Targeted 8-arm PEG Nanosystems for Localization of Choroidal Neovascularization Macular Degeneration Model.

8-arm PEG (polyethylene-glycol) is a highly promising nanoplatform due to its small size (<10 nm), ease-of-conjugation (many functionalized variants are readily available with "click-like" properties), biocompatibility, and optical inactivity. This study evaluates 8-arm PEG uptake into cells (in vitro) and localization and clearance in vasculature (in vivo) for targeting of choroidal neovascularization in mice, an animal model of macular degeneration. 8-arm PEG nanoparticles were labeled with fluorescein isothiocyanate (FITC) and functionalized in the absence or presence of pentameric Ar-Gly-Asp (RGD; 4 RGD motifs and a PGC linker), one of the most common peptide motifs used for active targeting. In vitro studies show that RGD-conjugated 8-arm PEG nanoparticles exhibit enhanced cellular uptake relative to non-RGD-conjugated control NPs at 34% ± 9%. Laser-induced choroidal neovascularization (CNV) was performed in a mouse model to measure 8-arm PEG localization and clearance to model macular degeneration lesions in vivo. It was determined that both RGD-conjugated and non-RGD-conjugated (nRGD) 8-arm PEG particles localized to CNV lesions, with a half-life around 24 h. In vivo experiments showed that RGD-conjugated nanoparticles exhibited enhanced localization by 15-20% relative to without RGD controls. Exhibiting a high rate of localization and fast clearance relative to larger nanoparticles, targeted 8-arm PEG nanoparticles with a conjugated RGD-peptide could be a promising modality for macular degeneration diagnosis and therapy.

Single-Molecule Stoichiometry of Supramolecular Complexes.

The use of single-molecule microscopy is introduced as a method to quantify the photophysical properties of supramolecular complexes rapidly at ultra low concentrations (<1 nM), previously inaccessible. Using a model supramolecular system based on the host-guest complexation of cucurbit[n]uril (CB[n]) macrocycles together with a fluorescent guest (Ant910Me), we probe fluorescent CB[n] host-guest complexes in the single molecule regime. We show quantification and differentiation of host-guest photophysics and stoichiometries, both in aqueous media and noninvasively in hydrogel, by thresholding detected photons. This methodology has wide reaching implications in aiding the design of next-generation materials with programmed and controlled properties.

On-Resin Recognition of Aromatic Oligopeptides and Proteins through Host-Enhanced Heterodimerization.

Peptide dimerization is ubiquitous in natural protein conjugates and artificial self-assemblies. A major challenge in artificial systems remains achieving quantitative peptide heterodimerization, critical for next-generation biomolecular purification and formulation of therapeutics. Here, we employ a synthetic host to simultaneously encapsulate an aromatic and a noncanonical l-perfluorophenylalanine-containing peptide through embedded polar-π interactions, constructing an unprecedented series of heteropeptide dimers. To demonstrate the utility, this heteropeptide dimerization strategy was applied toward on-resin recognition of N-terminal aromatic residues in peptides as well as insulin, both exhibiting high recycling efficiency (>95%). This research unveils a generic approach to exploit quantitative heteropeptide dimers for the design of supramolecular (bio)systems.

Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.

We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy.

Toward a Mobile Platform for Real-world Digital Measurement of Depression: User-Centered Design, Data Quality, and Behavioral and Clinical Modeling.

