µM) for 30 min. Nuclear protein fractions were prepared as described in Experimental Procedures and probed with anti-NF-κB-p65 Abs by Western blotting. Densitometry: Fibroblasts bTSH vs bTSH plus GFX, 0.826±0.196 AU vs 0.485±0.06 AU, bTSH vs bTSH plus AKTi, 0.253±0.02 AU vs 0.0 AU; Fibrocytes, 0.575±0.072 AU vs 0.391±0.056 AU and 0.485±0.03 AU vs 0.043±0.012 AU respectively. In 3 separate experiments, GFX reduced TSH-dependent p65 levels by 42±7% and 32±8% in fibroblasts and fibrocytes, respectively. AKTi reduced these levels by 91±1% in fibrocytes.</p

Involvement of PDK1 in the induction by TSH of IL-6.

<p>(A) Cultures were untreated (control) or bTSH (5 mIU/ml) was added to medium for 30 min. Cellular protein was subjected to Western blot analysis for PDK1 and pPDK1 and re-probed for β-actin. Results are representative of three separate experiments performed. Densitometric analysis; pPDK1, control fibroblasts, 16.25±2.85 AU; plus bTSH, 42.7±6.54 AU; control fibrocytes, 64.5±2.67 AU; plus TSH, 78.5±3.8 AU. In 3 separate experiments, TSH increased pPDK1 levels by 2.6±0.3-fold and 1.2±0.3-fold in fibroblasts and fibrocytes, respectively. (B) Sub- confluent cultures were transfected with either control siRNA or one targeting PDK1, followed by 48 h incubation. Cultures were treated with nothing or bTSH (5 mIU/mL) for 16 h, media collected and subjected to IL-6-specific ELISA and cell layers analyzed for protein content. Data are expressed as the mean ± SD of three independent determinations. Inset: Cell layers were subjected to Western blot analysis for PDK1 after transfection with control siRNA or that targeting PDK1. In 3 separate experiments, PDK1 siRNA reduced TSH-induced IL-6 levels by 73±4% in fibroblasts and 73±5% in fibrocytes. (C) Orbital fibroblasts, in this case from a patient with TAO, were transfected with PDK1siRNA while fibrocytes were treated with OSU-03012 (5 µM) for 6 h. Cultures were treated as indicated (bTSH, 5 mIU/mL) for 30 min. Cellular protein was subjected to Western blot analysis of PKCµ and pPKCµ in fibroblasts (left panel) and PKCβII and pPKCβII in fibrocytes (right panel). (D) Confluent cultures were pre-treated without or with OSU-03012 (5 µM) for 6 h, then treated with nothing (control) or bTSH (5 mIU/ml) for 30 min. Cellular proteins were subjected to Western blot analysis probing with AKT and pAKT antibodies. Inhibition of TSH-dependent pAKT by OSU-03012 in 3 separate experiments was 14.4±1.2% and 2.5±0.6% in fibroblasts and fibrocytes, respectively.</p

CREB and NF-κB sites are critical to activation of the IL-6 gene promoter by bTSH.

<p>(A). Schematic demonstrating <i>cis</i>-acting regulatory elements for CREB (TGACGA, −213 to −208 nt) and NF-κB (GGGATTTTCCCA, −73 to −62 nt) relative to the transcription start site (+1). These are underlined and base substitutions resulting from site-directed mutagenesis appear above those in the corresponding wild-type sequences (emboldened). (B) Orbital fibroblasts and (C) fibrocytes were transiently transfected with empty luciferase vector or one containing the 1171 nt fragment spanning −1168 to +3 nt of the human IL-6 gene promoter, or that fragment harboring a 3 base mutation in the CREB binding site (designated “m1”), or a 4 base mutation in the NF-κB binding site (designated “m2”). Sub-confluent cultures were then treated with nothing (control) or bTSH (5 mIU/mL) for 1 h., cell layers harvested and luciferase reporter activity assessed in a luminometer. Data are expressed as the mean ± SD of triplicate determinations from a single experiment, representative of three performed. ***, P<0.001 versus TSH-treated cells transfected with wt promoter fragment. In 3 separate experiments, the m1 mutation resulted in an 86±4% and 79±4% reduction in TSH-dependent promoter activity in fibroblasts and fibrocytes respectively compared to wild type. The m2 mutation resulted in an 87±4% and 84±4% reduction, respectively.</p

Knockdown of CREB with siRNA attenuates PGE₂-induced IL-6 protein expression in orbital fibroblasts.

