Wearable Ultrasound System Using Low-Voltage Time Delay Spectrometry for Dynamic Tissue Imaging.

OBJECTIVE: Wearable ultrasound is emerging as a new paradigm of real-time imaging in freely moving humans and has wide applications from cardiovascular health monitoring to human gesture recognition. However, current wearable ultrasound devices have typically employed pulse-echo imaging which requires high excitation voltages and sampling rates, posing safety risks, and requiring specialized hardware. Our objective was to develop and evaluate a wearable ultrasound system based on time delay spectrometry (TDS) that utilizes low-voltage excitation and significantly simplified instrumentation. METHODS: We developed a TDS-based ultrasound system that utilizes continuous, frequency-modulated sweeps at low excitation voltages. By mixing the transmit and receive signals, the system digitizes the ultrasound signal at audio frequency (kHz) sampling rates. Wearable ultrasound transducers were developed, and the system was characterized in terms of imaging performance, acoustic output, thermal characteristics, and applications in musculoskeletal imaging. RESULTS: The prototype TDS system is capable of imaging up to 6 cm of depth with signal-to-noise ratio of up to 42 dB at a spatial resolution of 0.33 mm. Acoustic and thermal radiation measurements were within clinically safe limits for continuous ultrasound imaging. We demonstrated the ability to use a 4-channel wearable system for dynamic imaging of muscle activity. CONCLUSION: We developed a wearable ultrasound imaging system using TDS to mitigate challenges with pulse echo-based wearable ultrasound imaging systems. Our device is capable of high-resolution, dynamic imaging of deep-seated tissue structures and is safe for long-term use. SIGNIFICANCE: This work paves the way for low-voltage wearable ultrasound imaging devices with significantly reduced hardware complexity.

Measurement and Modeling of Ship-Related Ultrafine Particles and Secondary Organic Aerosols in a Mediterranean Port City.

Maritime transport emerges as a major source of ultrafine particle (UFP) pollution in coastal regions with consequences for the health of people living in port cities. Inhalation of UFPs can cause inflammation and oxidative stress, which are starting points for further diseases. In addition to primary particles, secondary organic aerosol (SOA) may form through the photo-oxidation of volatile organic compounds emitted in ship exhaust. The characterization of size-segregated and chemical properties of particles is essential for assessing the health implications related to shipping. We applied a coupled regional-local chemistry transport modeling system to study the effects of ship emissions on atmospheric concentrations of UFP and SOA in the Mediterranean port city Marseille (France), which is characterized by the combination of high port activity, industrialized emissions, and active photochemistry in summer. Our results show that the average potential impact from local shipping in the port area was 6-9% for SOA and 27-51% for total particle number concentration in July 2020. The estimated oxidative potential of daily mean particulate organic matter related to shipping was lower than the oxidative potential reported for heavy fuel oil (HFO). The lower oxidative potential in this study is very likely due to the low share of ships using HFO during stopover.

Novel adhesive mineral-organic bone cements based on phosphoserine and magnesium phosphates or oxides.

Present surgical situations require a bone adhesive which has not yet been developed for use in clinical applications. Recently, phosphoserine modified cements (PMC) based on mixtures of o-phosphoserine (OPLS) and calcium phosphates, such as tetracalcium phosphate (TTCP) or α-tricalcium phosphate (α-TCP) as well as chelate setting magnesium phosphate cements have gained increasing popularity for their use as mineral bone adhesives. Here, we investigated new mineral-organic bone cements based on phosphoserine and magnesium phosphates or oxides, which possess excellent adhesive properties. These were analyzed by X-ray diffraction, Fourier infrared spectroscopy and electron microscopy and subjected to mechanical tests to determine the bond strength to bone after ageing at physiological conditions. The novel biomineral adhesives demonstrate excellent bond strength to bone with approximately 6.6-7.3 MPa under shear load. The adhesives are also promising due to their cohesive failure pattern and ductile character. In this context, the new adhesive cements are superior to currently prevailing bone adhesives. Future efforts on bone adhesives made from phosphoserine and Mg2+ appear to be very worthwhile.

Assessment of diagnostic capacity and decision-making based on the 2015 American Thyroid Association ultrasound classification system.

