A Journey with LGMD: From Protein Abnormalities to Patient Impact.

The limb-girdle muscular dystrophies (LGMD) are a collection of genetic diseases united in their phenotypical expression of pelvic and shoulder area weakness and wasting. More than 30 subtypes have been identified, five dominant and 26 recessive. The increase in the characterization of new genotypes in the family of LGMDs further adds to the heterogeneity of the disease. Meanwhile, better understanding of the phenotype led to the reconsideration of the disease definition, which resulted in eight old subtypes to be no longer recognized officially as LGMD and five new diseases to be added to the LGMD family. The unique variabilities of LGMD stem from genetic mutations, which then lead to protein and ultimately muscle dysfunction. Herein, we review the LGMD pathway, starting with the genetic mutations that encode proteins involved in muscle maintenance and repair, and including the genotype-phenotype relationship of the disease, the epidemiology, disease progression, burden of illness, and emerging treatments.

Two-stage classifiers that minimize PCA3 and the PSA proteolytic activity testing in the prediction of prostate cancer recurrence after radical prostatectomy.

INTRODUCTION: Early biochemical recurrence after prostate cancer surgery is associated with higher risk of aggressive disease and cancer specific death. Many new tests are being developed that will predict the presence of indicators of aggressive disease like early biochemical recurrence. Since recurrence occurs in less than 10% of patients treated for prostate cancer, validation of such tests will require expensive testing on large patient groups. Moreover, clinical application of the validated test requires that each new patient be tested. In this report we introduce a two-stage classifier system that minimizes the number of patients that must be tested in both the validation and clinical application of any new test for recurrence. MATERIALS AND METHODS: Expressed prostatic secretion specimens were prospectively collected from 450 patients prior to robot-assisted radical prostatectomy for prostate cancer. Patients were followed for 2.5 years for evidence of biochemical recurrence. Standard clinical parameters, the levels proteolytic activity of prostate specific antigen (PSA) and the levels of PCA3 RNA, PSA RNA and TMPRSS2:ERG fusion RNA were determined in each prospective patient specimen for subsequent correlation with biochemical recurrence. RESULTS: While levels of PCA3 and PSA proteolytic activity (PPA) in prostatic secretions provided an effective pre-surgical predictor of early biochemical recurrence in prostate cancer, application of the two-stage classifier shows that only 60% of the patients need these tests. CONCLUSION: Two-stage classifiers can provide a parsimonious approach to both the validation and clinical application of biomarker-based tests. Adoption of the two-stage neutral zone classifier can reduce unnecessary testing in prostate cancer treatment.

PSA enzymatic activity: a new biomarker for assessing prostate cancer aggressiveness.

BACKGROUND: With the advent of widespread prostate-specific antigen (PSA) testing in recent decades, prostate cancer (PCa) has emerged as the most frequently diagnosed non-skin cancer among men in the U.S. and Europe. Greater screening rates coupled with improved detection methods have caused a controversial upsurge in the number of men undergoing prostate biopsy and subsequent treatment. However, current diagnostic techniques generally suffer from limited ability to identify which seemingly indolent cancers are biologically aggressive. METHODS: We collected prostatic fluid from 778 post-radical prostatectomy specimens and randomly selected samples from both the clinically confirmed aggressive (n = 50) and non-aggressive (n = 50) prostate cancer populations. We measured the level of proteolytic enzyme activity of PSA (aPSA) in each sample and used receiver operating characteristic (ROC) analysis to correlate aPSA levels with prostate cancer aggressiveness. RESULTS: We found aPSA in prostatic fluid to be inversely proportional to disease stage, such that patients with the most aggressive PCa have on average significantly reduced aPSA compared to those with less aggressive disease. Significantly, our results suggest that many (22% in our study population) of the diagnosed patients with non-aggressive PCa could have averted or delayed radical prostatectomy. CONCLUSIONS: Given the high level of debate surrounding PSA screening effectiveness [3-5] and the recent U.S. Preventative Services Task Force recommendation to discontinue PSA screening [6], our results provide renewed hope that a clinical monitoring tool may emerge that truly refines PCa treatment decision-making.

Spectroscopic and redox properties of amine-functionalized K2[OsII(bpy)(CN)4] complexes.