<p><b>A</b>: siRNA specific to CREB (CREB si) or scrambled siRNA (Control si) was transfected into 80% confluent cultures. Representative Western blot analysis demonstrates the impact of the knockdown of CREB protein. <b>B</b>: Media were subjected to IL-6 ELISA. Data are expressed as the mean ± SD of three independent determinations These results were representative of those in two other orbital fibroblast strains.</p

bTSH induction of IL-6 is concentration- and time-dependent.

<p>(A) Orbital fibroblasts and fibrocytes were treated with escalating concentrations of bTSH for 16 h. (B) Cells were treated with bTSH (5 mIU/mL) for graded intervals indicated along the abscissas. Media were collected and subjected to ELISA or (C) IL-6 mRNA levels determined by real-time RT-PCR. Data are expressed as mean ± SD of three independent determinations. (**, p<0.01; ***, p<0.001 vs baseline). Studies were performed three times.</p

bTSH induces IL-6 in orbital fibroblasts and fibrocytes.

<p>(A) Confluent cultures were shifted to medium containing 1% FBS for 20 h and then treated without or with bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to IL-6-specific ELISA. Cell layers were analyzed for protein content. Data are expressed as mean ± SD of three independent determinations (***, p<0.001). In a total of 3 experiments, IL-6 induction by bTSH was 18.1±5.1-fold in fibroblasts and 24.1±4.4-fold in fibrocytes. (B) Cultures were treated without or with bTSH for 6 h. Cellular RNA from three strains each of TAO orbital fibroblasts, healthy orbital fibroblasts, fibrocytes from healthy donors, and those with GD. Real-time RT-PCR was performed using the comparative critical threshold (C_T) method. Ct values were normalized to respective GAPDH levels. Data are expressed as the mean±SD of three independent determinations. (C) IL-6 levels were determined as in panel A following treatment without or with M22 (2 µg/ml) for 16 h. (***, p<0.001). The induction of IL-6 by M22 in 3 experiments was 22.4±9.2-fold in fibroblasts and 47.1±10.1-fold in fibrocytes. (D) IL-6 levels were determined in fibroblasts from two individuals harboring a loss of function TSHR mutation (TSH-resistant) and two unaffected family members (control), treated without or with bTSH for 16 h as in panel A. Data are expressed as mean ± SD fold-change of three independent determinations (**, p<0.01; ***, p<0.001 vs untreated controls). In 3 separate experiments, TSH induced IL-6 by 12.7±1.6-fold and 14.5±1.6-fold in cultures from the healthy donors and 6.4±0.5 and 8.2±0.6-fold respectively in those from the two affected individuals.</p

Role of PKC in the induction by TSH of IL-6.

<p>(A) Confluent orbital fibroblasts and fibrocytes were treated with TSH alone or in combination with GFX (10 µM) for 16 h. Media were subjected to an IL-6 ELISA. Data are expressed as mean ± SD of triplicate independent determinations. (##, P<0.01 vs untreated controls **, P<0.01 vs TSH alone). In 3 separate experiments, GFX inhibited TSH-provoked IL-6 expression by 64.2±5.1% and 65.9±4.8% in fibroblasts and fibrocytes, respectively. (B) Cultures were treated with nothing, bTSH, 8-Br-cAMP (1 mM), PMA (50 ng/mL) or the combination indicated for 16 h. Media were analyzed for IL-6 content (**, P<0.01 vs untreated controls). (C). Cellular protein from three strains of each cell type were subjected to Western blot analysis of PKCµ and PKCβII. (D) RNA was extracted from the cell types indicated and subjected toRT-PCR for PKCµ and PKCβII mRNA by the C_T method. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. (E) Cultures were treated with nothing, bTSH (5 mIU/mL), or PMA (50 ng/mL) for 30 min, harvested, and proteins analyzed by Western blot for PKCµ, pPKCµ (Ser 916), PKCβII, and pPKCβII (Ser 660). Results are from a single experiment, representative of three performed. (F) Targeting siRNAs and their scrambled counterparts were transfected into sub- confluent monolayers as described in Experimental Procedures. After 48 h, they were treated with nothing or bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to an IL-6 ELISA. Data are expressed as the mean ± SD of three independent determinations from a single experiment, representative of three performed. **, p<0.01 vs TSH-treated cultures transfected with control siRNA. In 3 separate experiments, PKCµ siRNA inhibited TSH-provoked IL-6 by 48.5±1% in fibroblasts and PKCβII siRNA inhibited TSH-induced IL-6 by 59± % in fibrocytes.</p