Rapid and Sensitive Identification and Discrimination of Bound/Unbound Ligands on Colloidal Nanocrystals via Direct Analysis in Real-Time Mass Spectrometry.

Direct analysis in real-time mass spectrometry (DART-MS) has been applied to the characterization of colloidal nanocrystal surface ligands. The nanocrystals (NCs) in colloidal suspension were purified and deposited onto a solid substrate, and the solvent was allowed to evaporate. Ligand desorption was thermally stimulated using a temperature ramp from 30 °C up to 530 °C, and the desorbed ligands were introduced into a DART-MS instrument where metastable He atoms provide energy for ionization and fragmentation through the reaction with ambient vapors including O2 and H2O. The method allows the identification of ligand species with various functional groups, even in complex, mixed-ligand samples. Bound and unbound molecules can be distinguished based on the desorption temperature. In ideal cases, the desorption profile for a given molecule can be analyzed according to methods adapted from thermal desorption spectroscopy (TDS) to estimate desorption activation energy for NC-bound ligands. Results are presented and discussed for different nanocrystal and ligand types. The method is a promising complement to the range of existing tools for NC ligand analysis.

Improvement of Bi doping in ZnO nanocrystals by co-doping with Al: crystal geometry calculations and photocatalytic activity.

In this work we demonstrate enhancement in visible-light photocatalytic activity (PCA) of ZnO nanoparticles (NPs) with minimal attenuation of visible light transmittance. This approach can benefit numerous optoelectronic and photocatalytic applications. ZnO NPs were p-n co-doped with Al and Bi to improve Bi doping into the ZnO crystal. Al- and/or Bi-doped ZnO was coprecipitated by ammonia from aqueous nitrate solutions of Zn2+, Al3+, and Bi3+, followed by microwave heating. Doping concentrations in Al- and Bi- doped ZnO (AZO and BZO) and Al/Bi co-doped ZnO (ABZO) were 1, 3, 5, and 7 mole %. The resulting NPs were characterized by XRD, TEM, EDS, BET, and UV-visible absorption. While EDS shows that almost all added Bi was incorporated into the ZnO, XRD analysis of BZO reveals formation of α-Bi2O3 as a secondary phase due to the poor Bi solubility in ZnO. Co-doping of Al with Bi suppressed α-Bi2O3 formation and increased Bi solubility in ZnO. XRD-based calculations of the lattice constants and deformation strain, stress, and energy all show insertion of Al and/or Bi into the crystal with different extents according to the dopants' solubilities into ZnO. AZO and BZO NPs had E g lowered by 0.05-1.39 eV and 0.30-0.70 eV, respectively, relative to ZnO. On the other hand, ABZO had E g reductions of only 0.01-0.20 eV due to formation of acceptor-donor complex through co-doping. ABZO gave higher PCA enhancements with respect to E g reductions (Δk photo/-ΔE g) than either AZO and BZO, with values up to 370, 126, and 13 min-1 eV-1, respectively.

Vibronic Excitons and Conical Intersections in Semiconductor Quantum Dots.

Surface defects and organic surface-capping ligands affect the photoluminescence properties of semiconductor quantum dots (QDs) by altering the rates of competing nonradiative relaxation processes. In this study, broadband two-dimensional electronic spectroscopy reveals that absorption of light by QDs prepares vibronic excitons, excited states derived from quantum coherent mixing of the core electronic and ligand vibrational states. Rapidly damped coherent wavepacket motions of the ligands are observed during hot-carrier cooling, with vibronic coherence transferred to the photoluminescent state. These findings suggest a many-electron, molecular theory for the electronic structure of QDs, which is supported by calculations of the structures of conical intersections between the exciton potential surfaces of a small ammonia-passivated model CdSe nanoparticle.

Structural insights into the evolution of an antibody combining site.

The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 A resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarity.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Quenching mechanism of Zn(salicylaldimine) by nitroaromatics.

