Modeling spontaneous opioid withdrawal in male and female outbred mice using traditional endpoints and hyperalgesia.

Opioid withdrawal significantly impacts drug dependence cycles as hyperalgesia associated with withdrawal is often a reason for continued drug use. Animal models of addiction are important tools for studying how drug dependence and withdrawal impact not only normal neurocircuitry but also the effectiveness of potential treatments for dependence and withdrawal. We conducted a study of the time course of spontaneous morphine withdrawal in outbred male and female mice that can be used to examine sex differences in male and female mice using both traditional somatic endpoints and mechanical hyperalgesia as an endpoint of withdrawal. Male and female national institute of health (NIH) Swiss mice were made dependent upon morphine using an escalating dosing schedule. Injections were stopped after 5 days. Withdrawal behavior was assessed at time intervals up to 106 h after the final injection. Numbers of forepaw tremors, wet-dog shakes, jumps and other behaviors were scored to create a global score. Paw pressure readings were then also taken to track changes in sensitivity to a painful stimulus over time. Male and female mice had approximately similar withdrawal severity peaking at 24 h after the final injection as measured by composite global scores. Females did exhibit an earlier and greater frequency of tremors than males. Although males and females showed similar hyperalgesia during withdrawal, females recovered faster. Spontaneous opioid withdrawal peaking at 24 h was demonstrated in male and female NIH Swiss mice. We also successfully demonstrated that hyperalgesia is an endpoint that varies over the course of withdrawal.

Differentiating Live Versus Dead Gram-Positive and Gram-Negative Bacteria With and Without Oxidative Stress Using Buoyant Mass Measurements.

Expeditious and accurate determination of pathogenic bacteria cell viability is of great importance to public health for numerous areas including medical diagnostics, food safety, and environmental monitoring. In this work a cell buoyant mass classifier approach is presented to assess bacteria cell viability in real time. Buoyant mass measurements for live and dead Gram-positive and Gram-negative bacteria populations were acquired with a commercial suspended microchannel resonator, Archimedes, to generate receiver operating characteristic (ROC) curves. To quantitatively assess the difference in buoyant mass for live and dead bacteria populations, ROC curves were generated to demonstrate cell viability determination. The results are presented as a binary classifier with a decision boundary, above which cells are considered live and below which cells are considered dead. A decision threshold value is evaluated with consideration that a certain true positive rate (correct classification of a live cell) is maintained with an acceptable false positive rate. The potential for this approach to monitor cell viability in real time is significant, especially when considering multiple classifier dimensions such as buoyant mass and density. This classifier approach represents a next generation technique for rapid and label-free diagnostics based on cell feature measurements.

Psychological resilience is linked to motor strength and gait endurance in early multiple sclerosis.

BACKGROUND: Psychologically resilient persons persist despite obstacles and bounce back after adversity, leading to better outcomes in non-neurologic populations. It is unknown whether psychological resilience relates to objective functional outcomes in multiple sclerosis (MS). OBJECTIVE: To determine whether psychological resilience explains differential objective cognitive and motor functioning in persons with early MS. METHODS: Psychological resilience was assessed in 185 patients with early MS and 50 matched healthy controls with the Connors-Davidson Resilience Scale (CDRS-10). Subjects completed the MS Functional Composite (MSFC) and a comprehensive neurobehavioral evaluation. Correlations assessed links between CDRS-10 and MSFC, motor indices (Total, Fine Motor, Gross Motor), and cognitive indices (Total, Cognitive Efficiency, Memory). RESULTS: Higher CDRS-10 among patients was linked to better MSFC and motor outcomes (but not cognition), with the most robust relationships for gross motor function (grip strength, gait endurance). Findings were independent of mood and fatigue. CDRS-10 was unrelated to MS disease burden. CDRS-10 was also specifically linked to motor outcomes in healthy controls. CONCLUSION: Functional outcomes vary across persons with MS, even when disease burden and neurologic disability are low. These findings identify high psychological resilience as a non-disease-specific contributor to motor strength and endurance, which may explain differential outcomes across patients.

Word-finding difficulty is a prevalent disease-related deficit in early multiple sclerosis.