We report the first examples of amine-functionalized K(2)[Os(II)(bpy)(CN)(4)] (bpy = 2,2'-bipyridine) complexes. The tetracyanoosmate complexes were prepared by UV irradiation (λ = 254 nm) of K(4)[Os(II)(CN)(6)] and primary amine-functionalized bpy ligands in acidic aqueous media. The aqueous solution pH dependences of the spectroscopic and redox properties of 4,4'- and 5,5'-substituted complexes have been investigated. The pendant amine functional groups and coordinated cyanide ligands are basic sites that can be sequentially protonated, thereby allowing systematic tuning of electrochemical and optical spectroscopic properties.

Ferrocene and maleimide-functionalized disulfide scaffolds for self-assembled monolayers on gold.

A series of ferrocene-based electroactive molecules (EAMs) containing maleimide and disulfide groups in different asymmetric and branched architectures were designed and synthesized. Stable monolayers of each EAM on gold electrodes were confirmed by cyclic voltammetry. Importantly, these EAMs expand the repertoire of monolayer building blocks amenable to modular biofunctionalization for applications in electrochemical biosensor fabrication.

Protein binding and the electronic properties of iron(II) complexes: an electrochemical and optical investigation of outer sphere effects.

Metalloenzymes and electron transfer proteins influence the electrochemical properties of metal cofactors by controlling the second-sphere environment of the protein active site. Properties that tune this environment include the dielectric constant, templated charge structure, van der Waals interactions, and hydrogen bonds. By systematically varying the binding of a redox-active ligand with a protein, we can evaluate how these noncovalent interactions alter the electronic structure of the bound metal complex. For this study, we employ the well-characterized avidin-biotin conjugate as the protein-ligand system, and have synthesized solvatochromic biotinylated and desthiobiotinylated iron(II) bipyridine tetracyano complexes ([Fe(BMB)(CN)(4)](2-) (1) and [Fe(DMB)(CN)(4)](2-) (2)). The binding affinities of 1 and 2 with avidin are 3.5 × 10(7) M(-1) and 1.5 × 10(6) M(-1), respectively. The redox potentials of 1 and 2 (333 mV and 330 mV) shift to 193 mV and 203 mV vs Ag/AgCl when the complex is bound to avidin and adsorbed to a monolayer-coated gold electrode. Upon binding to avidin, the MLCT1 band red-shifts 20 nm for 1 and 10 nm for 2. Similarly, the MLCT2 band for 1 red-shifts 7 nm and the band for 2 red-shifts 6 nm. For comparison, the electronic properties of 1 and 2 were investigated in organic solvents, and similar shifts in the MLCT bands and redox potentials were observed. An X-ray crystal structure of 1 bound to avidin was obtained, and molecular dynamics simulations were performed to analyze the protein environment of the protein-bound transition metal complexes. Our studies demonstrate that changes in the binding affinity of a ligand-receptor pair influence the outer-sphere coordination of the ligand, which in turn affects the electronic properties of the bound complex.

Electroactive self-assembled monolayers on gold via bipodal dithiazepane anchoring groups.

Novel dithiazepane-functionalized ferrocenyl-phenylethynyl oligomers 1 and 2 have been synthesized. Self-assembled monolayers (SAMs) of these ferrocene derivatives have been studied by X-ray photoelectron spectroscopy, ellipsometry, and cyclic voltammetry. It has been shown by XPS that monolayers of the dithiazepane-anchored molecules on gold electrodes contain gold-thiolate species. Cyclic voltammetry of the SAMs were characteristic of stable electroactive monolayers even for single-component SAMs of 1 and 2, with the more ideal responses recorded for the two-component SAMs diluted with undecanethiol. The small variation in peak splittings at progressively higher scan rates in these SAMs makes dithiazepane-bridged redox species promising candidates for further studies on molecular wires with bipodal anchoring.

Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes.

Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.

A bio-barcode assay for on-chip attomolar-sensitivity protein detection.

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.

Structures of DNA-linked nanoparticle aggregates.

