Diminished prefrontal cortex activation in patients with binge eating disorder associates with trait impulsivity and improves after impulsivity-focused treatment based on a randomized controlled IMPULS trial.

BACKGROUND: Behavioral and cognitive control are vital for healthy eating behavior. Patients with binge eating disorder (BED) suffer under recurrent binge eating episodes accompanied by subjective loss of control that results, among other factors, from increased impulsivity. METHODS: In the current study, we investigated the frontal network using functional near-infrared spectroscopy (fNIRS) during a food specific go/nogo task to assess response inhibition in 24 patients with BED (BMI range 22.6-59.7 kg/m2) compared to 12 healthy controls (HC) (BMI range 20.9-27 kg/m2). Patients with BED were invited to undergo fNIRS measurements before an impulsivity-focused cognitive behavioral group treatment, directly after this treatment and 3 months afterwards. As this was a planned subgroup analysis of the randomized controlled IMPULS trial, patients with BED were randomized either to the treatment group (n = 14) or to a control group (n = 10). The treatment group received 8 weekly sessions of the IMPULS treatment. RESULTS: We found a significant response inhibition effect (nogo minus go), in terms of an increased oxygenated hemoglobin response in the bilateral prefrontal cortex in both groups. The greatest response was observed when participants were instructed to go for healthy and withhold their response to unhealthy high caloric food cues. The healthy nogo condition failed to show a significant prefrontal inhibitory response, which was probably related to the task design, as the condition was considered more demanding. BED patients, especially those with higher trait impulsivity, showed a weaker activation of the prefrontal cortex during response inhibition, predominantly in the right hemisphere. Interestingly, three months after the treatment, patients of the treatment group increased their right prefrontal cortex activity during response inhibition. Likewise, increased prefrontal cortex activation correlated with decreased trait impulsivity after treatment. CONCLUSIONS: Our results suggest that patients with BED have limited resources to activate the prefrontal cortex when asked to inhibit a reaction onto food-specific stimuli. However, this effect could be partly driven by differences in BMI between the HC and BED group. Cognitive-behavioral therapy targeting impulsive eating behavior may improve prefrontal cortex recruitment during response inhibition.

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry "unit operations" supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Metal Chelating Crosslinkers Form Nanogels with High Chelation Stability.

We present a series of hydrogel nanoparticles (nanogels) incorporating either acyclic or cyclic metal chelates as crosslinkers. These crosslinkers are used to formulate polyacrylamide-based nanogels (diameter 50 to 85 nm) yielding contrast agents with enhanced relaxivities (up to 6-fold greater than Dotarem®), because this nanogel structure slows the chelator's tumbling frequency and allows fast water exchange. Importantly, these nanogels also stabilize Gd3+ within the chelator thermodynamically and kinetically against metal displacement through transmetallation, which should reduce toxicity associated with release of free Gd3+. This chelation stability suggests that the chelate crosslinker strategy may prove useful for other applications of metal-chelating nanoparticles in medicine, including other imaging modalities and radiotherapy.

Biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide.

Oxidative stress is caused predominantly by accumulation of hydrogen peroxide and distinguishes inflamed tissue from healthy tissue. Hydrogen peroxide could potentially be useful as a stimulus for targeted drug delivery to diseased tissue. However, current polymeric systems are not sensitive to biologically relevant concentrations of H(2)O(2) (50-100 µM). Here we report a new biocompatible polymeric capsule capable of undergoing backbone degradation and thus release upon exposure to such concentrations of hydrogen peroxide. Two polymeric structures were developed differing with respect to the linkage between the boronic ester group and the polymeric backbone: either direct (1) or via an ether linkage (2). Both polymers are stable in aqueous solution at normal pH, and exposure to peroxide induces the removal of the boronic ester protecting groups at physiological pH and temperature, revealing phenols along the backbone, which undergo quinone methide rearrangement to lead to polymer degradation. Considerably faster backbone degradation was observed for polymer 2 over polymer 1 by NMR and GPC. Nanoparticles were formulated from these novel materials to analyze their oxidation triggered release properties. While nanoparticles formulated from polymer 1 only released 50% of the reporter dye after exposure to 1 mM H(2)O(2) for 26 h, nanoparticles formulated from polymer 2 did so within 10 h and were able to release their cargo selectively in biologically relevant concentrations of H(2)O(2). Nanoparticles formulated from polymer 2 showed a 2-fold enhancement of release upon incubation with activated neutrophils, while controls showed a nonspecific response to ROS producing cells. These polymers represent a novel, biologically relevant, and biocompatible approach to biodegradable H(2)O(2)-triggered release systems that can degrade into small molecules, release their cargo, and should be easily cleared by the body.

Iron oxide nanoparticle-based magnetic resonance method to monitor release kinetics from polymeric particles with high resolution.