BACKGROUND: Although effective mental health treatments exist, the ability to match individuals to optimal treatments is poor, and timely assessment of response is difficult. One reason for these challenges is the lack of objective measurement of psychiatric symptoms. Sensors and active tasks recorded by smartphones provide a low-burden, low-cost, and scalable way to capture real-world data from patients that could augment clinical decision-making and move the field of mental health closer to measurement-based care. OBJECTIVE: This study tests the feasibility of a fully remote study on individuals with self-reported depression using an Android-based smartphone app to collect subjective and objective measures associated with depression severity. The goals of this pilot study are to develop an engaging user interface for high task adherence through user-centered design; test the quality of collected data from passive sensors; start building clinically relevant behavioral measures (features) from passive sensors and active inputs; and preliminarily explore connections between these features and depression severity. METHODS: A total of 600 participants were asked to download the study app to join this fully remote, observational 12-week study. The app passively collected 20 sensor data streams (eg, ambient audio level, location, and inertial measurement units), and participants were asked to complete daily survey tasks, weekly voice diaries, and the clinically validated Patient Health Questionnaire (PHQ-9) self-survey. Pairwise correlations between derived behavioral features (eg, weekly minutes spent at home) and PHQ-9 were computed. Using these behavioral features, we also constructed an elastic net penalized multivariate logistic regression model predicting depressed versus nondepressed PHQ-9 scores (ie, dichotomized PHQ-9). RESULTS: A total of 415 individuals logged into the app. Over the course of the 12-week study, these participants completed 83.35% (4151/4980) of the PHQ-9s. Applying data sufficiency rules for minimally necessary daily and weekly data resulted in 3779 participant-weeks of data across 384 participants. Using a subset of 34 behavioral features, we found that 11 features showed a significant (P<.001 Benjamini-Hochberg adjusted) Spearman correlation with weekly PHQ-9, including voice diary-derived word sentiment and ambient audio levels. Restricting the data to those cases in which all 34 behavioral features were present, we had available 1013 participant-weeks from 186 participants. The logistic regression model predicting depression status resulted in a 10-fold cross-validated mean area under the curve of 0.656 (SD 0.079). CONCLUSIONS: This study finds a strong proof of concept for the use of a smartphone-based assessment of depression outcomes. Behavioral features derived from passive sensors and active tasks show promising correlations with a validated clinical measure of depression (PHQ-9). Future work is needed to increase scale that may permit the construction of more complex (eg, nonlinear) predictive models and better handle data missingness.

Abdominal functional electrical stimulation to enhance mechanical insufflation-exsufflation.

CONTEXT: Respiratory complications, attributed to the build-up of secretions in the airway, are a leading cause of rehospitalisation for the tetraplegic population. Previously, we observed that the application of Abdominal Functional Electrical Stimulation (AFES) improved cough function and increased demand for secretion removal, suggesting AFES may aid secretion clearance. Clinically, secretion clearance is commonly achieved by using Mechanical insufflation-exsufflation (MI-E) to simulate a cough. In this study the feasibility of combining AFES with MI-E is evaluated. FINDINGS: AFES was successfully combined with MI-E at eight fortnightly assessment sessions conducted with one sub-acute participant with tetraplegia. By using the signal from a pressure sensor, integrated with the MI-E device, AFES was correctly applied in synchrony with MI-E with an accuracy of 96.7%. Acute increases in exhaled volume and peak flow were observed during AFES assisted MI-E, compared to MI-E alone, at six of eight assessment sessions. CONCLUSION: The successful integration of AFES with MI-E at eight assessment sessions demonstrates the feasibility of this technique. The acute increases in respiratory function observed at the majority of assessment sessions generate the hypothesis that AFES assisted MI-E may be more effective for secretion clearance than MI-E alone.

Abdominal Functional Electrical Stimulation to Assist Ventilator Weaning in Acute Tetraplegia: A Cohort Study.

BACKGROUND: Severe impairment of the major respiratory muscles resulting from tetraplegia reduces respiratory function, causing many people with tetraplegia to require mechanical ventilation during the acute stage of injury. Abdominal Functional Electrical Stimulation (AFES) can improve respiratory function in non-ventilated patients with sub-acute and chronic tetraplegia. The aim of this study was to investigate the clinical feasibility of using an AFES training program to improve respiratory function and assist ventilator weaning in acute tetraplegia. METHODS: AFES was applied for between 20 and 40 minutes per day, five times per week on four alternate weeks, with 10 acute ventilator dependent tetraplegic participants. Each participant was matched retrospectively with a ventilator dependent tetraplegic control, based on injury level, age and sex. Tidal Volume (VT) and Vital Capacity (VC) were measured weekly, with weaning progress compared to the controls. RESULTS: Compliance to training sessions was 96.7%. Stimulated VT was significantly greater than unstimulated VT. VT and VC increased throughout the study, with mean VC increasing significantly (VT: 6.2 mL/kg to 7.8 mL/kg VC: 12.6 mL/kg to 18.7 mL/kg). Intervention participants weaned from mechanical ventilation on average 11 (sd: ± 23) days faster than their matched controls. CONCLUSION: The results of this study indicate that AFES is a clinically feasible technique for acute ventilator dependent tetraplegic patients and that this intervention may improve respiratory function and enable faster weaning from mechanical ventilation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02200393.