The room-temperature structure of DNA-linked gold nanoparticle aggregates is investigated using a combination of experiment and theory. The experiments involve extinction spectroscopy measurements and dynamic light scattering measurements of aggregates made using 60 and 80 nm gold particles and 30 base-pair DNA. The theoretical studies use calculated spectra for models of the aggregate structures to determine which structure matches the observations. These models include diffusion-limited cluster-cluster aggregation (DLCA), reaction-limited cluster-cluster aggregation (RLCA), and compact (nonfractal) cluster aggregation. The diameter of the nanoparticles used in the experiments is larger than has been considered previously, and this provides greater sensitivity of spectra to aggregate structure. We show that the best match between experiment and theory occurs for the RLCA fractal structures. This indicates that DNA hybridization takes place under irreversible conditions in the room-temperature aggregation. Some possible structural variations which might influence the result are considered, including the edge-to-edge distance between nanoparticles, variation in the diameter of the nanoparticles, underlying lattice structures of on-lattice compact clusters, and positional disorders in the lattice structures. We find that these variations do not change the conclusion that the room-temperature structure of the aggregates is fractal. We also examine the variation in extinction at 260 nm as temperature is increased, showing that the decrease in extinction at temperatures below the melting temperature is related to a morphological change from fractal toward compact structures.

Gold nanoparticle probes for the detection of nucleic acid targets.

BACKGROUND: Advances in nanoscience are having a significant impact on many scientific fields and are resulting in the development of a variety of important technologies. This impact is particularly large in the field of biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection, with DNA- or protein-functionalized gold nanoparticles used as the target-specific probes. METHODS: Assays provide an analysis of the unique biophysical properties displayed by gold nanoparticles and have advantages over conventional detection methods (e.g., molecular fluorophores, real-time polymerase chain reaction, RT-PCR, enzyme linked immunosorbent assays, ELISAs, gel electrophoresis, and microarray technologies). CONCLUSION: Some of the advantages include the assays' PCR-like sensitivity, their selectivity for target sequences, their capacity for massive multiplexing, their time efficiency, and most importantly, their ability to be performed at the point of care.

A bio-bar-code assay based upon dithiothreitol-induced oligonucleotide release.

The recently developed bio-bar-code assay for the PCR-less detection of protein and nucleic acid targets has been shown to be extraordinarily sensitive, exhibiting low attomolar sensitivity for protein targets and high zeptomolar sensitivity for nucleic acid targets. In the case of DNA detection, the original assay relies on three distinct oligonucleotide strands on a single nanoparticle for target identification and signal amplification. Herein, we report the development of a new nanoparticle probe that can be used in the bio-bar-code assay, which requires only one thiolated oligonucleotide strand. This new assay relies on the ability to liberate the adsorbed thiolated oligonucleotides from the gold nanoparticle surface with dithiothreitol (DTT), which simplifies the assay and increases its quantitative capabilities. The utility of this new DTT-based system is demonstrated by detecting a mock mRNA target using both fluorescent and scanometric assay readouts. When the scanometric readout is used, the sensitivity of the assay is 7 aM and quantification can be accomplished over the low-attomolar to the mid-femtomolar concentration range.

Supporting electrolyte and solvent effects on single-electron double layer capacitance charging of hexanethiolate-coated Au140 nanoparticles.

Sequential injections of single electrons (or holes) into the cores of Au(140) hexanethiolate monolayer-protected clusters (MPCs) occur at measurably different electrochemical potentials owing to the extremely small (subattofarad) values of the single MPC capacitance (C(MPC)) of the nanoparticle. The potential increment for each sequential injection is DeltaV = e/C(MPC). The dependence of DeltaV on the concentration of supporting electrolyte (from 1 to 100 mM), measured using square wave voltammetry, is shown to be caused, primarily, by changes in the diffuse double layer component (C(DIFFUSE)) of C(MPC). The dependence of C(DIFFUSE) on r(core), the radius of the nanoparticle, is considered. Additionally, significant changes in the magnitude of the compact double layer component (C(COMPACT), equivalent to the Stern layer) of C(MPC) were induced by adding hydrophobic solvent components such as hexane or dodecane or by introducing hydrophobic electrolyte ions (tetrabutyl-, tetrahexyl-, and tetraoctylammonium, perchlorate, and tetraphenylborate). These changes are interpreted as specific solvation and ion penetration of the hexanethiolate monolayer. For brevity we will refer to these phenomena as solvation/penetration phenomena.

Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease.