BACKGROUND: Persons with multiple sclerosis (MS) commonly report word-finding difficulty clinically, yet this language deficit remains underexplored. OBJECTIVE: To investigate the prevalence and nature of word-finding difficulty in persons with early MS on three levels: patient report, cognitive substrates, and neuroimaging. METHODS: Two samples of early MS patients (n = 185 and n = 55; ⩽5 years diagnosed) and healthy controls (n = 50) reported frequency/severity of cognitive deficits and underwent objective assessment with tasks of rapid automatized naming (RAN), measuring lexical access speed, memory, word generation, and cognitive efficiency. High-resolution brain magnetic resonance imaging (MRI) derived measurements of regional cortical thickness, global and deep gray matter volume, and T2 lesion volume. Relationships among patient-reported word-finding difficulty, cognitive performance, and neural correlates were examined. RESULTS: Word-finding difficulty was the most common cognitive complaint of MS patients and the only complaint reported more by patients than healthy controls. Only RAN performance discriminated MS patients with subjective word-finding deficits from those without subjective complaints and from healthy controls. Thinner left parietal cortical gray matter independently predicted impaired RAN performance, driven primarily by the left precuneus. CONCLUSION: Three levels of evidence (patient-report, objective behavior, regional gray matter) support word-finding difficulty as a prevalent, measurable, disease-related deficit in early MS linked to left parietal cortical thinning.

Dissociable cognitive patterns related to depression and anxiety in multiple sclerosis.

BACKGROUND: Individuals with multiple sclerosis (MS) frequently present with depression and anxiety, as well as cognitive impairment, challenging clinicians to disentangle interrelationships among these symptoms. OBJECTIVE: To identify cognitive functions associated with anxiety and depression in MS. METHODS: Mood and cognition were measured in 185 recently diagnosed patients (Reserve Against Disability in Early Multiple Sclerosis (RADIEMS) cohort), and an independent validation sample (MEM CONNECT cohort, n = 70). Partial correlations evaluated relationships of cognition to anxiety and depression controlling for age, sex, education, and premorbid verbal intelligence. RESULTS: In RADIEMS cohort, lower anxiety was associated with better nonverbal memory (rp = -0.220, p = 0.003) and lower depression to better attention/processing speed (rp = -0.241, p = 0.001). Consistently, in MEM CONNECT cohort, lower anxiety was associated with better nonverbal memory (rp = -0.271, p = 0.028) and lower depression to better attention/processing speed (rp = -0.367, p = 0.002). Relationships were unchanged after controlling for T2 lesion volume and fatigue. CONCLUSION: Consistent mood-cognition relationships were identified in two independent cohorts of MS patients, suggesting that cognitive correlates of anxiety and depression are separable. This dissociation may support more precise models to inform treatment development. Treatment of mood symptoms may mitigate effects on cognition and/or treatment of cognition may mitigate effects on mood.

Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas.

Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. (64)Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼ 2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ(2) = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors.

Data-driven optimized flip angle selection for T1 estimation from spoiled gradient echo acquisitions.

PURPOSE: Define criteria for selection of optimal flip angle sets for T1 estimation and evaluate effects on T1 mapping. THEORY AND METHODS: Flip angle sets for spoiled gradient echo-based T1 mapping were selected by minimizing T1 estimate variance weighted by the joint density of M0 and T1 in an initial acquisition. The effect of optimized flip angle selection on T1 estimate error was measured using simulations and experimental data in the human and rat brain. RESULTS: For two-point acquisitions, optimized angle sets were similar to those proposed by other groups and, therefore, performed similarly. For multipoint acquisitions, optimal angle sets for T1 mapping in the brain consisted of a repetition of two angles. Implementation of optimal angles reduced T1 estimate variance by 30-40% compared with a multipoint acquisition using a range of angles. Performance of the optimal angle set was equivalent to that of a repetition of the two-angle set selected using criteria proposed by other researchers. CONCLUSION: Repetition of two carefully selected flip angles notably improves the precision of resulting T1 estimates compared with acquisitions using a range of flip angles. This work provides a flexible and widely applicable optimization method of particular use for those who repeatedly perform T1 estimation. Magn Reson Med 76:792-802, 2016. © 2015 Wiley Periodicals, Inc.

Training voluntary motor suppression with real-time feedback of motor evoked potentials.

Training people to suppress motor representations voluntarily could improve response control. We evaluated a novel training procedure of real-time feedback of motor evoked potentials (MEPs) generated by transcranial magnetic stimulation (TMS) over motor cortex. On each trial, a cue instructed participants to use a mental strategy to suppress a particular finger representation without overt movement. A single pulse of TMS was delivered over motor cortex, and an MEP-derived measure of hand motor excitability was delivered visually to the participant within 500 ms. In experiment 1, we showed that participants learned to reduce the excitability of a particular finger beneath baseline (selective motor suppression) within 30 min of practice. In experiment 2, we performed a double-blind study with 2 training groups (1 with veridical feedback and 1 with matched sham feedback) to show that selective motor suppression depends on the veridical feedback itself. Experiment 3 further demonstrated the importance of veridical feedback by showing that selective motor suppression did not arise from mere mental imagery, even when incentivized with reward. Thus participants can use real-time feedback of TMS-induced MEPs to discover an effective mental strategy for selective motor suppression. This high-temporal-resolution, trial-by-trial-feedback training method could be used to help people better control response tendencies and may serve as a potential therapy for motor disorders such as Tourette's and dystonia.

