Cortico-cerebellar network involved in saccade adaptation.

Saccade adaptation is the learning process that ensures that vision and saccades remain calibrated. The central nervous system network involved in these adaptive processes remains unclear because of difficulties in isolating the learning process from the correlated visual and motor processes. Here we imaged the human brain during a novel saccade adaptation paradigm that allowed us to isolate neural signals involved in learning independent of the changes in the amplitude of corrective saccades usually correlated with adaptation. We show that the changes in activation in the ipsiversive cerebellar vermis that track adaptation are not driven by the changes in corrective saccades and thus provide critical supporting evidence for previous findings. Similarly, we find that activation in the dorsomedial wall of the contraversive precuneus mirrors the pattern found in the cerebellum. Finally, we identify dorsolateral and dorsomedial cortical areas in the frontal and parietal lobes that encode the retinal errors following inaccurate saccades used to drive recalibration. Together, these data identify a distributed network of cerebellar and cortical areas and their specific roles in oculomotor learning. NEW & NOTEWORTHY The central nervous system constantly learns from errors and adapts to keep visual targets and saccades in registration. We imaged the human brain while the gain of saccades adapted to a visual target that was displaced while the eye was in motion, inducing retinal error. Activity in the cerebellum and precuneus tracked learning, whereas parts of the dorsolateral and dorsomedial frontal and parietal cortex encoded the retinal error used to drive learning.

Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile.

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.

Skilled Bimanual Training Drives Motor Cortex Plasticity in Children With Unilateral Cerebral Palsy.

Background Intensive bimanual therapy can improve hand function in children with unilateral spastic cerebral palsy (USCP). We compared the effects of structured bimanual skill training versus unstructured bimanual practice on motor outcomes and motor map plasticity in children with USCP. Objective We hypothesized that structured skill training would produce greater motor map plasticity than unstructured practice. Methods Twenty children with USCP (average age 9.5; 12 males) received therapy in a day camp setting, 6 h/day, 5 days/week, for 3 weeks. In structured skill training (n = 10), children performed progressively more difficult movements and practiced functional goals. In unstructured practice (n = 10), children engaged in bimanual activities but did not practice skillful movements or functional goals. We used the Assisting Hand Assessment (AHA), Jebsen-Taylor Test of Hand Function (JTTHF), and Canadian Occupational Performance Measure (COPM) to measure hand function. We used single-pulse transcranial magnetic stimulation to map the representation of first dorsal interosseous and flexor carpi radialis muscles bilaterally. Results Both groups showed significant improvements in bimanual hand use (AHA; P < .05) and hand dexterity (JTTHF; P < .001). However, only the structured skill group showed increases in the size of the affected hand motor map and amplitudes of motor evoked potentials (P < .01). Most children who showed the most functional improvements (COPM) had the largest changes in map size. Conclusions These findings uncover a dichotomy of plasticity: the unstructured practice group improved hand function but did not show changes in motor maps. Skill training is important for driving motor cortex plasticity in children with USCP.

An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors.

Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization.

Posture-movement responses to stance perturbations and upper limb fatigue during a repetitive pointing task.

Localized muscle fatigue and postural perturbation have separately been shown to alter whole-body movement but little is known about how humans respond when subjected to both factors combined. Here we sought to quantify the kinematics of postural control and repetitive upper limb movement during standing surface perturbations and in the presence of fatigue. Subjects stood on a motion-based platform and repetitively reached between two shoulder-height targets until noticeably fatigued (rating of perceived exertion=8/10). Every minute, subjects experienced a posterior and an anterior platform translation while reaching to the distal target. Outcomes were compared prior to and with fatigue (first vs. final minute data). When fatigued, regardless of the perturbation condition, subjects decreased their shoulder abduction and increased contralateral trunk flexion, a strategy that may relieve the load on the fatiguing upper limb musculature. During perturbations, kinematic adaptations emerged across the trunk and arm to preserve task performance. In contrast to our expectation, the kinematic response to the perturbations did not alter in the presence of fatigue. Kinematic adaptations in response to the perturbation predominantly occurred in the direction of the reach whereas fatigue adaptations occurred orthogonal to the reach. These findings suggest that during repetitive reaching, fatigue and postural perturbation compensations organize so as to minimize interaction with each other and preserve the global task characteristics of endpoint motion.