The recently developed ultrasensitive bio-barcode assay was used to measure the concentration of amyloid-beta-derived diffusible ligands (ADDLs), a potential soluble pathogenic Alzheimer's disease (AD) marker, in the cerebrospinal fluid (CSF) of 30 individuals. ADDL concentrations for the subjects diagnosed with AD were consistently higher than the levels in the CSF taken from nondemented age-matched controls. Studies of ADDLs or for any other potential pathogenic AD markers in CSF have not been possible because of their low concentration in CSF (<1 pM). This study is a step toward a diagnostic tool, based on soluble pathogenic markers for the debilitating disease.

Electron hopping dynamics in monolayer-protected au cluster network polymer films by rotated disk electrode voltammetry.

Electrons are transported within polymeric films of alkanethiolate monolayer-protected Au clusters (MPCs) by electron hopping (self-exchange) between the metal cores. The surrounding monolayers, the molecular linkers that generate the network polymer film, or both, presumably serve as tunneling bridges in the electron transfers. This paper introduces a steady-state electrochemical method for measuring electron hopping rates in solvent-wetted and swollen, ionically conductive MPC films. The films are network polymer films of nanoparticles, coated on a rotated disk electrode that is contacted by a solution of a redox species (decamethylferrocene, CpFe). Controlling the electrode potential such that the film mediates oxidation of the redox probe can force control of the overall current onto the rate of electron hopping within the film, which is characterized as the apparent electron diffusion coefficient D(E). D(E) is translated into an apparent electron hopping rate k(ET) by a cubic lattice model. The experiment is applied to MPC network polymer films linked by alpha,omega-alkanedithiolates and by metal ion-carboxylate connections. We evaluate the dependencies of apparent hopping rate on CpFe concentration, film thickness, electrode potential relative to the CpFe formal potential, film-swelling solvent, and temperature. The apparent hopping rates are in the 10(4)-10(5) s(-)(1) range, which is slower than those for the same kind of MPC films, but in a dry (nonswollen) state measured by electronic conductivities.

Hexanethiolate monolayer protected 38 gold atom cluster.

The nucleation-growth-passivation Brust reaction has been modified so as to enrich the product in useful quantities of a 38-atom gold nanoparticle coated with a hexanethiolate monolayer. Two modifications are described, using -78 degrees C reduction temperature and a hyperexcess of thiol. Compositional evidence is presented that establishes the product as a Au38(C6)24 hexanethiolate monolayer protected cluster (MPC), based on transmission electron microscopy, laser ionization-desorption mass spectrometry, thermogravimetric analysis, and elemental analysis. Reverse phase HPLC confirms the relatively good monodispersity of the MPC products, but high-resolution double-column HPLC reveals that the MPCs are a mixture of closely related but chromatographically distinct products. Voltammetry, low energy spectrophotometry, and spectroelectrochemistry reveal, respectively, a 1.6 eV electrochemical energy gap between the first oxidation and the first reduction, an optical HOMO-LUMO energy absorbance edge at 1.3 eV, and a bleaching of optical absorbance near the 1.3 eV band edge that accompanies electrochemical oxidation of the nanoparticle.

Growth, conductivity, and vapor response properties of metal ion-carboxylate linked nanoparticle films.

Nanoparticles of metals (Au, Ag, Pd, alloys) in the size range 1-3 nm diameter can be stabilized against aggregation of the metal particles by coating the metal surface with a dense monolayer of ligands (thiolates). The stabilization makes it possible to analytically define the nanoparticle composition (for example, Au140(hexanethiolate)53, I) and to elaborate the chemical functionality of the protecting monolayer (for example, Au140(C6)35(MUA)18, II, where C6 = hexanethiolate and MUA = mercaptoundecanoic acid). Network polymer films (IIfilm) on interdigitated array electrodes can be prepared from II, based on cation coordination (i.e., Cu2+, Zn2+, Ag+, methyl viologen) by the carboxylates of MUA. The electronic conductivity of the IIfilm network polymer films occurs by electron hopping between the Au140 nanoparticle cores, and offers an avenue for investigation of metal-to-metal nanoparticle electron transfer chemistry. The report begins with a brief summary of what is known about metal nanoparticle electron transfer chemistry. The investigation goes on to assess factors that influence the dynamics of film formation as well as film conductivity, in the interest of better understanding the parameters affecting electron hopping rates in IIfilm network polymer films. Finally, sorption of organic vapors into IIfilm causes a decreased electronic conductivity and increased mass that can be assessed using quartz crystal microbalance measurements. The change in electronic conductivity can be exploited for the sensing of organic vapors.