n-type Ge/Si antennas for THz sensing.

Ge-on-Si plasmonics holds the promise for compact and low-cost solutions in the manipulation of THz radiation. We discuss here the plasmonic properties of doped Ge bow-tie antennas made with a low-point cost CMOS mainstream technology. These antennas display resonances between 500 and 700 GHz, probed by THz time domain spectroscopy. We show surface functionalization of the antennas with a thin layer of α-lipoic acid that red-shifts the antenna resonances by about 20 GHz. Moreover, we show that antennas protected with a silicon nitride cap layer exhibit a comparable red-shift when covered with the biolayer. This suggests that the electromagnetic fields at the hotspot extend well beyond the cap layer, enabling the possibility to use the antennas with an improved protection of the plasmonic material in conjunction with microfluidics.

Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells.

Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.

The receptor of the type I interferon family.

All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how a multitude of subtypes can generate similar signaling outputs but also govern specific cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.

Structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking.

The pleiotropic activity of type I interferons has been attributed to the specific interaction of IFN with the cell-surface receptor components ifnar1 and ifnar2. To date, the structure of IFN has been solved, but not that of the receptor or the complex. In this study, the structure of the IFN-alpha 2-ifnar2 complex was generated with a docking procedure, using nuclear Overhauser effect-like distance constraints obtained from double-mutant cycle experiments. The interaction free energy between 13 residues of the ligand and 11 of the receptor was measured by double-mutant cycles. Of the 100 pairwise interactions probed, five pairs of residues were found to interact. These five interactions were incorporated as distance constraints into the flexible docking program prodock by using fixed and movable energy-gradient grids attached to the receptor and ligand, respectively. Multistart minimization and Monte Carlo minimization docking of IFN-alpha 2 onto ifnar2 converged to a well-defined average structure, with the five distance constraints being satisfied. Furthermore, no structural artifacts or intraloop energy strain were observed. The mutual binding sites on IFN-alpha 2 and ifnar2 predicted from the model showed an almost complete superposition with the ones determined from mutagenesis studies. Based on this structure, differences in IFN-alpha 2 versus IFN-beta binding are discussed.

Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy.

Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constants over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFNalpha2) and the extracellular domain of its receptor ifnar2 (ifnar2-EC). We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, noncompetitive mAb provided stable, but still reversible, immobilization of ifnar2-EC. We measured kinetics and affinity of numerous of mutants of IFNalpha2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and association rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 microM were measured with protein concentrations up to 50 microM without no background signal or nonspecific binding. The instrument detection limit is approximately 10 pm without the need for temperature stabilization or referencing channels. The system proved effective for large-scale mutational analysis involving alanine scanning mutagenesis and double mutant cycles.

Operative experiences with thoracoabdominal aortic aneurysms.

During a 27-year period, resection of 690 aneurysms of the descending thoracic and/or abdominal aorta were performed. Thirty (4.3%) were thoracoabdominal aneurysms. Although the series of thoracoabdominal aneurysms is small, there was continued improvement in protection of the abdominal viscera and spinal cord from ischemic injury. Operative survivors experienced good late (68% at 5 yrs.) survival. Each of the last 12 pts. in the series survived the operation and 9 are still alive. The surgical results justify a more aggressive stance regarding resection of the thoracoabdominal aneurysms.

New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface.

Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an anti-viral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)alpha2 by 35 individual mutations to alanine and isosteric residues. Of the six "hot-spot" residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFNalpha2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential.

Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy.

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.

Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2.

Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling.

Is routine postaneurysmectomy hemodynamic assessment of the inferior mesenteric artery circulation helpful?

All patients with an abdominal aortic aneurysm treated during a 27-year period by one surgical group at the MidAmerica Heart Institute were included in this study. A prospective routine postaneurysmectomy hemodynamic assessment of the inferior mesenteric artery (IMA) circulation was performed in a test group of consecutive patients operated on by one surgeon. When a mean IMA stump pressure

Integrated optical surface plasmon resonance immunoprobe for simazine detection.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.

