Electromagnetic Dispersion in Periodic Structures

Photonic crystals are electromagnetic structures that affect the propagation of microwaves, clearly demonstrating nonlinear, band gap dispersion in the band theory of solids. Motivated partly by the development of an advanced physics lab in dispersion, we have compared the dispersion of microwaves in photonic crystals to the dispersion of electrons in semiconducting crystals. The transmission lines were fabricated to achieve periodicity with alternating widths of adjacent copper segments using photolithography. Three identical dispersion diagrams were constructed using different sets of values: the S-parameters measured by the vector network analyzer (V.N.A.), the S-parameters simulated using finite element analysis software, and the delay values measured by the V.N.A. All three methods showed close agreement in the dispersion with a band gap at the Brillioun zone edge. The values from the network analyzer were then used to examine the group velocity of the wave near the band gap. Near the edges of the band gap, the group velocity approached zero; inside the band gap, the evanescent waves tunneled through the crystal with superluminal group velocities. Periodic transmission lines with defects were also constructed; the defects engineered into the photonic crystals produced donor and acceptor states in the band gap. These results indicate that the microwave transmission lines successfully modeled the dispersion from band gaps in photonic crystals

Isolation and Genome Sequencing of Two Novel Mycobacteriophages, Optimus and Sassafras

Twenty new mycobacteriophages, capable of infecting Mycobacterium smegmatis, were isolated from soil samples collected on or nearby Hope College in Holland, Michigan. Collectively, the group displayed a variety of plaque morphologies indicating an assortment of different phages. Both lytic and temperate phages appear represented in this collection. Purified phage stocks were used to prepare genomic DNA samples for restriction digest analysis. Of 20 samples analyzed, a total of 13 phages produced just 4 types of restriction digest patterns indicating some degree of relatedness among some of our new phage isolates. Interestingly, one group of 4 phages (Optimus, Lynx, Aurora and TheCube14) that yielded a similar restriction digest pattern, were all isolated from mulch-covered soil at a depth of 4-8 cm. Two phages (Optimus and Sassafras) were chosen for complete genome sequencing and comparative genomic analyses. Both phages produced plaques of between 1-2 mm in diameter at 24 hours that enlarged to about 4 mm in diameter after 48 hours of incubation at 37°C. Whereas continued incubation of phage Optimus resulted in cessation of plaque growth by 72 hours, plaques produced by Sassafras continued to enlarge beyond 8 days, reaching a diameter of greater than 10 mm. Phage Optimus produced plaques that displayed a clear center surrounded by turbid rings. Phage Sassafras produced clear plaques with defined edges at 24 hours, but all subsequent growth was progressively more turbid in nature, resulting in plaques with a turbid ring around a center clear zone. Comparison of the restriction digest patterns for Optimus and Sassafras with more than 60 existing mycobacteriophage genomes indicates that Optimus may be a new representative of cluster H, while Sassafras shows some similarity to the F cluster of mycobacteriophages. Results of our analyses of both genomes are reported

Effect of thalidomide on CUL5 and NEDD8 localization in HUVEC after 24 hrs of treatment.

<p><b>A.</b> Expression of CUL5 and NEDD8 in Control, Thalidomide (20 μg/ml) and PMA (10 nM) treated HUVEC for 24 hours. Immunostaining with anti-VACM-1/CUL5 and anti NEDD8 antibodies was performed as described in the Methods. Images are from CUL5 staining (FITC-green) and NEDD8 staining (Texas red) and merged pictures. Magnification is 40X. <b>B.</b> Control and thalidomide treated cells expressing nuclear CUL5 and NEDD8 were quantitated (n = 10 and n = 3, respectively; *, p <0.05).</p

CUL5 protein localization in HUVEC treated with increasing doses of thalidomide.

<p><b>A.</b> The control and thalidomide-treated HUVEC (20 and 50 μg/ml). <b>B.</b> Examples of HUVEC treated with 100 μg/ml thalidomide. Cells were immunostained using anti- CUL5 specific antibody as described in the Methods. Magnification is 100X.</p

Thalidomide treatment inhibits growth in RAMEC but not in RAMEC transfected with a dominant negative mutant of CUL5 (^S730ACUL5) cDNA.

