Extracellular Vesicles Slow Down Aß(1-42) Aggregation by Interfering with the Amyloid Fibril Elongation Step.

Formation of amyloid-ß (Aß) fibrils is a central pathogenic feature of Alzheimer's disease. Cell-secreted extracellular vesicles (EVs) have been suggested as disease modulators, although their exact roles and relations to Aß pathology remain unclear. We combined kinetics assays and biophysical analyses to explore how small (<220 nm) EVs from neuronal and non-neuronal human cell lines affected the aggregation of the disease-associated Aß variant Aß(1-42) into amyloid fibrils. Using thioflavin-T monitored kinetics and seeding assays, we found that EVs reduced Aß(1-42) aggregation by inhibiting fibril elongation. Morphological analyses revealed this to result in the formation of short fibril fragments with increased thicknesses and less apparent twists. We suggest that EVs may have protective roles by reducing Aß(1-42) amyloid loads, but also note that the formation of small amyloid fragments could be problematic from a neurotoxicity perspective. EVs may therefore have double-edged roles in the regulation of Aß pathology in Alzheimer's disease.

An Extraordinary Voice Expressed Through Humor: A Tribute to Casey Quinlan.

The Journal of Participatory Medicine introduces Extraordinary Lives, a new journal section celebrating the voices and work of steadfast advocates of participatory medicine that we have lost. This inaugural essay spotlights Casey Quinlan, a patient activist who effectively used her humor and incisive analysis of health care to encourage others to strive for meaningful change. A first-generation "professional patient," Casey served as a role model who inspired many to share their stories and achieve genuine partnerships in care delivery. A maker of "good trouble," her voice and stance were part of her power and influence in disrupting the status quo. We present her fight for personal access to health data, her aspiration for personally customized evidence, and her drive for all people to control their health and their health care.

Ginsenoside Rg3 Reduces the Toxicity of Graphene Oxide Used for pH-Responsive Delivery of Doxorubicin to Liver and Breast Cancer Cells.

Doxorubicin (DOX) is extensively used in chemotherapy, but it has serious side effects and is inefficient against some cancers, e.g., hepatocarcinoma. To ameliorate the delivery of DOX and reduce its side effects, we designed a pH-responsive delivery system based on graphene oxide (GO) that is capable of a targeted drug release in the acidic tumor microenvironment. GO itself disrupted glutathione biosynthesis and induced reactive oxygen species (ROS) accumulation in human cells. It induced IL17-directed JAK-STAT signaling and VEGF gene expression, leading to increased cell proliferation as an unwanted effect. To counter this, GO was conjugated with the antioxidant, ginsenoside Rg3, prior to loading with DOX. The conjugation of Rg3 to GO significantly reduced the toxicity of the GO carrier by abolishing ROS production. Furthermore, treatment of cells with GO-Rg3 did not induce IL17-directed JAK-STAT signaling and VEGF gene expression-nor cell proliferation-suggesting GO-Rg3 as a promising drug carrier. The anticancer activity of GO-Rg3-DOX conjugates was investigated against Huh7 hepatocarcinoma and MDA-MB-231 breast cancer cells. GO-Rg3-DOX conjugates significantly reduced cancer cell viability, primarily via downregulation of transcription regulatory genes and upregulation of apoptosis genes. GO-Rg3 is an effective, biocompatible, and pH responsive DOX carrier with potential to improve chemotherapy-at least against liver and breast cancers.

Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles.

Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.

Chemically Synthesized, Self-Assembling Small Interfering RNA-Prohead RNA Molecules Trigger Dicer-Independent Gene Silencing.

RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5' of the 3WJ (5'-siRNA-3WJ) relative to 3' of the 3WJ (3'-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.

High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes.

We present a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection at ultra-high analytical throughput. The imaging platform is capable of multiparametric fluorescence quantification and sub-cellular localization of these structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of the approach through the analysis and localization of P-bodies and stress granules in yeast and human cells using fluorescence and bright-field detection at analytical throughputs in excess of 60,000 and 400,000 cells/s, respectively. Results highlight the utility of our imaging flow cytometer in directly investigating phase-separated compartments within cellular environments and screening rare events at the sub-cellular level for a range of diagnostic applications.

Influence of phase of respiratory cycle on ultrasound imaging of deep abdominal muscle thickness.