Nitroaromatics and nitroalkanes quench the fluorescence of Zn(Salophen) (H2Salophen = N,N'-phenylene-bis-(3,5-di- tert-butylsalicylideneimine); ZnL(R)) complexes. A structurally related family of ZnL(R) complexes (R = OMe, di-tBu, tBu, Cl, NO2) were prepared, and the mechanisms of fluorescence quenching by nitroaromatics were studied by a combined kinetics and spectroscopic approach. The fluorescent quantum yields for ZnL(R) were generally high (Phi approximately 0.3) with sub-nanosecond fluorescence lifetimes. The fluorescence of ZnL(R) was quenched by nitroaromatic compounds by a mixture of static and dynamic pathways, reflecting the ZnL(R) ligand bulk and reduction potential. Steady-state Stern-Volmer plots were curved for ZnL(R) with less-bulky substituents (R = OMe, NO2), suggesting that both static and dynamic pathways were important for quenching. Transient Stern-Volmer data indicated that the dynamic pathway dominated quenching for ZnL(R) with bulky substituents (R = tBu, DtBu). The quenching rate constants with varied nitroaromatics (ArNO2) followed the driving force dependence predicted for bimolecular electron transfer: ZnL* + ArNO2 --> ZnL(+) + ArNO2(-). A treatment of the diffusion-corrected quenching rates with Marcus theory yielded a modest reorganization energy (lambda = 25 kcal/mol), and a small self-exchange reorganization energy for ZnL*/ZnL(+) (ca. 20 kcal/mol) was estimated from the Marcus cross-relation, suggesting that metal phenoxyls may be robust biological redox cofactors. Electronic structure calculations indicated very small changes in bond distances for the ZnL --> ZnL(+) oxidation, suggesting that solvation was the dominant contributor to the observed reorganization energy. These mechanistic insights provide information that will be helpful to further develop ZnL(R) as sensors, as well as for potential photoinduced charge transfer chemistry.

Evolution of a cytokine using DNA family shuffling.

DNA shuffling of a family of over 20 human interferon-alpha (Hu-IFN-alpha) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-alpha2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-alpha2a and 185-fold relative to Hu-IFN-alpha1. Remarkably, the three most active clones were more active than the native murine IFN-alphas. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.

Crystal structures of the free and liganded form of an esterolytic catalytic antibody.

The crystal structure of the esterase catalytic antibody 48G7 has been determined in the presence of hapten at 2.0 A resolution and in the absence of hapten at 2.7 A resolution. The root-mean-square difference between the two structures is 0.6 A for the variable domain and 0.7 A for the constant domain. Comparison of the active site shows that no significant changes occur upon hapten binding as main-chain and side-chain displacements are negligible. Complex formation occurs as hapten fits into a pre-formed pocket about 10 A deep. Although 151 water molecules were modeled into the 48G7-hapten structure, none are bound in the active site. Comparison of the 48G7 structures with those of other published ester hydrolysis antibodies illustrates an emerging theme used by esterolytic antibodies in binding their (nitro-)phenyl haptens and in hydrolysing their cognate esters and carbonates: hapten is bound with the aryl end buried deep in the binding pocket, and the phosphonate moiety is responsible for the majority of the binding energy to the antibody-hapten interaction.

Applications of DNA shuffling to pharmaceuticals and vaccines.

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.

Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins.

Recent developments in directed evolution technologies combined with innovations in robotics and screening methods have revolutionized protein engineering. These methods are being applied broadly to many fields of biotechnology, including chemical engineering, agriculture and human therapeutics. More specifically, DNA shuffling and other methods of genetic recombination and mutation have resulted in the improvement of proteins of therapeutic interest. Optimizing genetic diversity and fitness through iterative directed evolution will accelerate improvements in engineered protein therapeutics.

Optic nerve head drusen and pseudotumor cerebri.