A new method to precisely monitor rapid release kinetics from polymeric particles using super paramagnetic iron oxide nanoparticles, specifically by measuring spin-spin relaxation time (T(2)), is reported. Previously, we have published the formulation of logic gate particles from an acid-sensitive poly-ß-aminoester ketal-2 polymer. Here, a series of poly-ß-aminoester ketal-2 polymers with varying hydrophobicities were synthesized and used to formulate particles. We attempted to measure fluorescence of released Nile red to determine whether the structural adjustments could finely tune the release kinetics in the range of minutes to hours; however, this standard technique did not differentiate each release rate of our series. Thus, a new method based on encapsulation of iron oxide nanoparticles was developed, which enabled us to resolve the release kinetics of our particles. Moreover, the kinetics matched the relative hydrophobicity order determined by octanol-water partition coefficients. To the best of our knowledge, this method provides the highest resolution of release kinetics to date.

An extracellular MRI polymeric contrast agent that degrades at physiological pH.

Macromolecular contrast agents have the potential to assist magnetic resonance imaging (MRI) due to their high relaxivity, but are not clinically useful because of toxicity due to poor clearance. We have prepared a biodegradable ketal-based polymer contrast agent which is designed to degrade rapidly at physiological pH by hydrolysis, facilitating renal clearance. In vitro, the agent degraded more rapidly at lower pH, with complete fragmentation after 24 h at pH 7.4. In vitro relaxivity measurements showed a direct correlation between molecular weight and relaxivity. We compared our polymer contrast agent with commercially available Magnevist in vivo by MRI imaging, as well as measuring the Gd concentration in blood. Our results show that our polymer contrast agent gives a higher contrast and intensity in the same organs and areas as Magnevist and is cleared from the blood at a similar rate. We aim to improve our polymer contrast agent design to develop it for use as a MRI contrast agent, and explore its use as a platform for other imaging modalities.

Protein nanopatterns by oxime bond formation.

Patterning proteins on the nanoscale is important for applications in biology and medicine. As feature sizes are reduced, it is critical that immobilization strategies provide site-specific attachment of the biomolecules. In this study, oxime chemistry was exploited to conjugate proteins onto nanometer-sized features. Poly(Boc-aminooxy tetra(ethylene glycol) methacrylate) was synthesized by free radical polymerization. The polymer was patterned onto silicon wafers using an electron beam writer. Trifluoroacetic acid removal of the Boc groups provided the desired aminooxy functionality. In this manner, patterns of concentric squares and contiguous bowtie shapes were fabricated with 150-170-nm wide features. Ubiquitin modified at the N-terminus with an α-ketoamide group and N(ε)-levulinyl lysine-modified bovine serum albumin were subsequently conjugated to the polymer nanopatterns. Protein immobilization was confirmed by fluorescence microscopy. Control studies on protected surfaces and using proteins presaturated with O-methoxyamine indicated that attachment occurred via oxime bond formation.

tuberculosis detection via rolling circle amplification.

Hybridization-based assays for DNA detection often use single-stranded DNA (ssDNA) probes to capture ssDNA targets in solution. Unfortunately, these assays are often not able to detect double-stranded DNA (dsDNA). Here, we achieve highly sensitive dsDNA target detection by including short oligonucleotide sequences during denaturing and cooling. After performing an isothermal nucleic acid amplification technique (Rolling Circle Amplification, RCA), these captured dsDNA targets are labeled, allowing single amplified molecules to be imaged and counted. This detection method was first applied to the detection of PCR-generated (polymerase chain reaction) dsDNA targets, yielding a limit of detection of 4.25 fM. As an application of the developed assay, the detection of extracted Mycobacterium tuberculosis (M. tb.) genomic DNA was attempted. A M. tb.-specific target was detected with high specificity compared to similar bacteria, and a detection limit of 10 000 colony forming units (cfu) ml-1 was achieved, close to the sensitivity required for clinical diagnosis.

Attomole DNA detection assay via rolling circle amplification and single molecule detection.

A bead-based assay was developed for highly sensitive single molecule DNA detection. Rolling circle amplification (RCA), an isothermal amplification technique that creates tandem repeated sequences, was used in combination with a fluorescent complementary DNA to create dense clusters of fluorescence. These clusters, each corresponding to a single target molecule, can be detected unambiguously due to their high signal/noise ratios. The limit of detection of this assay is approximately 1 amol. This simple single molecule assay allows high detection sensitivity without the use of complex equipment.

Directed carbon nanotube assembly using a pyrene-functionalized polymer.

A pyrene-functionalized polymer was patterned via electron beam lithography onto a silicon wafer and shown to selectively bind with carbon nanotubes.

Spatially controlled assembly of nanomaterials at the nanoscale.