A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold.

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.

Time-dependent adaptations to posture and movement characteristics during the development of repetitive reaching induced fatigue.

Repetitive movements are common to many daily activities but often lead to the development of fatigue. We have previously shown that fatigue leads to changes in tridimensional spatial characteristics of the whole body. However, temporal aspects of these posture and movement adaptations have yet to be investigated. Healthy subjects (N = 14) performed a continuous reaching task by pointing between two targets placed at shoulder height, at 100 and 30% arm's length, anterior to the subject's midline until fatigue (assessed using the Borg CR-10 scale). Whole body kinematics and upper Trapezius EMG were recorded and analyzed at 1-min intervals to document the progression of fatigue on outcome variables. For all upper limb and postural variables analyzed, changes began to occur approximately midway to fatigue and were followed by an increase in Trapezius activity from baseline. Reach-to-reach variability of joint average positions and range of motion (ROM) increased in multiple directions for shoulder and elbow parameters. Reach-to-reach variability of the center-of-mass ROM also increased in several directions. Changes were also observed in within-movement inter-segmental timing. The peak velocities of elbow and endpoint occurred closer together in time during fatigue while the shoulder peak velocity occurrence showed a greater reach-to-reach variability. Our results suggest that the effects of fatigue on repetitive movement kinematics can be observed across three temporal dimensions of the task: (1) within individual movements, (2) from one movement to the next, and (3) as fatigue develops. Each observed change is discussed as a potential contributor to task-specific control strategies to prolong task performance.

Posture-movement changes following repetitive motion-induced shoulder muscle fatigue.

Repetitive motion-induced fatigue not only alters local motion characteristics but also provokes global reorganization of movement. However, the three-dimensional (3D) characteristics of these reorganization patterns have never been documented in detail. The goal of this study was to assess the effects of repetitive reaching-induced arm fatigue on the whole-body, 3D biomechanical task characteristics. Healthy subjects (N=14) stood and performed a continuous reaching task (RRT) between two targets placed at shoulder height to fatigue. Whole-body kinematic (Vicon), kinetic (AMTI force platforms) and electromyographic (EMG, Noraxon) characteristics were recorded. Maximal voluntary isometric efforts (MVIE) of the shoulder and elbow were measured pre- and post-RRT. Post-RRT shoulder elevation MVIE was reduced by 4.9+/-8.3% and trapezius EMG amplitude recorded during the RRT increased by 46.9+/-49.9% from the first to last minute of the RRT, indicating that arm fatigue was effectively induced. During fatigued reaching, subjects elevated their shoulder (11.7+/-10.5 mm) and decreased their average shoulder abduction angle by 8.3+/-4.4 degrees. These changes were accompanied by a lateral shift of the body's center of mass towards the non-reaching arm. These findings suggest a compensatory strategy to decrease the load on the fatigued shoulder musculature.

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes.

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16+/-0.2 and 0.32+/-0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06+/-0.02 and 0.14+/-0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.

Age-related modifications in steering behaviour: effects of base-of-support constraints at the turn point.

This study investigated the effects of altering the base of support (BOS) at the turn point on anticipatory locomotor adjustments during voluntary changes in travel direction in healthy young and older adults. Participants were required to walk at their preferred pace along a 3-m straight travel path and continue to walk straight ahead or turn 40 degrees to the left or right for an additional 2-m. The starting foot and occasionally the gait starting point were adjusted so that participants had to execute the turn using a cross-over step with a narrow BOS or a lead-out step with a wide BOS. Spatial and temporal gait variables, magnitudes of angular segmental movement, and timing and sequencing of body segment reorientation were similar despite executing the turn with a narrow or wide BOS. A narrow BOS during turning generated an increased step width in the step prior to the turn for both young and older adults. Age-related changes when turning included reduced step velocity and step length for older compared to young adults. Age-related changes in the timing and sequencing of body segment reorientation prior to the turn point were also observed. A reduction in walking speed and an increase in step width just prior to the turn, combined with a delay in motion of the center of mass suggests that older adults used a more cautious combined foot placement and hip strategy to execute changes in travel direction compared to young adults. The results of this study provide insight into mobility constraints during a common locomotor task in older adults.