BACKGROUND: It is difficult to evaluate the transversus abdominis (TrA) and internal oblique (IO) due to their dual role in both trunk control and breathing. OBJECTIVES: To investigate whether TrA and IO thickness as measured by ultrasound differs across the respiratory cycle in upright standing. DESIGN: Observational study. METHODS: Thickness of TrA and IO was measured with ultrasound in 67 subjects in upright standing. Measures were performed 3 times and by 2 assessors, at the end of relaxed expiration, at the end of a full inspiration, and at the end of full expiration. Differences were assessed by ANOVA. Intra- and inter-rater reliability (of a single measure and the average of 3 measures) were assessed by intra-class correlation (ICC). RESULTS: Thickness of the TrA and IO was higher at full expiration than at the end of relaxed expiration (p < 0.001), and in turn compared to at full inspiration (p < 0.001). Intra-rater reliability was excellent at all respiratory phases (ICC 0.76-0.87). Whereas inter-rater reliability for a single measure was only fair to good for TrA (ICC 0.52-0.71) and good to excellent for IO (ICC 0.61-0.78), the inter-rater reliability of the average was excellent at all respiratory phases (ICC 0.75-0.90). CONCLUSIONS: Thickness of TrA and IO increases when lung volume decreases. The intra- and inter-rater reliability of an average measure were excellent at the end of relaxed expiration, full inspiration and full expiration. This provides new opportunities to evaluate the deep abdominal muscles, and their role in respiration, in a physiotherapeutic setting.

Oscillatory Viscoelastic Microfluidics for Efficient Focusing and Separation of Nanoscale Species.

The ability to precisely control particle migration within microfluidic systems is essential for focusing, separating, counting, and detecting a wide range of biological species. To date, viscoelastic microfluidic systems have primarily been applied to the focusing, separation, and isolation of micrometer-sized species, with their use in nanoparticle manipulations being underdeveloped and underexplored, due to issues related to nanoparticle diffusivity and a need for extended channel lengths. To overcome such issues, we herein present sheathless oscillatory viscoelastic microfluidics as a method for focusing and separating both micrometer and sub-micrometer species. To highlight the efficacy of our approach, we segment our study into three size regimes, namely, micrometer (where characteristic particle dimensions are above 1 µm), sub-micrometer (where characteristic dimensions are between 1 µm and 100 nm), and nano (where characteristic dimensions are below 100 nm) regimes. Based on the ability to successfully manipulate particles in all these regimes, we demonstrate the successful isolation of p-bodies from biofluids (in the micrometer regime), the focusing of λ-DNA (in the sub-micrometer regime), and the focusing of extracellular vesicles (in the nanoregime). Finally, we characterize the physics underlying viscoelastic microflows using a dimensionless number that relates the lateral velocity (due to elastic effects) to the diffusion constant of the species within the viscoelastic carrier fluid. Based on the ability to precisely manipulate species in all three regimes, we expect that sheathless oscillatory viscoelastic microfluidics may be used to good effect in a range of biological and life science applications.

Environmental Fate of RNA Interference Pesticides: Adsorption and Degradation of Double-Stranded RNA Molecules in Agricultural Soils.

Double-stranded RNA (dsRNA) pesticides are a new generation of crop protectants that interfere with protein expression in targeted pest insects by a cellular mechanism called RNA interference (RNAi). The ecological risk assessment of these emerging pesticides necessitates an understanding of the fate of dsRNA molecules in receiving environments, among which agricultural soils are most important. We herein present an experimental approach using phosphorus-32 (32P)-radiolabeled dsRNA that allows studying key fate processes of dsRNA in soils with unprecedented sensitivity. This approach resolves previous analytical challenges in quantifying unlabeled dsRNA and its degradation products in soils. We demonstrate that 32P-dsRNA and its degradation products are quantifiable at concentrations as low as a few nanograms of dsRNA per gram of soil by both Cerenkov counting (to quantify total 32P-activity) and by polyacrylamide gel electrophoresis followed by phosphorimaging (to detect intact 32P-dsRNA and its 32P-containing degradation products). We show that dsRNA molecules added to soil suspensions undergo adsorption to soil particle surfaces, degradation in solution, and potential uptake by soil microorganisms. The results of this work on dsRNA adsorption and degradation advance a process-based understanding of the fate of dsRNA in soils and will inform ecological risk assessments of emerging dsRNA pesticides.