A molecular monolayer of 4-nitrothiophenol ongold electrodes is reduced electrochemically when its nitro groups are converted into amino groups by potentiometric scans. The protonated amine with its NH3+ functions can be employed to induce the self-assembly of gold nanoparticles at the surface of the electrodes. The electrochemical reaction and the induced assembly process can be controlled at the nanoscale level on the electrodes with a high degree of selectivity. The technology opens up the possibility of fabricating complex multi-nanomaterial nanostructures on the basis of a two-step electrochemical assembly process.

Positioning multiple proteins at the nanoscale with electron beam cross-linked functional polymers.

Constructing multicomponent protein structures that match the complexity of those found in nature is essential for the next generation of medical materials. In this report, a versatile method for precisely arranging multicomponent protein nanopatterns in two-dimensional single-layer or three-dimensional multilayer formats using electron beam lithography is described. Eight-arm poly(ethylene glycol)s (PEGs) were modified at the chain ends with either biotin, maleimide, aminooxy, or nitrilotriacetic acid. Analysis by 1H NMR spectroscopy revealed that the reactions were efficient and that end-group conversions were 91-100%. The polymers were then cross-linked onto Si surfaces using electron beams to form micron-sized patterns of the functional groups. Proteins with biotin binding sites, a free cysteine, an N-terminal alpha-oxoamide, and a histidine tag, respectively, were then incubated with the substrate in aqueous solutions without the addition of any other reagents. By fluorescence microscopy experiments it was determined that proteins reacted site-specifically with the exposed functional groups to form micropatterns. Multicomponent nanoscale protein patterns were then fabricated. Different PEGs with orthogonal reactivities were sequentially patterned on the same chip. Simultaneous assembly of two different proteins from a mixture of the biomolecules formed the multicomponent two-dimensional patterns. Atomic force microscopy demonstrated that nanometer-sized polymer patterns were formed, and fluorescence microscopy demonstrated that side-by-side patterns of the different proteins were obtained. Moreover, multilayer PEG fabrication produced micron- and nanometer-sized patterns of one functional group on top of the other. Precise three-dimensional arrangements of different proteins were then realized.

Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification.

We report a sensitive and efficient magnetic bead-based assay for viral DNA identification using isothermal amplification of a reporting probe.

Nanoscale growth factor patterns by immobilization on a heparin-mimicking polymer.

In this study, electrostatic interactions between sulfonate groups of an immobilized polymer and the heparin binding domains of growth factors important in cell signaling were exploited to nanopattern the proteins. Poly(sodium 4-styrenesulfonate-co-poly(ethylene glycol) methacrylate) (pSS-co-pPEGMA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using ethyl S-thiobenzoyl-2-thiopropionate as a chain transfer agent and 2,2'-azoisobutyronitrile (AIBN) as the initiator. The resulting polymer (1) was characterized by 1H NMR, GPC, FT-IR, and UV-vis and had a number average molecular weight (Mn) of 24,000 and a polydispersity index (PDI) of 1.17. The dithioester end group of 1 was reduced to the thiol, and the polymer was subsequently immobilized on a gold substrate. Binding of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) to the polymer via the heparin binding domains was then confirmed by surface plasmon resonance (SPR). The interactions were stable at physiological salt concentrations. Polymer 1 was cross-linked onto silicon wafers using an electron beam writer forming micro- and nanopatterns. Resolutions of 100 nm and arbitrary nanoscale features such as concentric circles and contiguous squares and triangles were achieved. Fluorescence microscopy confirmed that bFGF and VEGF were subsequently immobilized to the polymer micro- and nanopatterns.

Electrochemically controllable conjugation of proteins on surfaces.

The rational design of surfaces for immobilization of proteins is essential to a variety of biological and medical applications ranging from molecular diagnostics to advanced platforms for fundamental studies of molecular and cell biology. We have developed an advanced electrochemically based approach for site-selective and reaction-controlled immobilization of proteins on surfaces. When a molecular monolayer of 4-nitrothiophenol on gold electrode surfaces is reduced electrochemically in a selective fashion at its nitro groups, to afford amino groups by potentiometric scans, the amine can be employed to orchestrate the immobilization of proteins to the surface. This protein immobilization strategy could allow one to fabricate intricate protein structures on surfaces for addressing fundamental and applied problems in biology and medicine.

Dynamically configurable biomolecular nanoarrays.

Nanoarrays of distinct DNA and protein biomolecules were fabricated by electrochemically controlling their assembly/release from Au nanoelectrodes on a chip. The surface density, ratio, and activity of the biomolecules assembled on each nanoelectrode in the array can be configured quantitatively and temporally by adjusting the electrochemical potential applied on the nanoelectrode. The dynamically configurable biomolecular nanoarray can potentially activate combinatorial interactions with microbiosystems under the control of an electronic circuit for biological and medical applications.