Small-molecule microarrays as tools in ligand discovery.

Small molecules that bind and modulate specific protein targets are increasingly used as tools to decipher protein function in a cellular context. Identifying specific small-molecule probes for each protein in the proteome will require miniaturized assays that permit screening of large collections of compounds against large numbers of proteins in a highly parallel fashion. Simple and general binding assays involving small-molecule microarrays can be used to identify probes for nearly any protein in the proteome. The assay may be used to identify ligands for proteins in the absence of knowledge about structure or function. In this tutorial review, we introduce small-molecule microarrays (SMMs) as tools for ligand discovery; discuss methods for manufacturing SMMs, including both non-covalent and covalent attachment strategies; and provide examples of ligand discovery involving SMMs.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

A comparison between polymeric microsphere and bacterial vectors for macrophage P388D1 gene delivery.

PURPOSE: The purpose of this study was to compare bacterial and polymeric gene delivery devices for the ability to deliver plasmid DNA to a murine macrophage P388D1 cell line. METHODS: An 85:15 ratio of poly(lactic-co-glycolic acid) (PLGA) and poly(beta-amino ester) polymers were formulated into microspheres that physically entrapped plasmid DNA encoding for the firefly luciferase reporter gene; whereas, the same plasmid was biologically transformed into a strain of Escherichia coli engineered to produce recombinant listeriolysin O. The two delivery devices were then tested for gene delivery and dosage effects using a macrophage cell line with both assays taking advantage of a 96-well high throughput format to quantify and compare each vector type. RESULTS: Gene delivery was comparable for both vectors at higher vector dosages while lower dosages showed an improved delivery for the microsphere vectors. Delivery efficiency (defined as luciferase measurement/mg cellular protein/ng DNA delivered) was 881 luminescence mg(-1) ng(-1) for polymeric microspheres compared to 171 luminescence mg(-1) ng(-1) for the bacterial vectors. CONCLUSION: A first head-to-head comparison between polymeric and bacterial gene delivery vectors shows a delivery advantage for polymeric microspheres that must also be evaluated in light of vector production, storage, and future potential.

Intracellular delivery of core-shell fluorescent silica nanoparticles.

Highly fluorescent core-shell silica nanoparticles made by the modified Stöber process (C dots) are promising as tools for sensing and imaging subcellular agents and structures but will only be useful if they can be easily delivered to the cytoplasm of the subject cells. This work shows that C dots can be electrostatically coated with cationic polymers, changing their surface charge and enabling them to escape from endosomes and enter the cytoplasm and nucleus. As an example of cellular delivery, we demonstrate that these particles can also be complexed with DNA and mediate and trace DNA delivery and gene expression.

Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells.

We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF(165). The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including alpha-fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.

Strategies used by older adults to change travel direction.

INTRODUCTION: A distinct body reorientation strategy during steering tasks has been reported in young adults. As challenges to whole-body stability in older adults occur when navigating complex environments, this study was designed to examine control strategies used by older adults to initiate a voluntary change in travel direction. METHODS: Thirteen older adults, recruited from an independent living division of a local retirement residence, were instrumented with reflective markers and whole-body kinematic data were monitored using a video camera (30 Hz). Participants executed self-paced walking trials 3-m along a straight path and were instructed prior to the trial to continue either straight ahead or randomly turn 40 degrees left or right and continue walking for an additional 2-m. Timing of changes with respect to when the trunk crossed the turning point were calculated for deviations in head and trunk position and foot rotation in the medial-lateral plane. RESULTS: Older adults reoriented themselves into the new travel direction in a top-down, segmental sequence, beginning with head reorientation followed by trunk reorientation, foot rotation and foot displacement into the new travel direction. These changes were initiated over two or more steps 69% of the time and over one step 31% of the time. A significant relationship between turning strategy used and balance confidence was observed; the frequency of using a turning strategy involving two or more steps to initiate a change in travel direction increased as balance confidence decreased. DISCUSSION AND CONCLUSION: Older adults made segmental changes to voluntarily reorient themselves in a new travel direction in a similar sequence to that observed in young adults. Older adults chose primarily to initiate these changes two or more steps prior to the turn; the selection of this strategy was related to balance confidence.