BACKGROUND: This study evaluates the American Thyroid Association (ATA) ultrasound (US) classification system for the initial assessment of thyroid nodules to determine if it indeed facilitates clinical decision-making. AIM: To perform a systematic review and meta-analysis of the diagnostic value of the ATA US classification system for the initial assessment of thyroid nodules. METHODS: In accordance with the PRISMA statement for diagnostic test accuracy, we selected articles that evaluated the 2015 ATA US pattern guidelines using a diagnostic gold standard. We analyzed these cases using traditional diagnostic parameters, as well as the threshold approach to clinical decision-making and decision curve analysis. RESULTS: We reviewed 13 articles with 8445 thyroid nodules, which were classified according to 2015 ATA patterns. Of these, 46.62% were malignant. No cancer was found in any of the ATA benign pattern nodules. The Bayesian analysis post-test probability for cancer in each classification was: (1) Very-low suspicion, 0.85%; (2) Low, 2.6%; (3) Intermediate, 6.7%; and (4) High, 40.9%. The net benefit (NB), expressed as avoided interventions, indicated that the highest capacity to avoid unnecessary fine needle aspiration biopsy (FNAB) in the patterns that we studied was 42, 31, 35, and 43 of every 100 FNABs. The NB calculation for a probability threshold of 11% for each of the ATA suspicion patterns studied is less than that of performing FNAB on all nodules. CONCLUSION: These three types of analysis have shown that only the ATA high-suspicion diagnostic pattern is clinically useful, in which case, FNAB should be performed. However, the curve decision analysis has demonstrated that using the ATA US risk patterns to decide which patients need FNAB does not provide a greater benefit than performing FNAB on all thyroid nodules. Therefore, it is likely that a better way to approach the assessment of thyroid nodules would be to perform FNAB on all non-cystic nodules, as the present analysis has shown the ATA risk patterns do not provide an adequate clinical decision-making framework.

City Scale Modeling of Ultrafine Particles in Urban Areas with Special Focus on Passenger Ferryboat Emission Impact.

Air pollution by aerosol particles is mainly monitored as mass concentrations of particulate matter, such as PM10 and PM2.5. However, mass-based measurements are hardly representative for ultrafine particles (UFP), which can only be monitored adequately by particle number (PN) concentrations and are considered particularly harmful to human health. This study examines the dispersion of UFP in Hamburg city center and, in particular, the impact of passenger ferryboats by modeling PN concentrations and compares concentrations to measured values. To this end, emissions inventories and emission size spectra for different emission sectors influencing concentrations in the city center were created, explicitly considering passenger ferryboat traffic as an additional emission source. The city-scale chemical transport model EPISODE-CityChem is applied for the first time to simulate PN concentrations and additionally, observations of total particle number counts are taken at four different sampling sites in the city. Modeled UFP concentrations are in the range of 1.5-3 × 104 cm-3 at ferryboat piers and at the road traffic locations with particle sizes predominantly below 50 nm. Urban background concentrations are at 0.4-1.2 × 104 cm-3 with a predominant particle size in the range 50-100 nm. Ferryboat traffic is a significant source of emissions near the shore along the regular ferry routes. Modeled concentrations show slight differences to measured data, but the model is capable of reproducing the observed spatial variation of UFP concentrations. UFP show strong variations in both space and time, with day-to-day variations mainly controlled by differences in air temperature, wind speed and wind direction. Further model simulations should focus on longer periods of time to better understand the influence of meteorological conditions on UFP dynamics.

5,5-Dialkylluciferins are thermal stable substrates for bioluminescence-based detection systems.

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.

Integrating Modes of Transport in a Dynamic Modelling Approach to Evaluate Population Exposure to Ambient NO2 and PM2.5 Pollution in Urban Areas.