Biophysical analysis of the interaction of human ifnar2 expressed in E. coli with IFNalpha2.

Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNalpha2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFNalpha2. The stoichiometry of binding IFNalpha2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor. No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectorscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence de-quenching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The surface properties of the IFNalpha2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction.

Coronary artery bypass for isolated disease of the left anterior descending artery. Late survival of 648 patients.

We studied a series of 648 consecutive patients who underwent coronary artery bypass grafting for isolated primary disease of the anterior descending coronary artery. We evaluated the patients periodically during a long-term follow-up period of up to 17 years. We studied factors such as survival, survival without acute event (i.e., acute myocardial infarction, repeat coronary artery bypass, and percutaneous transluminal coronary angioplasty), and asymptomatic survival (i.e., survival without acute event or angina). We further analyzed these factors as they occurred in patients who received only saphenous vein grafts versus their occurrence in patients who received internal mammary artery grafts. There was 1 death in the early postoperative period (defined as 30 days or earlier after the operation). The 5-, 10-, and 15-year survival rates were 94.8%, 86.6%, and 72.2%, respectively. These survival rates are slightly better than those of an age- and sex-matched United States census population. In our series, the rates of survival, event-free survival, and asymptomatic survival were better, although not significantly so, in the group of 108 patients in whom the internal mammary artery was used as the bypass conduit. We conclude that patients who undergo coronary artery bypass grafting for isolated disease of the left anterior descending coronary artery enjoy normal survival rates, in comparison with the survival rates of an age- and sex-matched United States census population, through at least the 1st 16 postoperative years. Additionally, patients who receive an internal mammary artery bypass graft have slightly better rates of survival, event-free survival, and asymptomatic survival than do those who receive only saphenous vein grafts.

25-year trends in resection of abdominal aortic aneurysms.

An attempt was made to document trends that have occured over a 25-year period in clinical presentation, preoperative evaluation, operative management, and patient outcome in patients with an abdominal aortic aneurysm. The experience (574 aneurysmectomies) of one cardiovascular surgical group was analyzed by retrospective review of hospital and office records. Changes over time of patients' ages, aneurysm sizes and statuses, prior myocardial revascularization, operative mortality, and certain other parameters were evaluated. During the period of study, there was a significant decrease in aneurysm size, increase in patients' ages, and an increased incidence of previous coronary artery bypass. No ruptured aneurysm was < 5 cm in diameter. The incidence of rupture and the operative mortality in patients with a ruptured aneurysm did not change significantly. There was a significantly (p = 0.03) lower operative mortality of 0.4% in the latter half of the series for elective aneurysmectomy. Increased utilization of preoperative cardiologic evaluation, and myocardial revascularization, has been associated with a decreased operative mortality in patients undergoing elective aneurysmectomy even though the patients are now older and have more age-related comorbidities. Elective aneurysmectomy should be offered to most patients when an abdominal aortic aneurysm is > or =5 cm in diameter.

Label-free monitoring of DNA-ligand interactions.

We report on the label- and isotope-free monitoring of DNA interactions with low-molecular-weight ligands. An optical technique based on interference at thin layers was used to monitor in real time binding of ligands at DNA which was immobilized by Coulomb interactions at a positively charged surface. Approximately 2 ng DNA/m2 was irreversibly bound to the surface, which remained stable over several days. This result was confirmed by characterization of the layer using spectroscopic ellipsometry. During incubation of immobilized DNA with a variety of intercalators and other DNA-binding compounds in a flow system, interactions were monitored by reflectometric interference spectroscopy. Binding effects between 10 and 400 pg/ mm2 were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charged reference surface. Variation of intercalator concentration allowed the characterization of interaction with respect to kinetics and thermodynamics by the evaluation of binding rate and equilibrium coverage. The affinity constants were determined in the range between 10(5) and 10(6) M-1, in good agreement to those obtained by homogeneous phase assays. Association rate constants between 10(3) and 10(5) M-1 s-1 and dissociation rate constants between 10(-1) and 10(-2) s-1 were determined by evaluation of the binding curves. Both the fast and simple test format and a universal applicability make the new technique described attractive for detecting and characterizing interaction of low-molecular-weight molecules with DNA.

