Characterization of an Algorithm for Autonomous, Closed-Loop Neuromodulation During Motor Rehabilitation.

BACKGROUND: Recent evidence demonstrates that manually triggered vagus nerve stimulation (VNS) combined with rehabilitation leads to increased recovery of upper limb motor function after stroke. This approach is premised on studies demonstrating that the timing of stimulation relative to movements is a key determinant in the effectiveness of this approach. OBJECTIVE: The overall goal of the study was to identify an algorithm that could be used to automatically trigger VNS on the best movements during rehabilitative exercises while maintaining a desired interval between stimulations to reduce the burden of manual stimulation triggering. METHODS: To develop the algorithm, we analyzed movement data collected from patients with a history of neurological injury. We applied 3 different algorithms to the signal, analyzed their triggering choices, and then validated the best algorithm by comparing triggering choices to those selected by a therapist delivering VNS therapy. RESULTS: The dynamic algorithm triggered above the 95th percentile of maximum movement at a rate of 5.09 (interquartile range [IQR] = 0.74) triggers per minute. The periodic algorithm produces stimulation at set intervals but low movement selectivity (34.05%, IQR = 7.47), while the static threshold algorithm produces long interstimulus intervals (27.16 ± 2.01 seconds) with selectivity of 64.49% (IQR = 25.38). On average, the dynamic algorithm selects movements that are 54 ± 3% larger than therapist-selected movements. CONCLUSIONS: This study shows that a dynamic algorithm is an effective strategy to trigger VNS during the best movements at a reliable triggering rate.

Comparison of Illumination Methods for Flow-Through Optofluidic Biosensors.

Optofluidic biosensors have become an important medical diagnostic tool because they allow for rapid, high-sensitivity testing of small samples compared to standard lab testing. For these devices, the practicality of use in a medical setting depends heavily on both the sensitivity of the device and the ease of alignment of passive chips to a light source. This paper uses a model previously validated by comparison to physical devices to compare alignment, power loss, and signal quality for windowed, laser line, and laser spot methods of top-down illumination.

Usage of RePlay as a Take-Home System to Support High-Repetition Motor Rehabilitation After Neurological Injury.

Stroke is a leading cause of chronic motor disability. While physical rehabilitation can promote functional recovery, several barriers prevent patients from receiving optimal rehabilitative care. Easy access to at-home rehabilitative tools could increase patients' ability to participate in rehabilitative exercises, which may lead to improved outcomes. Toward achieving this goal, we developed RePlay: a novel system that facilitates unsupervised rehabilitative exercises at home. RePlay leverages available consumer technology to provide a simple tool that allows users to perform common rehabilitative exercises in a gameplay environment. RePlay collects quantitative time series force and movement data from handheld devices, which provide therapists the ability to quantify gains and individualize rehabilitative regimens. RePlay was developed in C# using Visual Studio. In this feasibility study, we assessed whether participants with neurological injury are capable of using the RePlay system in both a supervised in-office setting and an unsupervised at-home setting, and we assessed their adherence to the unsupervised at-home rehabilitation assignment. All participants were assigned a set of 18 games and exercises to play each day. Participants produced on average 698 ± 36 discrete movements during the initial 1 hour in-office visit. A subset of participants who used the system at home produced 1593 ± 197 discrete movements per day. Participants demonstrated a high degree of engagement while using the system at home, typically completing nearly double the number of assigned exercises per day. These findings indicate that the open-source RePlay system may be a feasible tool to facilitate access to rehabilitative exercises and potentially improve overall patient outcomes.

Performance Comparison of Flow-Through Optofluidic Biosensor Designs.

Optofluidic flow-through biosensors are being developed for single particle detection, particularly as a tool for pathogen diagnosis. The sensitivity of the biosensor chip depends on design parameters, illumination format (side vs. top), and flow configuration (parabolic, two- and three-dimensional hydrodynamic focused (2DHF and 3DHF)). We study the signal differences between various combinations of these design aspects. Our model is validated against a sample of physical devices. We find that side-illumination with 3DHF produces the strongest and consistent signal, but parabolic flow devices process a sample volume more quickly. Practical matters of optical alignment are also discussed, which may affect design choice.

Optofluidic Flow-Through Biosensor Sensitivity - Model and Experiment.

We present a model and simulation for predicting the detected signal of a fluorescence-based optical biosensor built from optofluidic waveguides. Typical applications include flow experiments to determine pathogen concentrations in a biological sample after tagging relevant DNA or RNA sequences. An overview of the biosensor geometry and fabrication processes is presented. The basis for the predictive model is also outlined. The model is then compared to experimental results for three different biosensor designs. The model is shown to have similar signal statistics as physical tests, illustrating utility as a pre-fabrication design tool and as a predictor of detection sensitivity.

Greatly Enhanced Single Particle Fluorescence Detection Using High Refractive Index Liquid-Core Waveguides.