To evaluate the effectiveness of alternative policies and measures to reduce air pollution effects on urban citizen's health, population exposure assessments are needed. Due to road traffic emissions being a major source of emissions and exposure in European cities, it is necessary to account for differentiated transport environments in population dynamics for exposure studies. In this study, we applied a modelling system to evaluate population exposure in the urban area of Hamburg in 2016. The modeling system consists of an urban-scale chemistry transport model to account for ambient air pollutant concentrations and a dynamic time-microenvironment-activity (TMA) approach, which accounts for population dynamics in different environments as well as for infiltration of outdoor to indoor air pollution. We integrated different modes of transport in the TMA approach to improve population exposure assessments in transport environments. The newly developed approach reports 12% more total exposure to NO2 and 19% more to PM2.5 compared with exposure estimates based on residential addresses. During the time people spend in different transport environments, the in-car environment contributes with 40% and 33% to the annual sum of exposure to NO2 and PM2.5, in the walking environment with 26% and 30%, in the cycling environment with 15% and 17% and other environments (buses, subway, suburban, and regional trains) with less than 10% respectively. The relative contribution of road traffic emissions to population exposure is highest in the in-car environment (57% for NO2 and 15% for PM2.5). Results for population-weighted exposure revealed exposure to PM2.5 concentrations above the WHO AQG limit value in the cycling environment. Uncertainties for the exposure contributions arising from emissions and infiltration from outdoor to indoor pollutant concentrations range from -12% to +7% for NO2 and PM2.5. The developed "dynamic transport approach" is integrated in a computationally efficient exposure model, which is generally applicable in European urban areas. The presented methodology is promoted for use in urban mobility planning, e.g., to investigate on policy-driven changes in modal split and their combined effect on emissions, population activity and population exposure.

Modeling of the Concentrations of Ultrafine Particles in the Plumes of Ships in the Vicinity of Major Harbors.

Marine traffic in harbors can be responsible for significant atmospheric concentrations of ultrafine particles (UFPs), which have widely recognized negative effects on human health. It is therefore essential to model and measure the time evolution of the number size distributions and chemical composition of UFPs in ship exhaust to assess the resulting exposure in the vicinity of shipping routes. In this study, a sequential modelling chain was developed and applied, in combination with the data measured and collected in major harbor areas in the cities of Helsinki and Turku in Finland, during winter and summer in 2010-2011. The models described ship emissions, atmospheric dispersion, and aerosol dynamics, complemented with a time-microenvironment-activity model to estimate the short-term UFP exposure. We estimated the dilution ratio during the initial fast expansion of the exhaust plume to be approximately equal to eight. This dispersion regime resulted in a fully formed nucleation mode (denoted as Nuc2). Different selected modelling assumptions about the chemical composition of Nuc2 did not have an effect on the formation of nucleation mode particles. Aerosol model simulations of the dispersing ship plume also revealed a partially formed nucleation mode (Nuc1; peaking at 1.5 nm), consisting of freshly nucleated sulfate particles and condensed organics that were produced within the first few seconds. However, subsequent growth of the new particles was limited, due to efficient scavenging by the larger particles originating from the ship exhaust. The transport of UFPs downwind of the ship track increased the hourly mean UFP concentrations in the neighboring residential areas by a factor of two or more up to a distance of 3600 m, compared with the corresponding UFP concentrations in the urban background. The substantially increased UFP concentrations due to ship traffic significantly affected the daily mean exposures in residential areas located in the vicinity of the harbors.

Moving Object Detection Using a Parallax Shift Vector Algorithm.

There are various algorithms currently in use to detect asteroids from ground-based observatories, but they are generally restricted to linear or mildly curved movement of the target object across the field of view. Space based sensors in high inclination, low Earth orbits can induce significant parallax in a collected sequence of images, especially for objects at the typical distances of asteroids in the inner Solar System. This results in a highly non-linear motion pattern of the asteroid across the sensor, which requires a more sophisticated search pattern for detection processing. Both the classical pattern matching used in ground based asteroid search and the more sensitive matched filtering and synthetic tracking techniques, can be adapted to account for highly complex parallax motion. A new shift vector generation methodology is discussed along with its impacts on commonly used detection algorithms, processing load, and responsiveness to asteroid track reporting. The matched filter, template generator, and pattern matcher source code for the software described herein are available via GitHub.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 µM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 ß-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and ß-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

[Cervical lymphangioma in the adult. A report of 2 cases]. / Linfangioma de cuello en el adulto. Reporte de 2 casos.

BACKGROUND: Cervical lymphangiomas are uncommon benign congenital malformations usually present in children, and are rare in adults. Currently, complete resection is still the standard care. Two cases are presented of a cervical lymphangioma in an adult. The diagnosis and surgical approach is also discussed. CLINICAL CASE: Case 1. The first case is a 23 year old male with chief complaint of a tumour in the posterior triangle of the neck, which showed a substantial increase in size in the last 9 months. No associated signs or symptoms, or any trauma history was reported. CT scan of the neck showed images suggestive of a posterior cervical lymphangioma. Exploratory cervical surgery was performed, with complete resection of a cystic tumour located in the posterior triangle of the neck. Surgery was performed without complications and postoperative care was unremarkable. CASE 2: The second case is a 28 woman with a cystic tumour in submandibular space. She had history of a previous incomplete operation in another institution 2 years ago, with recurrence of the tumour. A second surgery was performed with complete resection without complications, and with a good outcome. CONCLUSIONS: Cervical lymphangioma is a very rare benign disease, surgical treatment is preferred, but sclerotherapy can be used as alternative treatment.

