Background. Walking-related disability is the most frequent reason for inpatient stroke rehabilitation. Task-related practice is a critical component for improving patient outcomes. Objective. To test the feasibility of providing quantitative feedback about daily walking performance and motivating greater skills practice via remote sensing. Methods. In this phase III randomized, single blind clinical trial, patients participated in conventional therapies while wearing wireless sensors (triaxial accelerometers) at both ankles. Activity-recognition algorithms calculated the speed, distance, and duration of walking bouts. Three times a week, therapists provided either feedback about performance on a 10-meter walk (speed only) or walking speed feedback plus a review of walking activity recorded by the sensors (augmented). Primary outcomes at discharge included total daily walking time, derived from the sensors, and a timed 15-meter walk. Results. Sixteen rehabilitation centers in 11 countries enrolled 135 participants over 15 months. Sensors recorded more than 1800 days of therapy, 37 000 individual walking bouts, and 2.5 million steps. No significant differences were found between the 2 feedback groups in daily walking time (15.1 ± 13.1 vs 16.6 ± 14.3 minutes, P = .54) or 15-meter walking speed (0.93 ± 0.47 vs 0.91 ± 0.53 m/s, P = .96). Remarkably, 30% of participants decreased their total daily walking time over their rehabilitation stay. Conclusions. In this first trial of remote monitoring of inpatient stroke rehabilitation, augmented feedback beyond speed alone did not increase the time spent practicing or improve walking outcomes. Remarkably modest time was spent walking. Wireless sensing, however, allowed clinicians to audit skills practice and provided ground truth regarding changes in clinically important, mobility-related activities.
Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1? maturation and resistance to fungal dissemination in Candida albicans infection. ?-Glucans are major components of fungal cell walls that trigger IL-1? secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ?-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1? production in response to ?-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ?-glucan-induced IL-1? processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ?-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1? maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1? production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ?-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ?-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.
Today, the bicycle is utilized as a daily commute tool, a physical rehabilitation asset and sporting equipment, prompting studies into the biomechanics of cycling. Of the number of important parameters that affect cycling efficiency, the foot angle profile is one of the most important as it correlates directly with the effective force applied to the bike. However, there has been no compact and portable solution for measuring the foot angle and for providing the cyclist with real-time feedback due to a number of difficulties of the current tracking and sensing technologies¬ and the myriad types of bikes available. This paper presents a novel sensing and mobile computing system for classifying the foot angle profiles during cycling and for providing real-time guidance to the user to achieve the correct profile. Continuous foot angle tracking is firstly converted into a discrete problem requiring only recognition of acceleration profiles of the foot using a single shoe mounted tri-axial accelerometer during each pedaling cycle. A classification method is then applied to identify the pedaling profile. Finally, a mobile solution is presented to provide realtime signal processing and guidance.
RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-?B activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.
The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.
A number of pathogens cause host cell death upon infection, and Yersinia pestis, infamous for its role in large pandemics such as the "Black Death" in medieval Europe, induces considerable cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinase-caspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis, influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-? (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1?, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-?B; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1? and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1-caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-?B and inflammasome activation, and host resistance after Y. pestis infection.
Postoperative ileus (POI) can worsen outcomes, increase cost, and prolong hospitalization. An objective marker could help identify POI patients who should not be prematurely fed. We developed a disposable, non-invasive acoustic gastro-intestinal surveillance (AGIS) biosensor. We tested whether AGIS can distinguish healthy controls from patients recovering from abdominal surgery.
The pivot shift has been correlated with patient-reported outcomes and knee function following ACL injury and reconstruction. Tibial rotation has been recognized as an important component to the pivot shift motion path. However, few methodologies exist to quantify tibial rotation in the clinical setting. The purpose of this study was to validate the use of a wireless gyroscopic sensor to measure axial rotation of the tibia during a manually simulated pivot shift manoeuvre in cadaveric specimens. We hypothesized that integrated gyroscopic measurements of tibial rotation velocity (tibial rotation) would be highly correlated with tibial rotations simultaneously recorded with a rotary potentiometer during a simulated pivot shift motion under intact and ACL-deficient conditions.