High sensitivity and easy integration with microfabrication techniques has made silicon photonics one of the leading technologies used to build biosensors for diagnostic applications. Here we introduce a new silicon dioxide based optofluidic platform having a planar solid-core (SC) waveguide orthogonally intersecting a liquid-core (LC) waveguide with high refractive index ZnI2 salt solution as core. This enables both more uniform collection of particle fluorescence by the core mode and its propagation to an off-chip detector. This approach results in ultra-high sensitivity performance, demonstrated by achieving 8X enhancement in signal-to-noise ratio, a 45x increase in detection efficiency, and a 100x lower detection limit of 80 aM of fluorescent nanobeads. This represents a key step towards an ultrasensitive biosensor system for analyzing pathogens at clinical concentrations.

A Quality Improvement Initiative to Reduce the Frequency of Delays in Initiation and Restarts of Continuous Renal Replacement Therapy.

BACKGROUND: Continuous renal replacement therapy (CRRT) is a lifesaving intervention for critically ill patients. Delays in initiation, or an inability to resume CRRT following a temporary suspension in therapy (CRRT restart), can result in suboptimal CRRT delivery. LOCAL PROBLEM: Intensive care units across the health care system were experiencing significant delays in CRRT initiation and restarts. APPROACH: A multimodal quality improvement initiative was implemented across 7 adult intensive care units, which allowed unit-based staff nurses to initiate and restart CRRT, a task that had previously been delegated to non-unit-based dialysis nurses. OUTCOMES: A 75% reduction in CRRT initiation delays and a 90% reduction in CRRT restart delays were observed in the 12 months following the initiative. There were no adverse events or increased disposable CRRT circuit usage following the initiative. CONCLUSIONS: Implementation of CRRT initiation and restarts by unit-based nurses were achievable and resulted in substantial improvements in timeliness of CRRT delivery.

3D Hydrodynamic Focusing in Microscale Optofluidic Channels Formed with a Single Sacrificial Layer.

Optofluidic devices are capable of detecting single molecules, but greater sensitivity and specificity is desired through hydrodynamic focusing (HDF). Three-dimensional (3D) hydrodynamic focusing was implemented in 10-µm scale microchannel cross-sections made with a single sacrificial layer. HDF is achieved using buffer fluid to sheath the sample fluid, requiring four fluid ports to operate by pressure driven flow. A low-pressure chamber, or pit, formed by etching into a substrate, enables volumetric flow ratio-induced focusing at a low flow velocity. The single layer design simplifies surface micromachining and improves device yield by 1.56 times over previous work. The focusing design was integrated with optical waveguides and used in order to analyze fluorescent signals from beads in fluid flow. The implementation of the focusing scheme was found to narrow the distribution of bead velocity and fluorescent signal, giving rise to 33% more consistent signal. Reservoir effects were observed at low operational vacuum pressures and a balance between optofluidic signal variance and intensity was achieved. The implementation of the design in optofluidic sensors will enable higher detection sensitivity and sample specificity.

Effects of Post-Etch Microstructures on the Optical Transmittance of Silica Ridge Waveguides.

Silica waveguides are often etched by reactive ion etch (RIE) processes. These processes can leave residual topography that can increase optical loss. We investigated the relation between optical loss and various RIE etch. A wet etch step meant to remove microstructures was also considered and compared. Ridge waveguides were fabricated in plasma enhanced chemical vapor deposited films by three different RIE processes, each with a different gas composition, pressure setting, and applied power setting. Half of each set of waveguides were also subjected to a hydrofluoric acid (HF) solution. The waveguides were tested for optical transmission via the cutback method. The transmission vs waveguide length measurements were plotted to fit an exponential curve and the optical loss and measurement uncertainty for each waveguide set was calculated. Clear distinctions in optical loss were found between the different RIE processes. The HF treatment also has an effect, significantly reducing optical loss for two processes and increasing it for the third. Of the tested RIE processes, one can be suggested for silica waveguides. It results in the lowest optical loss and coincidently has the fastest etch rate.

3D hydrodynamic focusing in microscale channels formed with two photoresist layers.

3D hydrodynamic focusing was implemented with channel cross-section dimensions smaller than 10 µm. Microchannels were formed using sacrificial etching of two photoresist layers on a silicon wafer. The photoresist forms a plus-shaped prismatic focusing fluid junction which was coated with plasma-enhanced chemical-vapor-deposited oxide. Buffer fluid carried to the focusing junction envelopes an intersecting sample fluid, resulting in 3D focusing of the sample stream. The design requires four fluid ports and operates across a wide range of fluid velocities through pressure-driven flow. The focusing design was integrated with optical waveguides to interrogate fluorescing particles and confirm 3D focusing. Particle diffusion away from a focused stream was characterized.

Pet ownership and blood pressure in old age.