Functioning Without Cartilage: Older People With Radiographic Knee Osteoarthritis Who Self-Report No Functional Limitations Do Score Lower on a Performance Battery.

The majority of the older population shows signs of radiographic knee osteoarthritis. However, many remain without functional complaints for a long period. This study aims to find early functional changes associated with stages of radiographic knee osteoarthritis. A group of older people without self-reported complaints was divided in two groups: knee osteoarthritis (K&L = 2-4, N = 29) and control (K&L = 0-1, N = 31). Muscle function was assessed with voluntary and electrically-stimulated isometric knee contractions, including a fatigue test. Physical functioning was assessed with a 6-min walk test (6MWT), a stair climb test (SCT), and a short performance battery. There were no differences in muscle function parameters, 6MWT, and SCT between groups. A clinically relevant lower score on the performance battery was found in participants with knee osteoarthritis. In conclusion, even when older people indicate to have no functional limitations, a decline in functional outcome can be measured with a physical performance battery.

Communication at Transitions: One Audacious Bite at a Time.

To be audacious and take significant steps toward achieving the Quadruple Aim (improving the patient experience of care; improving the health of populations; reducing the per capita cost of health care; and improving the work life of clinicians and staff), we patients and caregivers need to better understand key features of our health journeys. When on that health journey, we are patients interacting with a series of care teams: our home team (social network), our community agency teams, our emergency care team, our hospital teams, and on and on. These care teams include ourselves, our caregivers, clinicians, other professionals, and direct care and support staff-people at the center of care. The actions taken by people at the center of care to improve, maintain, or adapt to our health or illness represents our health care. Actions can be diagnostic, taking medications, undergoing procedures, learning, living life and getting help living life. So, our health journey is teams of people at the center of care taking such actions to provide healthcare and service to us. During this journey, we transition from one setting to another, from one team to another, repeatedly. Communication knits this maze of actions, interactions, and transitions together. At its core communication is two or more people or parties sharing some information via some channel (voice, paper, digital, dramatic), one time or several times in a particular setting, hoping to accomplish something that moves us along in our health journey. One of the most persistent and ubiquitous frustrations in health care is that of poor communication. Poor communication at transitions is at the root of much overuse, underuse, and misuse of health resources, and results in the inability of patients to complete recommended treatment. For the patient and their family this means unnecessary delays in returning to health or worse. For those professionals on the care team the incidents of harm, burnout, stress, and frustration cause financial, emotional and career-ending consequences. Poor communication at transitions impacts each of the Quadruple Aims. The potential return for the investment in communication may cross over one or more organizational boundaries. Organization Boards and the C-Suite customarily focus on activities within their institutions, not between. The daunting nature of the challenge, caused by the shear volume and variety of transition nodes, can paralyze those in decision making roles, leading to smaller, more manageable local solutions. I support building a more holistic solution that includes the necessary governance, infrastructure, habits, and relationships. This leads to systematically applied common standards for local, node-specific solutions. Development should include all persons at the center of care in governance, design, operations and learning for systemic and local solutions. Refined clinical work flow should be constructed to respect patient and care partner life flow. Solutions should use interoperable technology to aid, not replace, communication. Transition information and processes should be transparent to patients and their care partners.

Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus.

BACKGROUND: Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear. METHODS: In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes. RESULTS: In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus. CONCLUSIONS: We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.

Effects of self-reported osteoarthritis on physical performance: a longitudinal study with a 10-year follow-up.

BACKGROUND AND AIMS: Osteoarthritis (OA) of the knee or hip is associated with limitations in activities of daily life. There are only a few long-term studies on how knee or hip OA affects the course of physical performance. The aim of this study was to investigate the effects of knee or hip OA on physical performance during a follow-up period of 10 years. METHODS: Participants in the Longitudinal Aging Study Amsterdam with self-reported hip or knee OA (N = 155) were prospectively followed for 10 years on 4 occasions from the onset of OA and compared to participants without OA (N = 1004). Physical performance was tested with walk, chair stand and balance tests. Scores for each test were summed to a total performance score (range 0-12), higher scores indicating better performance. Generalized estimating equations were used to analyze differences between participants with and without OA, unadjusted as well as adjusted for confounders. RESULTS: There was a significant interaction between OA and sex (P = 0.068). Both in men and women, total performance was lower for participants with OA, with greater differences in men. Chair stand and walking performance (P < 0.05), but not balance, were lower in participants with OA. After adjustment for confounders, these associations remained significant in men but not in women. Additional analyses correcting for follow-up duration and attrition showed lower performance scores for men and women with OA. CONCLUSIONS: OA negatively affected physical performance 3-6 years after it was first reported. Performance in men with OA was more affected than in women.