Decreases in bone mineral density at cortical and trabecular sites in the tibia and femur during the first year of spinal cord injury.

BACKGROUND: Disuse osteoporosis occurs in response to long-term immobilization. Spinal cord injury (SCI) leads to a form of disuse osteoporosis that only affects the paralyzed limbs. High rates of bone resorption after injury are evident from decreases in bone mineral content (BMC), which in the past have been attributed in the main to loss of trabecular bone in the epiphyses and cortical thinning in the shaft through endocortical resorption. METHODS: Patients with motor-complete SCI recruited from the Queen Elizabeth National Spinal Injuries Unit (Glasgow, UK) were scanned within 5weeks of injury (baseline) using peripheral Quantitative Computed Tomography (pQCT). Unilateral scans of the tibia, femur and radius provided separate estimates of trabecular and cortical bone parameters in the epiphyses and diaphyses, respectively. Using repeat pQCT scans at 4, 8 and 12months post-injury, changes in BMC, bone mineral density (BMD) and cross-sectional area (CSA) of the bone were quantified. RESULTS: Twenty-six subjects (5 female, 21 male) with SCI (12 paraplegic, 14 tetraplegic), ranging from 16 to 76years old, were enrolled onto the study. Repeated-measures analyses showed a significant effect of time since injury on key bone parameters at the epiphyses of the tibia and femur (BMC, total BMD, trabecular BMD) and their diaphyses (BMC, cortical BMD, cortical CSA). There was no significant effect of gender or age on key outcome measures, but there was a tendency for the female subjects to experience greater decreases in cortical BMD. The decreases in cortical BMD in the tibia and femur were found to be statistically significant in both men and women. CONCLUSIONS: By carrying out repeat pQCT scans at four-monthly intervals, this study provides a uniquely detailed description of the cortical bone changes that occur alongside trabecular bone changes in the first year of complete SCI. Significant decreases in BMD were recorded in both the cortical and trabecular bone compartments of the tibia and femur throughout the first year of injury. This study provides evidence for the need for targeted early intervention to preserve bone mass within this patient group.

Isometric hip and knee torque measurements as an outcome measure in robot assisted gait training.

BACKGROUND: Strength changes in lower limb muscles following robot assisted gait training (RAGT) in subjects with incomplete spinal cord injury (ISCI) has not been quantified using objective outcome measures. OBJECTIVE: To record changes in the force generating capacity of lower limb muscles (recorded as peak voluntary isometric torque at the knee and hip), before, during and after RAGT in both acute and subacute/chronic ISCI subjects using a repeated measures study design. METHODS: Eighteen subjects with ISCI participated in this study (Age range: 26-63 years mean age = 49.3 ± 11 years). Each subject participated in the study for a total period of eight weeks, including 6 weeks of RAGT using the Lokomat system (Hocoma AG, Switzerland). Peak torques were recorded in hip flexors, extensors, knee flexors and extensors using torque sensors that are incorporated within the Lokomat. RESULTS: All the tested lower limb muscle groups showed statistically significant (p < 0.001) increases in peak torques in the acute subjects. Comparison between the change in peak torque generated by a muscle and its motor score over time showed a non-linear relationship. CONCLUSIONS: The peak torque recorded during isometric contractions provided an objective outcome measure to record changes in muscle strength following RAGT.

The spinal cord-injured patient in the medical ward.

People with spinal cord injury (SCI) suffer from the complications of paralysis in addition to the diseases of the general population. Spinal injury centres in the UK are tasked primarily with looking after newly injured patients and patients with established SCI must often turn to their local hospitals and general physicians for inpatient medical care. This paper outlines investigation and management of some of the common conditions which the physician on the general ward might expect to come across in patients with SCI and also how best to maintain the general health of the SCI patient in hospital.

Two-photon fluorescence spectroscopy and imaging of 4-dimethylaminonaphthalimide peptide and protein conjugates.