<p><b>A.</b> A representative growth assay results from CMV vector transfected RAMEC control cells treated with increasing doses of thalidomide. VEGF (50 nM) was used as a control. <b>B.</b> Western blot analysis of cell lysates from CMV vector transfected RAMEC treated with increasing doses of thalidomide for 24 hours. To ascertain equal protein loading blots were stripped and re-probed with anti-GAPDH specific antibody as described in <i>Methods</i>. <b>C.</b> The signal intensities shown in B above, were quantitated. <b>D.</b> A representative wound assay in CUL5 cDNA transfected cells treated with thalidomide. Arrows indicate space in the wound assay at time 0 and 18 hrs. <b>E.</b> Growth data shown in (D) was quantitated and expressed as a percent (%) regrowth from time 0. The effects of 0 μg/mL (black bars), 10 μg/ml (gray bars), and 50 μg/ml (white bars) of thalidomide were examined (*, p<0.05). <b>F.</b> A representative growth assay results from ^S730ACUL5 cDNA transfected RAMEC cells treated with increasing doses of thalidomide. VEGF (50 nM) was used as a control. <b>G.</b> Dose dependent effect of thalidomide on CUL5 in ^S730ACUL5 cDNA transfected RAMEC as detected with anti CUL5 protein specific antibody. <b>H</b>. Data shown in G were quantitated and expressed as mean ± standard error. (RAMEC CMV n = 2 and RAMEC−^S730AVACM-1 (<i>n</i> = 3, * = <i>p</i> < 0.05). <b>I.</b> A representative light microscopy experiment using the wound assay in ^S730ACUL5 cDNA transfected cells. Arrows indicate space in the wound assay. <b>J.</b> Growth data shown in (I) were quantitated and expressed as a percent regrowth from time 0. The effects of 0 (black bars), 10 μg/ml (gray bars), and 50 μg/ml (white bars) of thalidomide were examined (* = <i>p</i> < 0.05).</p

Effect of thalidomide on CUL5 and NEDD8 localization in control and si-transfected HUVEC at 24 hours after treatment.

<p><b>A.</b> CUL5 (green) and NEDD8 (red) localization in control and si-CUL5 transfected HUVEC and treated with thalidomide (50 μg/mL) for 24 hrs. Magnification is 100X. <b>B.</b> HUVEC treated with thalidomide (50 μg/mL) immunostained with anti NEDD8 Ab. Magnification is 40X. <b>C.</b> NEDD8 signal in Control and Thalidomide treated cells shown in B was quantitated (<i>n</i> = 3; error bars are S.E.M., *, <i>p</i> < 0.05). D. Western blot analysis of neddylated CUL5 (upper band) and free NEDD8 (lower band) in control, thalidomide and PMA-treated HUVEC (24 hours).</p

Effect of thalidomide on expression and cellular localization of CUL5 in control RAMEC and RAMEC transfected with ^S730ACUL5.

<p><b>A.</b> Immunocytochemistry of control RAMEC treated with thalidomide and immunostained with anti-CUL5 (VACM-1) Ab. <b>B.</b> ^S730ACUL5 cDNA transfected RAMEC treated with thalidomide and immunostained with anti- CUL5 Ab. Cells were mounted in Vectashield^® containing DAPI (magnification, 100X).</p

Effect of thalidomide on CUL5 and NEDD8 localization in HUVEC.

<p>CUL5 and NEDD8 colocalization in control cells treated with thalidomide (50 μg/mL) for 15 and 45 min, respectively. Immunostaining with anti- CUL5 and anti NEDD8 antibodies and nuclear DAPI staining was performed as described in the <i>Methods</i>. Images are of merged pictures from CUL5 staining (FITC-green) and NEDD8 staining (Texas red). Magnification is 40X.</p

siRNA-mediated depletion of CUL5 in HUVEC prevents thalidomide-dependent decrease in cellular proliferation.

<p><b>A.</b> A representative immunocytochemistry results showing CUL5 distribution in control and thalidomide (50 μg/ml) treated HUVEC that were sham transfected or transfected with anti-VACM-1 specific siRNA. Magnification is 100X. <b>B.</b> Western blot analysis of cell lysates from control (Cont), thalidomide (thal) and siRNA-GAPDH and siCUL5 transfected HUVEC probed with anti-VACM-1/CUL5 Ab A. A sample of lysate from ^S730AVACM-1/CUL5 transfected RAMEC was used as a positive control (Cont(+ve)). Blots were subsequently re-probed using anti-actin antibody. <b>C.</b> DAPI nuclear staining of control-siRNA and anti-CUL5 si-RNA transfected HUVEC treated with thalidomide (50 μg/ml) for 24 hours. Magnification is 10X. <b>D.</b> Cellular count using DAPI-stain signal for the experiment shown in C (*, p<0.05 for cell growth in thalidomide treated vs control cells. #, p<0.05 when cell counts in control-siRNA and anti-CUL5 si-RNA transfected cells were compared). <b>E.</b> Effects of thalidomide on siRNA-transfected HUVEC growth using alamarBlue^® assay. PMA (10⁻⁷ M) was used as a control (n = 3, *, p<0.05 when compared to control). <b>F.</b> Control and siRNA-transfected HUVEC grown on Matrigel^® coated plates. Magnification is 20X. <b>G.</b> A representative experiment showing effects of thalidomide in control and siRNA-transfected HUVEC grown on Matrigel^® coated plates. Magnification is 10X.</p