Effect of stimulation intensity on assessment of voluntary activation.

INTRODUCTION: The interpolated twitch technique is often used to assess voluntary activation (VA) of skeletal muscles. We investigated VA and the voluntary torque-superimposed torque relationship using either supramaximal nerve stimulation or better tolerated submaximal muscle stimulation, which is often used with patients. METHODS: Thirteen healthy subjects performed maximal and submaximal isometric knee extensions with superimposed maximal or submaximal doublets (100 Hz). RESULTS: Superimposed torque relative to potentiated resting doublets was smaller with maximal nerve than with submaximal muscle stimulation. Maximal VA was 87 ± 7% and 93 ± 5% for submaximal muscle and maximal nerve stimulation, respectively. The individual voluntary torque-superimposed torque relationships were more linear for submaximal muscle stimulation, possibly leading to less overestimation of VA. CONCLUSIONS: Submaximal muscle stimulation can be used to estimate VA in the knee extensors. It is less painful, and overestimation of VA may be less compared with maximal nerve stimulation.

Fast unilateral isometric knee extension torque development and bilateral jump height.

PURPOSE: We hypothesized that the initial rate (first 40 ms) of unilateral knee extensor torque development during a maximally fast isometric contraction would depend on the subjects' ability for fast neural activation and that it would predict bilateral jumping performance. METHODS: Nine males (21.8 +/- 0.9 yr, means +/- SD) performed unilateral fast isometric knee extensions (120 degrees knee angle) without countermovement on a dynamometer and bilateral squat jumps (SJ) and countermovement jumps (CMJ) starting from 90 and 120 degrees knee angles (full extension = 180 degrees ). The dynamometer contractions started either from full relaxation or from an isometric pre-tension (15% maximal isometric torque, Tmax). Torque time integral for the first 40 ms after torque onset (TTI-40, normalized to Tmax) and averaged normalized rectified knee extensor EMG for 40 ms before fast torque onset (EMG-40) were used to quantify initial torque rise and voluntary muscle activation. RESULTS: TTI-40 without pre-tension (range: 0.02-0.19% Tmax per second) was significantly lower than TTI-40 with pre-tension, and both were significantly (r = 0.81 and 0.80) related to EMG-40. During jumping, similar significant positive relations were found between jump height and knee extensor EMG during the first 100 ms of the rise in ground reaction force. There also were significant positive linear relations between dynamometer TTI-40 and jump height (r = 0.75 (SJ 90), 0.84 (SJ 120), 0.76 (CMJ 90), and 0.86 (CMJ 120)) but not between dynamometer Tmax and jump height (-0.16 < r < 0.02). CONCLUSION: One-legged TTI-40 to a large extent explained the variation in jump height. The ability to produce a high efferent neural drive before muscle contraction seemed to dominate performance in both the simple single-joint isometric task and the complex multijoint dynamic task.

Improving behavioral health satisfaction assessment: measuring patients' perceptions.

In order to focus on and improve key aspects of patient satisfaction in its behavioral health programs, Catholic Health East (CHE) enhanced its measurement methodology. In an effort to be consistent with the federal government's movement from measuring patient advocacy programs to measuring patients' perceptions, CHE transitioned to behavior-based questions. These questions give clear targets for program goals and initiatives by objectively measuring whether certain events and desired staff behaviors occurred during treatment, rather than subjectively ranking attributes of institution-defined service. Through this change in approach, CHE may better align its care and services with patients' wants and needs, as illustrated by four case examples.

Developing a prioritization matrix for improving breast care--a complex project.

Prioritization is an ongoing challenge for quality management professionals. A previous companion Brief Report described a tool for prioritizing improvement projects (Pelletier, Beaudin, and van Leeuwen, 1999). This tool provides a framework for prioritizing opportunities/solutions for major institutional improvement projects. By separating and categorizing opportunities and solutions and then weighing their importance and impact, the organization can graphically evaluate and then select interventions to initiate.