Novel Use of Ultrasound Elastography to Quantify Muscle Tissue Changes After Dry Needling of Myofascial Trigger Points in Patients With Chronic Myofascial Pain.

OBJECTIVES: To compare a mechanical heterogeneity index derived from ultrasound vibration elastography with physical findings before and after dry-needling treatment of spontaneously painful active myofascial trigger points in the upper trapezius muscle. METHODS: Forty-eight patients with chronic myofascial pain enrolled in a prospective interventional trial of 3 weekly dry-needling treatments for active myofascial trigger points. Trigger points were evaluated at baseline and at treatment completion using palpation, the pressure-pain threshold, and the mechanical heterogeneity index. Thirty patients were reevaluated at 8 weeks. Trigger points that "responded" changed to tissue that was no longer spontaneously painful, with or without the presence of a palpable nodule. Trigger points that "resolved" changed to tissue without a palpable nodule. The mechanical heterogeneity index was defined as the proportion of the upper trapezius muscle that appeared mechanically stiffer on elastography. Statistical significance for comparisons was determined at P < .05. RESULTS: Following 3 dry needle treatments, the mechanical heterogeneity index decreased significantly for the 38 myofascial trigger points (79% of 48) that responded to treatment. Among these, the baseline mechanical heterogeneity index was significantly lower for the 13 trigger points (27% of 38) that resolved, but the decrease after 3 dry needle treatments did not reach significance. The pressure-pain threshold improved significantly for both groups. At 8 weeks, the mechanical heterogeneity index decreased significantly for the 22 trigger points (73% of 30) that responded and for the 10 (45% of 22) that resolved. The pressure-pain threshold improvement was significant for trigger points that responded but did not reach significance for resolved trigger points. CONCLUSIONS: The mechanical heterogeneity index identifies changes in muscle tissue properties that correlate with changes in the myofascial trigger point status after dry needling.

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease.

NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions.

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.

Dry Needling Alters Trigger Points in the Upper Trapezius Muscle and Reduces Pain in Subjects With Chronic Myofascial Pain.

OBJECTIVE: To determine whether dry needling of an active myofascial trigger point (MTrP) reduces pain and alters the status of the trigger point to either a non-spontaneously tender nodule or its resolution. DESIGN: A prospective, nonrandomized, controlled, interventional clinical study. SETTING: University campus. PARTICIPANTS: A total of 56 subjects with neck or shoulder girdle pain of more than 3 months duration and active MTrPs were recruited from a campus-wide volunteer sample. Of these, 52 completed the study (23 male and 33 female). Their mean age was 35.8 years. INTERVENTIONS: Three weekly dry needling treatments of a single active MTrP. PRIMARY OUTCOMES: Baseline and posttreatment evaluations of pain using a verbal analogue scale, the Brief Pain Inventory, and the status of the MTrP as determined by digital palpation. Trigger points were rated as active (spontaneously painful), latent (requiring palpation to reproduce the characteristic pain), or resolved (no palpable nodule). SECONDARY OUTCOMES: Profile of Mood States, Oswestry Disability Index, and Short Form 36 scores, and cervical range of motion. PRIMARY OUTCOMES: A total of 41 subjects had a change in trigger point status from active to latent or resolved, and 11 subjects had no change (P < .001). Reduction in all pain scores was significant (P < .001). SECONDARY OUTCOMES: Significant improvement in posttreatment cervical rotational asymmetry in subjects as follows: unilateral/bilateral MTrPs (P = .001 and P = 21, respectively); in pain pressure threshold in subjects with unilateral/bilateral MTrPs, (P = .006 and P = .012, respectively); improvement in the SF-36 mental health and physical functioning subscale scores (P = .019 and P = .03), respectively; and a decrease in the Oswestry Disability Index score (P = .003). CONCLUSIONS: Dry needling reduces pain and changes MTrP status. Change in trigger point status is associated with a statistically and clinically significant reduction in pain. Reduction of pain is associated with improved mood, function, and level of disability.