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.

Changes in pulmonary function measures following a passive abdominal functional electrical stimulation training program.

OBJECTIVE: To demonstrate the effect of a passive abdominal functional electrical stimulation (AFES) training program on unassisted respiratory measures in tetraplegia. DESIGN: Longitudinal feasibility study. SETTING: National spinal injuries unit in a university teaching hospital. PARTICIPANTS: Twelve patients with tetraplegic spinal cord injury, who could breathe independently, with reduced vital capacity and no visible abdominal movement. INTERVENTION: Three weeks of abdominal muscle conditioning using transcutaneous AFES. MAIN OUTCOME MEASURES: Forced vital capacity (FVC), forced exhaled volume in 1 second (FEV1), peak expiratory flow rate (PEF), and maximum exhaled pressure (MEP). RESULTS: Mean (SD) FVC increased by 0.36 l (0.23) during training (P = 0.0027). Mean (SD) FEV1 and PEF tended to increase by 0.18 l (0.16) and 0.39 l/seconds (0.35), respectively, but this was not significant. No significant change was found in the outcome measures during a 1-week pre-training control phase and during a 3-week post-training phase. CONCLUSIONS: The increase in FVC over the training period and the absence of change before or after training suggest that passive abdominal FES training can be used for respiratory rehabilitation in tetraplegia.

Neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness.

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -ß, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335(+), TCR-ß(+) cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti-IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.

Role of peripheral quantitative computed tomography in identifying disuse osteoporosis in paraplegia.

OBJECTIVE: Disuse osteoporosis is a major long-term health consequence of spinal cord injury (SCI) that still needs to be addressed. Its management in SCI should begin with accurate diagnosis, followed by targeted treatments in the most vulnerable subgroups. We present data quantifying disuse osteoporosis in a cross-section of the Scottish paraplegic population to identify subgroups with lowest bone mineral density (BMD). MATERIALS AND METHODS: Forty-seven people with chronic SCI at levels T2-L2 were scanned using peripheral quantitative computed tomography at four tibial sites and two femoral sites, at the Queen Elizabeth National Spinal Injuries Unit, Glasgow (UK). At the distal epiphyses, trabecular BMD (BMDtrab), total BMD, total bone cross-sectional area (CSA) and bone mineral content (BMC) were determined. In the diaphyses, cortical BMD, total bone CSA, cortical CSA and BMC were calculated. Bone, muscle and fat CSAs were estimated in the lower leg and thigh. RESULTS: BMDtrab decreased exponentially with time since injury at different rates in the tibia and femur. At most sites, female paraplegics had significantly lower BMC, total bone CSA and muscle CSA than male paraplegics. Subjects with lumbar SCI tended to have lower bone values and smaller muscle CSAs than in thoracic SCI. CONCLUSION: At the distal epiphyses of the tibia and femur, there is generally a rapid and extensive reduction in BMDtrab after SCI. Female subjects, and those with lumbar SCI, tend to have lower bone values than males or those with thoracic SCI, respectively.

A randomized controlled trial of intranasal fentanyl vs intravenous morphine for analgesia in the prehospital setting.

STUDY OBJECTIVE: The objective of the study was to compare intranasal fentanyl (INF) with intravenous morphine (IVM) for prehospital analgesia. METHODS: This was a randomized, controlled, open-label trial. Consecutive adult patients (n = 258) requiring analgesia (Verbal Rating Score [VRS] >2/10 noncardiac or >5/10 cardiac) were recruited. Patients received INF 180 mug +/- 2 doses of 60 mug at > or =5-minute intervals or IVM 2.5 to 5 mg +/- 2 doses of 2.5 to 5 mg at > or =5-minute intervals. The end point was the difference in baseline/destination VRS. RESULTS: Groups were equivalent (P = not significant) for baseline VRS [mean (SD): INF 8.3 (1.7), IVM 8.1 (1.6)] and minutes to destination [mean (SD): INF 27.2 (15.5), IVM 30.6 (19.1)]. Patients had a mean (95% confidence interval) VRS reduction as follows: INF 4.22 (3.74-4.71), IVM 3.57 (3.10-4.03); P = .08. Higher baseline VRS (P < .001), no methoxyflurane use (P < .01), and back pain (P = .02) predicted VRS reduction. Safety and acceptability were comparable. CONCLUSIONS: There was no significant difference in the effectiveness of INF and IVM for prehospital analgesia.