Mass and density measurements of live and dead Gram-negative and Gram-positive bacterial populations.

Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10(5) and 10(8) cells/ml, while growth was not observed with optical density measurements until the concentration was 10(7) cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.

Synergistic effects of GDNF and VEGF on lifespan and disease progression in a familial ALS rat model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.

Priority influences task selection decisions in multi-task management.

The role of task priority on task selection in multi-task management is unclear based on prior work, leading to a common finding of 'priority neglect'. However, properties such as urgency and conflict may influence whether operators weigh priority in their decision. We examined the role of instructed task prioritization, bolstered by more urgent and conflicting conditions, on how operators select among emergent, concurrent tasks when multitasking. Using the Multi-Attribute Task Battery (MATB) multitasking platform we tested both an auditory communications task and a manual tracking task as the priority tasks. Results showed that instructed priority significantly increased target task selection under the conflicting task conditions for both tasks. Urgency itself may modulate whether instructions to prioritize affect task selection choices when multitasking, and therefore counter to prior results instructions may yet be useful for helping operators select a higher priority task under conflict, a generalizable effect to be further explored.

Examining post-error performance in a complex multitasking environment.

Previous work on indices of error-monitoring strongly supports that errors are distracting and can deplete attentional resources. In this study, we use an ecologically valid multitasking paradigm to test post-error behavior. It was predicted that after failing an initial task, a subject re-presented with that task in conflict with another competing simultaneous task, would more likely miss their response opportunity for the competing task and stay 'tunneled' on the initially errored task. Additionally, we predicted that an error's effect on attention would dissipate after several seconds, making error cascades less likely when subsequent conflict tasks are delayed. A multi-attribute task battery was used to present tasks and collect measures of both post-error and post-correct performance. Results supported both predictions: post-error accuracy on the competing task was lower compared to post-correct accuracy, and error-proportions were higher at shorter delays, dissipating over time. An exploratory analysis also demonstrated that following errors (as opposed to post-correct trials), participants clicked more on the task panel of the initial error regardless of delay; this continued task-engagement provides preliminary support for errors leading to a cognitive tunneling effect.

Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection.

Microfluidic fabrication of hydrogel microparticles containing functionalized viral nanotemplates.

We demonstrate rapid microfluidic fabrication of hybrid microparticles composed of functionalized viral nanotemplates directly embedded in polymeric hydrogels. Specifically, genetically modified tobacco mosaic virus (TMV) templates were covalently labeled with fluorescent markers or metalized with palladium (Pd) nanoparticles (Pd-TMV) and then suspended in a poly(ethylene glycol)-based solution. Upon formation in a flow-focusing device, droplets were photopolymerized with UV light to form microparticles. Fluorescence and confocal microscopy images of microparticles containing fluorescently labeled TMV show uniform distribution of TMV nanotemplates throughout the microparticles. Catalytic activity, via the dichromate reduction reaction, is also demonstrated with microparticles containing Pd-TMV complexes. Additionally, Janus microparticles were fabricated containing viruses embedded in one side and magnetic nanoparticles in the other, which enabled simple separation from bulk solution. These results represent a facile route to directly harness the advantages of viral nanotemplates into a readily usable and stable 3D assembled format.

Unique and overlapping gene expression patterns driven by IL-4 and IL-13 in the mouse lung.

BACKGROUND: Allergic asthma results from inappropriate T(H)2-mediated inflammation. Both IL-4 and IL-13 contribute to asthma pathogenesis, but IL-4 predominantly drives T(H)2 induction, whereas IL-13 is necessary and sufficient for allergen-induced airway hyperresponsiveness and goblet cell hyperplasia. Although these 2 cytokines share signaling components, the molecular mechanisms by which they mediate different phases of the allergic asthmatic response remain elusive. OBJECTIVE: We sought to clarify the role or roles of IL-4 and IL-13 in asthma-pathogenesis. METHODS: We used DNA Affymetrix microarrays to profile pulmonary gene expression in BALB/c mice inoculated intratracheally with ragweed pollen, house dust mite, IL-4, IL-13, or both cytokines. IL-13 dependence was confirmed by comparing pulmonary gene expression in house dust mite-inoculated wild-type and IL-13 knockout mice. RESULTS: A signature gene expression profile consisting of 23 genes was commonly induced by means of inoculation with house dust mite, ragweed pollen, or IL-4 plus IL-13. Although rIL-4 and rIL-13 treatment induced an overlapping set of genes, IL-4 uniquely induced 21 genes, half of which were interferon response genes and half of which were genes important in immunoregulation. IL-13 uniquely induced 8 genes, most of which encode proteins produced by epithelial cells. CONCLUSIONS: IL-4 and IL-13 together account for most allergen-induced pulmonary genes. Selective IL-4 induction of IFN-gamma response genes and other genes that might negatively regulate allergic inflammation could partially explain the greater importance of IL-13 in the effector phase of allergic airway disease.