A systematic comparison between subjects with no pain and pain associated with active myofascial trigger points.

OBJECTIVE: To determine whether standard evaluations of pain distinguish subjects with no pain from those with myofascial pain syndromes (MPS) and active myofascial trigger points (MTrPs) and to assess whether self-reports of mood, function, and health-related quality of life differ between these groups. DESIGN: A prospective, descriptive study. SETTING: University. PATIENTS: Adults with and without neck pain. METHODS: We evaluated adults with MPS and active (painful) MTrPs and those without pain. Subjects in the "active" (A) group had at least one active MTrP with spontaneous pain that was persistent, lasted longer than 3 months, and had characteristic pain on palpation. Subjects in the "no pain" (NP) group had no spontaneous pain. However, some of these subjects had discomfort upon MTrP palpation (latent MTrP), whereas others in the NP group had no discomfort upon palpation of nodules or had no nodules. OUTCOME MEASURES: Each participant underwent range of motion measurement, a 10-point manual muscle test, and manual and algometric palpation. The latter determined the pain/pressure threshold using an algometer of 4 predetermined anatomic sites along the upper trapezius. Participants rated pain using a verbal analog scale (0-10) and completed the Brief Pain Inventory and Oswestry Disability Scale (which included a sleep subscale), the Short -Form 36 Health Survey, and the Profile of Mood States. RESULTS: The A group included 24 subjects (mean age 36 years; 16 women), and the NP group included 26 subjects (mean age 26 years; 12 women). Group A subjects differed from NP subjects in the number of latent MTrPs (P = .0062), asymmetrical cervical range of motion (P = .01 for side bending and P = .002 for rotation), and in all pain reports (P < .0001), algometry (P < .03), Profile of Mood States (P < .038), Short Form 36 Health Survey (P < .01), and Oswestry Disability Scale (P < .0001). CONCLUSION: A systematic musculoskeletal evaluation of people with MPS reliably distinguishes them from subjects with no pain. The 2 groups are significantly different in their physical findings and self-reports of pain, sleep disturbance, disability, health status, and mood. These findings support the view that a "local" pain syndrome has significant associations with mood, health-related quality of life, and function.

Imaging proteolytic activity in live cells and animal models.

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

Ultrasonic characterization of the upper trapezius muscle in patients with chronic neck pain.

Myofascial trigger points (MTrPs) are palpable, tender nodules in taut bands of skeletal muscle that are painful on compression. MTrPs are characteristic findings in myofascial pain syndrome (MPS). The role of MTrPs in the pathophysiology of MPS is unknown. Localization, diagnosis, and clinical outcome measures of painful MTrPs can be improved by objectively characterizing and quantitatively measuring their properties. The goal of this study was to evaluate whether ultrasound imaging and elastography can differentiate symptomatic (active) MTrPs from normal muscle. Patients with chronic (>3 months) neck pain with spontaneously painful, palpable (i.e., active) MTrPs and healthy volunteers without spontaneous pain (having palpably normal muscle tissue) were recruited for this study. The upper trapezius muscles in all subjects were imaged, and the echotexture was analyzed using entropy filtering of B-mode images. Vibration elastography was performed by vibrating the muscle externally at 100 Hz. Color Doppler variance imaging was used to quantify the regions of color deficit exhibiting low vibration amplitude. The imaging measures were compared against the clinical findings of a standardized physical exam. We found that sites with active MTrPs (n = 14) have significantly lower entropy (p < 0.05) and significantly larger nonvibrating regions (p < 0.05) during vibration elastography compared with normal, uninvolved muscle (n = 15). A combination of both entropy analysis and vibration elastography yielded 69% sensitivity and 81% specificity in discriminating active MTrPs from normal muscle. These results suggest that active MTrPs have more homogeneous texture and heterogeneous stiffness when compared with normal, unaffected muscle. Our methods enabled us to improve the imaging contrast between suspected MTrPs and surrounding muscle. Our results indicate that in subjects with chronic neck pain and active MTrPs, the abnormalities are not confined to discrete isolated nodules but instead affect the milieu of the muscle surrounding palpable MTrPs. With further refinement, ultrasound imaging can be a promising objective method for characterizing soft tissue abnormalities associated with active MTrPs and elucidating the role of MTrPs in the pathophysiology of MPS.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.