A 42-year-old woman presented with a history of headache. Results of funduscopic examination revealed elevated disc margins and bilateral optic nerve head drusen. Lumbar puncture, head computed tomography, and fluorescein fundus angiography results were consistent with the diagnosis of pseudotumor cerebri and coexistent disc drusen. Visual loss was demonstrated by formal perimetry. Headaches were unresponsive to a medical regimen that included prednisone, glycerol, acetazolamide, furosemide, and repeated lumbar punctures. A lumbar peritoneal shunt was performed, with immediate resolution of headache. Optic disc drusen can be associated with pseudotumor cerebri and can lead to diagnostic confusion.

Torsion and bending analysis of internal fixation techniques for femoral neck fractures: the role of implant design and bone density.

To evaluate pin/screw/plate fixation for management of femoral neck fractures, 39 proximal femora were tested in both torsion and flexion under physiological loading conditions. Three, four, or five implants of six commonly used multiple-fixation devices, and a sliding hip screw with and without an additional 6.5-mm cancellous screw were examined in paired femora. The intact and postfixation femora were initially subjected to a single applied moment, and the torsion and bending stiffness were determined from the load-deformation data. Postfixation femora were also subjected to cyclic loading in flexion at three load ranges, and fixation was judged successful if no failure occurred on or before 1,500 cycles of 667 to 2,000 N of a combined compressive force and moment. Anterior-posterior and lateral radiographs of each specimen were taken after fixation in order to evaluate Singh's index of bone density, fracture reduction, implant placement, and cross-sectional diameter of the femoral neck. Bone density was also evaluated by computed tomography (CT) and physical measurement of core samples obtained from the femoral head. The results indicate that there appears to be no justification for the use of more than three pin/screw implants for management of femoral neck fractures. Bone density was found to correlate with fracture stability and may be a useful predictor of fixation success.

The immunogenicity of biopharmaceuticals. Lessons learned and consequences for protein drug development.

Most biopharmaceuticals, including those proteins that are more or less identical to native human proteins, induce antibodies in a significant fraction of patients. The main factors contributing to immunogenicity are impurities and the presence of aggregates. Sequence divergence from the native proteins only plays a minor role except in proteins from microbial, plant or distant vertebrate origin. In the majority of cases the antibodies have no biological or clinical effects. The most common clinical effect is the loss of efficacy of the biopharmaceutical. Serious complications of immunogenicity are rare. The best method to prevent immunogenicity is optimizing production, purification and formulation of the biopharmaceutical protein to generate soluble, non-aggregated, native protein free of contaminating adjuvants. The best way to predict immunogenicity in humans is evaluation in immune tolerant transgenic mice.

The role of endovascular therapy in lower extremity revascularization. Lessons learned and current strategies.

Percutaneous endovascular therapy has emerged as an important modality in the treatment of lower extremity ischemia. Its role is relatively small but better defined at present. Balloon angioplasty remains as the most useful and versatile of all catheter interventions. Common iliac artery lesions, if short and stenotic, are best managed with angioplasty. Percutaneous therapy of femoral-popliteal lesions is less satisfactory but applicable in a small subset of patients with favorable lesions. Intravascular stents and thrombolysis are viewed as major developments in the field. Critically ischemic limbs are seldom amenable to endovascular recanalization. The subspecialty of endovascular therapy should become increasingly the focus of attention by the contemporary vascular surgeon. Catheter technology is destined to influence profoundly current strategies and techniques in the treatment of vascular diseases.

The talent endoluminal AAA stent-graft system. Report of the phase I USA trial, and summary of worldwide experience.

In the United States, the Phase I Feasibility Study under IDE G970065 was approved by the Food and Drug Administration on 04/11/97. The approved protocol called for implantation of the bifurcated Talent spring stent-graft system on patients who are high-risk candidates for conventional surgery because of cardio-respiratory, medical, general, or local anatomical reasons which would likely complicate the technical execution of the operation or be accompanied by a high expected mortality rate. Patient enrollment was complete with 16 cases as of September 26, 1997. This was a multicenter experience involving five different sites. This is an ongoing study and patients, of course, will continue to be followed longitudinally. Phase II will likely be approved by the FDA for initiation in January or February of 1998. Standard-risk AAA patients will be entered into the study at this time; comparison with concurrent controls will be used for comparison with conventional surgery.