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Early repair of postinfarction ventricular septal rupture.

BACKGROUND: Postinfarction rupture of the interventricular septum is usually fatal without surgical intervention. The optimal timing and the most appropriate technique of surgical repair remain unsettled. METHODS: The results of surgical closure of postinfarction ventricular septal defect in a consecutive series of patients seen over a 24-year period were reviewed and analyzed. Late follow-up was obtained in all patients who survived the operation. RESULTS: Sixty of 76 patients treated surgically exhibited cardiogenic shock, low cardiac output syndrome, or both at the time of operation. A plan of early operative intervention was followed in these unstable patients, with 60% of them undergoing repair within 24 hours of septal rupture. For the entire series of patients, the hospital mortality rate was 40.8%; survival was 41.5% at 5 years and 25.6% at 10 years postoperatively. CONCLUSIONS: Significant trends observed during the period of study were a more aggressive stance regarding surgical intervention in all patients who presented with hemodynamic instability and improved survival in those patients who presented with septal rupture complicating an inferior myocardial infarction.

Specific binding of low molecular weight ligands with direct optical detection.

The characterization of low molecular weight ligand interaction with receptor molecules is of importance for the investigation of biological processes and for drug research. We report on the investigation of the binding of low molecular weight ligands to immobilized receptors by label-free detection. Reflectometric interference spectroscopy, an optical transducer which allows the monitoring of a few picograms per square millimetre changes in surface coverage, was used to study two model systems. In both cases detection of the binding event was successful. High affinity binding of biotin to immobilized streptavidin was clearly detectable at receptor surface concentrations as low as 1-2 x 10(10) binding sites/mm2. Linear correlation between the receptor surface concentration and the response to biotin binding was observed. Using immobilized DNA, we investigated the binding of common intercalators with respect to kinetics and thermodynamics by evaluation of the association and the dissociation part of the binding curve. Bi-exponential increase and decrease of intercalator loading was observed, indicating complex interaction kinetics. The four structurally different intercalators showed significant distinction in binding kinetics and equilibrium signals. Improvement of experimental parameters is required to obtain more reliable kinetic data.

Characterization of grating couplers for affinity-based pesticide sensing.

The reflection grating coupler for direct affinity sensing is characterized in detail. The performance of this device and its potential in affinity sensing application are investigated with two affinity-based systems: A self-assembling protein-multilayer system based on avidin-biotin interaction was used to compare the response of the device with theoretical expectations. The analytical performance was characterized by a pesticide immunoassay carried out in an indirect test format with a covalently immobilized triazine derivative. Experimentally determined parameters were in good agreement with model calculations. During the binding of 12 protein monolayers at the surface, the change in effective refractive index Dn(eff) detected for a single layer decreased from approximately 8 x 10(-4) to less than 4 x10(-5) by more than 95%, indicating a filling of the evanescent field. By comparison with bulk refractive-index measurements, a refractive index n(D) approximately 1.38 of the protein multilayer was estimated. Fitting of the model gave a refractive index n(D) = 1.377 of the protein multilayer and an average thickness of 11 nm for a single protein layer. An average noise of Dn(eff) = 8.5 x 10(-7) was detected, corresponding to approximately 1% of the maximum response for a protein monolayer. At a triazine derivative attached to the surface through dextran-based surface chemistry, a maximum antibody loading that corresponds to an Dn(eff) of 1.5 x 10(-3) was observed. In an indirect immunoassay of the herbicide simazine, a detection limit of 0.25 mug/1 of simazine was reached with polyclonal Fab fragments in a concentration of 1 mug/ml.