Clamshell structure of Sc(biphenyl) from high resolution photoelectron spectroscopy.

Sc(biphenyl) is produced in a laser vaporization molecular beam source and characterized by pulsed field ionization-zero electron kinetic energy (ZEKE) photoelectron spectroscopy and density functional theory. The theory predicts two low-energy isomers: a clamshell structure with Sc binding to both phenyl rings and a half-sandwich structure with Sc binding to single ring. The joint experimental and theoretical study shows that the Sc(biphenyl) complex prefers the clamshell structure with a twelve-fold binding mode. From the free ligand to the clamshell structure of the complex, phenyl rings first rotate to become coplanar, then bend toward the Sc atom, and finally are clamped in place by Sc binding.

Pulsed-field ionization electron spectroscopy and binding energies of alkali metal-dimethyl ether and -dimethoxyethane complexes.

Lithium and sodium complexes of dimethyl ether (DME) and dimethoxyethane (DXE) were produced by reactions of laser-vaporized metal atoms with organic vapors in a pulsed nozzle cluster source. The mono-ligand complexes were studied by photoionization and pulsed field ionization zero electron kinetic energy (ZEKE) spectroscopy. Vibrationally resolved ZEKE spectra were obtained for Li(DME), Na(DME) and Li(DXE) and a photoionization efficiency spectrum for Na(DXE). The ZEKE spectra were analyzed by comparing with the spectra of other metal-ether complexes and with electronic structure calculations and spectral simulations. Major vibrations measured for the M(DME) (M=Li,Na) ions were M-O and C-O stretches and M-O-C and C-O-C bends. These vibrations and additional O-Li-O and O-C-C-O bends were observed for the Li(DXE) ion. The M(DME) complexes were in C2v symmetry with the metal atom binding to oxygen, whereas Li(DXE) was in a C2 ring configuration with the Li atom attaching to both oxygen atoms. Moreover, the ionization energies of these complexes were measured from the ZEKE or photoionization spectra and bond dissociation energies were derived from a thermodynamic cycle.

Photoelectron spectroscopy and density functional theory of puckered ring structures of Group 13 metal-ethylenediamine.

The ethylenediamine (en) complexes of Al, Ga, and In atoms were prepared in laser-vaporization supersonic molecular beams and studied with pulsed field ionization zero electron kinetic energy photoelectron spectroscopy and density functional theory. Several conformers of each metal complex are obtained by B3LYP calculations, and a five-membered cyclic structure is identified by combining the experimental measurements and theoretical calculations. Adiabatic ionization potentials, vibrational frequencies, and bond dissociation energies are determined for the ring structure. The ionization potentials of the Al, Ga, and In species are measured to be 32 784 (5), 33 324 (5), and 33 637 (7) cm(-1), respectively, and metal-ligand dissociation energies of the ionic and neutral complexes are calculated to be 60.2/16.2 (Al(+)/Al), 55.5/13.0 (Ga(+)/Ga), and 50.0/11.4 (In(+)/In) kcal mol(-1). Metal-ligand stretch and bend as well as a number of ligand-based vibrations are measured. Harmonic frequencies and anharmonicities of the M(+)-N (M=Al,Ga,In) stretch are determined for all three M(+)-en ions and the C-C-N bend of Ga(+)-en and In(+)-en. In comparison to monodentate methylamine, the bidentate binding of ethylenediamine leads to a significantly lower ionization potential and higher metal-ligand bond strength of the metal complexes.