Improving respiration in patients with tetraplegia by functional electrical stimulation: an anatomical perspective.

Patients with tetraplegia often have respiratory complications because of paralysis of the abdominal and intercostal muscles. Functional electrical stimulation (FES) has been used to improve breathing in these patients by applying surface stimulation to the abdominal muscles. We aimed to find the best nerves to stimulate directly to increase tidal volume and make cough more effective. Surface electrodes were placed on a patient's abdominal wall to find the optimum points for surface stimulation. These positions were plotted on a transparent sheet. The abdomino-intercostal nerves were dissected in five male dissecting room cadavers matched for size with the patient. The plastic sheet was then superimposed over each of the dissections to clarify the relationship between optimum surface stimulation points and the underlying nerves. Results show that the optimum surface stimulation points overlie the course of abdomino-intercostal nerves T9, 10, and 11. The success with selecting stimulation points associated with T9, 10, and 11 is probably because of the large mass of abdominal muscle supplied by these nerves. The constant position of the nerves below the ribs makes the intercostal space a possible site for direct stimulation of the abdomino-intercostal nerves.

Spinal injuries in Scottish mountaineers.

OBJECTIVE: To identify the frequency and pattern of spinal injury sustained in mountaineering accidents. METHODS: All patients with spinal injuries sustained while mountaineering who were treated at the Queen Elizabeth National Spinal Injuries Unit from 1992 to 2001 were studied. Information was obtained from hospital notes, and each patient completed a questionnaire. RESULTS: Twenty-one patients with spinal injuries sustained in mountaineering accidents were identified. Fourteen patients (67.7%) were experienced mountaineers. Four (19.0%) were rock climbing, 6 (28.6%) were winter climbing, 6 (28.6%) were hill walking, and 5 (23.8%) were winter walking. Ten patients (47.6%) sustained cervical injuries, 5 (23.8%) sustained thoracic injuries, 5 (23.8%) sustained lumbar injuries, and 1 (4.8%) sustained cervical and lumbar injuries. Nineteen patients (90.5%) sustained fractures, 1 (4.8%) sustained a dislocation, and 1 (4.8%) sustained a fracture dislocation. Seventeen patients (81.0%) were neurologically intact, 2 (9.5%) sustained complete cord injury, and 2 (9.5%) sustained incomplete cauda equina lesions. Ten patients (47.6%) required internal fixation of their fractures. Fourteen patients (57.1%) sustained other significant injuries. CONCLUSIONS: This is the first study specifically aimed to describe patterns of spinal injuries and their outcomes in mountaineers. The group we studied had sustained fractures at differing spinal levels with no specific injury pattern. The incidence of cord injury was lower than in other spinal injury patient groups. The majority had also sustained other significant and potentially distracting injuries. Prehospital care providers should maintain a high suspicion of spinal injury in this group.

Prophylaxis of thromboembolism in spinal injuries--survey of practice in spinal units in the British Isles.

AIM: Survey and discussion of the current thromboembolic prophylaxis practice in spinal injury units within the British Isles. BACKGROUND: Deep vein thrombosis and pulmonary embolism are major causes of morbidity and mortality in patients with spinal injuries. A wide range of thromboprophylactic measures have been proposed. The optimum treatment and duration for thromboprophylaxis in spinal injuries is unknown. Different spinal units within the British Isles use different measures despite similar training backgrounds. The present study was performed to review practice and make suggestions on best practice using literature review. METHODS: All the 13 regional and national spinal injury referral centres within the British Isles were contacted to find out their protocols for thromboembolic prophylaxis in patients with acute spinal injuries. RESULTS: All units replied. A wide variation in methods used was found in different spinal units ranging from no chemical prophylaxis to oral anticoagulation with warfarin and contrasting views on the use of antithromboembolic stockings. CONCLUSION: All units recognised the risk of thromboembolism after spinal injuries. A wide variety of thromboprophylactic measures are used.