Disease-specific gene expression profiling in multiple models of lung disease.

RATIONALE: Microarray technology is widely employed for studying the molecular mechanisms underlying complex diseases. However, analyses of individual diseases or models of diseases frequently yield extensive lists of differentially expressed genes with uncertain relationships to disease pathogenesis. OBJECTIVES: To compare gene expression changes in a heterogeneous set of lung disease models in order to identify common gene expression changes seen in diverse forms of lung pathology, as well as relatively small subsets of genes likely to be involved in specific pathophysiological processes. METHODS: We profiled lung gene expression in 12 mouse models of infection, allergy, and lung injury. A linear model was used to estimate transcript expression changes for each model, and hierarchical clustering was used to compare expression patterns between models. Selected expression changes were verified by quantitative polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: A total of 24 transcripts, including many involved in inflammation and immune activation, were differentially expressed in a substantial majority (9 or more) of the models. Expression patterns distinguished three groups of models: (1) bacterial infection (n = 5), with changes in 89 transcripts, including many related to nuclear factor-kappaB signaling, cytokines, chemokines, and their receptors; (2) bleomycin-induced diseases (n = 2), with changes in 53 transcripts, including many related to matrix remodeling and Wnt signaling; and (3) T helper cell type 2 (allergic) inflammation (n = 5), with changes in 26 transcripts, including many encoding epithelial secreted molecules, ion channels, and transporters. CONCLUSIONS: This multimodel dataset highlights novel genes likely involved in various pathophysiological processes and will be a valuable resource for the investigation of molecular mechanisms underlying lung disease pathogenesis.

Emerging biomarkers in autism spectrum disorder: a systematic review.

Autism spectrum disorder (ASD) affects approximately 2% of children in the United States (US) yet its etiology is unclear and effective treatments are lacking. Therapeutic interventions are most effective if started early in life, yet diagnosis often remains delayed, partly because the diagnosis of ASD is based on identifying abnormal behaviors that may not emerge until the disorder is well established. Biomarkers that identify children at risk during the pre-symptomatic period, assist with early diagnosis, confirm behavioral observations, stratify patients into subgroups, and predict therapeutic response would be a great advance. Here we underwent a systematic review of the literature on ASD to identify promising biomarkers and rated the biomarkers in regards to a Level of Evidence and Grade of Recommendation using the Oxford Centre for Evidence-Based Medicine scale. Biomarkers identified by our review included physiological biomarkers that identify neuroimmune and metabolic abnormalities, neurological biomarkers including abnormalities in brain structure, function and neurophysiology, subtle behavioral biomarkers including atypical development of visual attention, genetic biomarkers and gastrointestinal biomarkers. Biomarkers of ASD may be found prior to birth and after diagnosis and some may predict response to specific treatments. Many promising biomarkers have been developed for ASD. However, many biomarkers are preliminary and need to be validated and their role in the diagnosis and treatment of ASD needs to be defined. It is likely that biomarkers will need to be combined to be effective to identify ASD early and guide treatment.

Epi-allelic Erk1 and Erk2 knockdown series for quantitative analysis of T cell Erk regulation and IL-2 production.

Erk activation is often used as a downstream pathway indicator of TCR signaling, generally in terms of both Erk1 and Erk2 isoforms measured together. In order to investigate potential distinctions between Erk1 and Erk2 regulation and effects downstream of TCR ligation, we generated a series of stable and independent Erk1 and Erk2 shRNA knockdown lines in the 1B6 T cell hybridoma. We observed no compensatory effect by opposite isoform upregulation, and found similar fractions of total phosphorylated Erk1/2 across this epi-allelic series in response to both anti-CD3 and peptide-MHC stimulation of TCR. Moreover, a previous prediction of an isoform-independent linear relationship between Erk activation and IL-2 production was confirmed. The effect of the shRNA-mediated knockdowns in reducing IL-2 production was observed to be stronger than that arising from pharmacological MEK inhibition at comparable degrees of ERK1/2 phosphorylation levels.

Integrated Methods to Manufacture Hydrogel Microparticles with High Protein Conjugation Capacity and Binding Kinetics via Viral Nanotemplate Display.

Genetically modified tobacco mosaic virus (TMV) can serve as a potent nanotemplate for high capacity protein conjugation through covalent coupling to its coat proteins with precise nanoscale spacing. TMV's own genomic RNA can also be exploited for orientationally controlled assembly onto various platforms with sequence and spatial selectivity via nucleic acid hybridization. Here we describe detailed methods for fabrication of hydrogel microparticles with capture DNA sequences, chemical activation and programming of TMV templates, TMV assembly with the microparticles and protein conjugation via bio-orthogonal click reactions.