BACKGROUND: It has been proposed that pet ownership improves cardiovascular health. This study examines the relation of pet ownership with systolic and diastolic blood pressure, pulse pressure, mean arterial pressure, and hypertension in a large sample of older men and women. METHODS: Participants were 1179 community-dwelling men (n = 498) and women (n = 681) age 50-95 years. Participants responded to a 1991-1992 mailed questionnaire ascertaining pet ownership, and they attended a 1992-1996 clinic visit at which systolic (SBP) and diastolic (DBP) blood pressures were measured and use of antihypertensive medication was validated. Pulse pressure was calculated as SBP minus DBP. Mean arterial pressure was calculated as (SBP+DBP)/2. Body mass index, waist-hip ratio, and information on other potential confounders were obtained. RESULTS: Average age of participants was 70.4 +/- 10.8 years; 30.0% reported current pet ownership. Mean SBP was 137.5 +/- 21.4 mm Hg, and DBP was 76.1 +/- 9.3 mm Hg; 55.6% were hypertensive (SBP >or= 140, DBP >or= 90 or taking hypertension medication). Pet owners were younger and slightly more overweight and they exercised less than nonowners; owners were somewhat more likely to have diabetes and to use beta-blockers. In unadjusted analyses, pet owners had lower SBP, pulse pressure, and mean arterial pressure, and a reduced risk of hypertension (odds ratio = 0.62; 95% confidence interval = 0.49-0.80). However, after adjustment for age and other confounders, pet ownership was not associated with systolic or diastolic blood pressure, pulse pressure, mean arterial pressure or risk of hypertension. CONCLUSIONS: Results suggest that pet ownership is not independently associated with blood pressure, vascular reactivity, or hypertension.

Synthesis and in vitro antifolate activity of rotationally restricted aminopterin and methotrexate analogues.

Heretofore unknown analogues of aminopterin (AMT) and methotrexate (MTX) in which free rotation of the amide bond between the phenyl ring and amino acid side chain is prevented by a CH(2) bridge were synthesized and tested for in vitro antifolate activity. The K(i) of the AMT analogue (9) against human dihydrofolate reductase (DHFR) was 34 pM, whereas that of the MTX analogue (10) was 2100 pM. Both compounds were less potent than the parent drugs. However, although the difference between AMT and MTX was <2-fold, the difference between 9 and 10 was 62-fold, suggesting that the effect of N(10)-methyl substitution is amplified in the bridged compounds. The K(i) values of 9 and 10 as inhibitors of [(3)H]MTX influx into CCRF-CEM human leukemia cells via the reduced folate carrier (RFC) were 0.28 and 1.1 muM, respectively. The corresponding K(i) and K(t) values determined earlier for AMT and MTX were 5.4 and 4.7 muM, respectively. Thus, in contrast to its unfavorable effect on DHFR binding, the CH(2) bridge increased RFC binding. In a 72 h growth assay with CCRF-CEM cells, the IC(50) values of 9 and 10 were 5.1 and 140 nM, respectively, a 27-fold difference that was qualitatively consistent with the observed combination of weaker DHFR binding and stronger RFC binding. Although rotationally restricted inhibitors of other enzymes of folate pathway enzymes have been described previously, 9 and 10 are the first reported examples of DHFR inhibitors of this type.

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity.

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.

Synthesis and in vitro antitumor activity of new deaza analogues of the nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523).

Details are disclosed for the synthesis of N(alpha)-[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (2) and N(alpha)-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-yn-4-yl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (6) as analogues of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (1, PT523), a nonpolyglutamatable antifolate currently in advanced preclinical development. In a 72 h growth inhibition assay against cultures of CCRF-CEM human leukemic lymphoblasts, the IC(50) of 2 and 6 was 0.69 +/- 0.044 nM and 1.3 +/- 0.35 nM, respectively, as compared with previously reported values 4.4 +/- 0.10 nM for aminopterin (AMT) and 1.5 +/- 0.39 nM for PT523. In a spectrophotometric assay of dihydrofolate reductase (DHFR) inhibition using dihydrofolate and NADPH as the cosubstrates, the previously unreported compounds 2 and the mixed 10R and 10S diastereomers of 6 had K(i) values of 0.21 +/- 0.05 pM and 0.60 +/- 0.02 pM, respectively, as compared with previously reported values of 3.70 +/- 0.35 pM for AMT and 0.33 +/- 0.04 pM for PT523. Thus, while they were comparable to 1 and several of its previously studied analogues in their ability to bind to DHFR and inhibit the growth of CCRF-CEM cells, 2 and the mixed diastereomers of 6 were several times more active than AMT despite the fact that they cannot form gamma-polyglutamylated metabolites of the type formed in cells from AMT and other classical antifolates with a glutamate side chain.

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid.

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.

Synthesis and enzymatic activation of N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithiny]-L-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT).

N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithinyl]-L-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (K(i)>10 microM). In a 72 h growth inhibition assay against cultured CCRF-CEM human leukemic lymphoblasts, the growth inhibitory activities of 1+bCPA, 2+bCPA, and 2 alone were the same (IC(50) 1.3-1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC(50) 155 nM).