Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

Clinical decision-making: physicians' preferences and experiences

BACKGROUND: Shared decision-making has been advocated; however there are relatively few studies on physician preferences for, and experiences of, different styles of clinical decision-making as most research has focused on patient preferences and experiences. The objectives of this study were to determine 1) physician preferences for different styles of clinical decision-making; 2) styles of clinical decision-making physicians perceive themselves as practicing; and 3) the congruence between preferred and perceived style. In addition we sought to determine physician perceptions of the availability of time in visits, and their role in encouraging patients to look for health information. METHODS: Cross-sectional survey of a nationally representative sample of U.S. physicians. RESULTS: 1,050 (53% response rate) physicians responded to the survey. Of these, 780 (75%) preferred to share decision-making with their patients, 142 (14%) preferred paternalism, and 118 (11%) preferred consumerism. 87% of physicians perceived themselves as practicing their preferred style. Physicians who preferred their patients to play an active role in decision-making were more likely to report encouraging patients to look for information, and to report having enough time in visits. CONCLUSION: Physicians tend to perceive themselves as practicing their preferred role in clinical decision-making. The direction of the association cannot be inferred from these data; however, we suggest that interventions aimed at promoting shared decision-making need to target physicians as well as patients

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.Optical Imaging; Biological Services; Biophysic

A Multitrait–Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic. Parent and child ratings of symptom severity were assessed using the Multidimensional Anxiety Scale for Children (MASC). Diagnostician ratings were obtained from the Anxiety Disorders Interview Schedule for Children and Parents (ADIS: C/P). Discriminant and convergent validity were assessed using confirmatory factor analytic techniques to test a multitrait–multimethod model. Confirmatory factor analyses supported the current classification of these child anxiety disorders. The disorders demonstrated statistical independence from each other (discriminant validity of traits), the model fit better when the anxiety syndromes were specified than when no specific syndromes were specified (convergent validity), and the methods of assessment yielded distinguishable, unique types of information about child anxiety (discriminant validity of methods). Using a multi-informant approach, these findings support the distinctions between childhood anxiety disorders as delineated in the current classification system, suggesting that disagreement between informants in psychometric studies of child anxiety measures is not due to poor construct validity of these anxiety syndromes

Distinguishing four components underlying physical activity: a new approach to using physical activity questionnaire data in old age

<p>Abstract</p> <p>Background</p> <p>It is evident that physical activity has many benefits, but it often remains unclear which types of activity are optimal for health and functioning in old age. The aim of this methodological study was to propose a method for distinguishing four components underlying self reported physical activity of older adults: intensity, muscle strength, turning actions and mechanical strain.</p> <p>Methods</p> <p>Physical activity was assessed by the validated LAPAQ questionnaire among 1699 older adults of the Longitudinal Aging Study Amsterdam. Based on expert consultation and literature review, the four component scores for several individual daily and sports activities were developed. Factor analysis was performed to confirm whether the developed components indeed measured different constructs of physical activity.</p> <p>Results</p> <p>Based on the factor analyses, three components were distinguished: 1. intensity and muscle strength loaded on the same factor, 2. mechanical strain and 3. turning actions. Analyses in gender, age and activity level subgroups consistently distinguished three factors.</p> <p>Conclusion</p> <p>Future research using these components may contribute to our understanding of how specific daily and sports activities may have a different influence on health and physical functioning in old age.</p

Functional Protein Network Activation Mapping Reveals New Potential Molecular Drug Targets for Poor Prognosis Pediatric BCP-ALL

Background: In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy. Methodology/Principal Findings: We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis. Conclusions/Significance: We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies

Observations on comatose survivors of cardiopulmonary resuscitation with generalized myoclonus

BACKGROUND: There is only limited data on improvements of critical medical care is resulting in a better outcome of comatose survivors of cardiopulmonary resuscitation (CPR) with generalized myoclonus. There is also a paucity of data on the temporal dynamics of electroenephalographic (EEG) abnormalities in these patients. METHODS: Serial EEG examinations were done in 50 comatose survivors of CPR with generalized myoclonus seen over an 8 years period. RESULTS: Generalized myoclonus occurred within 24 hours after CPR. It was associated with burst-suppression EEG (n = 42), continuous generalized epileptiform discharges (n = 5), alpha-coma-EEG (n = 52), and low amplitude (10 μV <) recording (n = 1). Except in 3 patients, these EEG-patterns were followed by another of these always nonreactive patterns within one day, mainly alpha-coma-EEG (n = 10) and continuous generalized epileptiform discharges (n = 9). Serial recordings disclosed a variety of EEG-sequences composed of these EEG-patterns, finally leading to isoelectric or flat recordings. Forty-five patients died within 2 weeks, 5 patients survived and remained in a permanent vegetative state. CONCLUSION: Generalized myoclonus in comatose survivors of CPR still implies a poor outcome despite advances in critical care medicine. Anticonvulsive drugs are usually ineffective. All postanoxic EEG-patterns are transient and followed by a variety of EEG sequences composed of different EEG patterns, each of which is recognized as an unfavourable sign. Different EEG-patterns in anoxic encephalopathy may reflect different forms of neocortical dysfunction, which occur at different stages of a dynamic process finally leading to severe neuronal loss

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds-approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure-reducing some bacteria while selecting for others

Quantitative Assessment of Whole-Body Tumor Burden in Adult Patients with Neurofibromatosis

Patients with neurofibromatosis 1 (NF1), NF2, and schwannomatosis are at risk for multiple nerve sheath tumors and premature mortality. Traditional magnetic resonance imaging (MRI) has limited ability to assess disease burden accurately. The aim of this study was to establish an international cohort of patients with quantified whole-body internal tumor burden and to correlate tumor burden with clinical features of disease.We determined the number, volume, and distribution of internal nerve sheath tumors in patients using whole-body MRI (WBMRI) and three-dimensional computerized volumetry. We quantified the distribution of tumor volume across body regions and used unsupervised cluster analysis to group patients based on tumor distribution. We correlated the presence and volume of internal tumors with disease-related and demographic factors.WBMRI identified 1286 tumors in 145/247 patients (59%). Schwannomatosis patients had the highest prevalence of tumors (P = 0.03), but NF1 patients had the highest median tumor volume (P = 0.02). Tumor volume was unevenly distributed across body regions with overrepresentation of the head/neck and pelvis. Risk factors for internal nerve sheath tumors included decreasing numbers of café-au-lait macules in NF1 patients (P = 0.003) and history of skeletal abnormalities in NF2 patients (P = 0.09). Risk factors for higher tumor volume included female gender (P = 0.05) and increasing subcutaneous neurofibromas (P = 0.03) in NF1 patients, absence of cutaneous schwannomas in NF2 patients (P = 0.06), and increasing age in schwannomatosis patients (p = 0.10).WBMRI provides a comprehensive phenotype of neurofibromatosis patients, identifies distinct anatomic subgroups, and provides the basis for investigating molecular biomarkers that correlate with unique disease manifestations