Programmed necrosis mediated by receptor interacting protein kinase (RIP)3 (also called RIPK3) has emerged as an alternate death pathway triggered by TNF family death receptors, pathogen sensors, IFNRs, Ag-specific TCR activation, and genotoxic stress. Necrosis leads to cell leakage and acts as a "trap door," eliminating cells that cannot die by apoptosis because of the elaboration of pathogen-encoded caspase inhibitors. Necrotic signaling requires RIP3 binding to one of three partners-RIP1, DAI, or TRIF-via a common RIP homotypic interaction motif. Once activated, RIP3 kinase targets the pseudokinase mixed lineage kinase domain-like to drive cell lysis. Although necrotic and apoptotic death can enhance T cell cross-priming during infection, mice that lack these extrinsic programmed cell death pathways are able to produce Ag-specific T cells and control viral infection. The entwined relationship of apoptosis and necrosis evolved in response to pathogen-encoded suppressors to support host defense and contribute to inflammation.
The identification of noncanonical (caspase-1-independent) pathways for IL-1? production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1? processing and release in murine bone marrow-derived dendritic cells (BMDC) costimulated with TLR4 agonists and proapoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17-kDa) IL-1? was nearly equivalent in wild-type (WT) BMDC, Casp1(-/-)Casp11(-/-) BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1? was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8(-/-)Rip3(-/-) or Trif(-/-) BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1? processing and release in Casp1(-/-)Casp11(-/-) BMDC that was IETD sensitive. These data suggest that TLR4 induces assembly of caspase-8-based signaling complexes that become licensed as IL-1?-converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cellular inhibitor of apoptosis protein 1, suggesting suppressive roles for this and likely other inhibitor of apoptosis proteins on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, proapoptotic chemotherapeutic agents stimulate the caspase-8-mediated processing and release of IL-1?, implicating direct effects of such drugs on a noncanonical inflammatory cascade that may modulate immune responses in tumor microenvironments.
Enabling large scale monitoring and classification of a range of motion activities is of primary importance due to the need by healthcare and fitness professionals to monitor exercises for quality and compliance. Past work has not fully addressed the unique challenges that arise from scaling. This paper presents a novel end-to-end system solution to some of these challenges. The system is built on prescription based context-driven activity classification methodology. First, we show that by refining the definition of context, and introducing the concept of scenarios, a prescription model can provide personalized activity monitoring. Second, through a flexible architecture constructed from interface models, we demonstrate the concept of a context-driven classifier. Context classification is achieved through a classification committee approach, and activity classification follows by means of context specific activity models. Then, the architecture is implemented in an end-to-end system featuring an Android application running on a mobile device, and a number of classifiers as core classification components. Finally, we use a series of experimental field evaluations to confirm the expected benefits of the proposed system in terms of classification accuracy, rate, and sensor operating life.
Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-?B together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-? (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.
Platelets in the circulation are triggered by vascular damage to activate, aggregate and form a thrombus that prevents excessive blood loss. Platelet activation is stringently regulated by intracellular signalling cascades, which when activated inappropriately lead to myocardial infarction and stroke. Strategies to address platelet dysfunction have included proteomics approaches which have lead to the discovery of a number of novel regulatory proteins of potential therapeutic value. Global analysis of platelet proteomes may enhance the outcome of these studies by arranging this information in a contextual manner that recapitulates established signalling complexes and predicts novel regulatory processes. Platelet signalling networks have already begun to be exploited with interrogation of protein datasets using in silico methodologies that locate functionally feasible protein clusters for subsequent biochemical validation. Characterization of these biological systems through analysis of spatial and temporal organization of component proteins is developing alongside advances in the proteomics field. This focused review highlights advances in platelet proteomics data mining approaches that complement the emerging systems biology field. We have also highlighted nucleated cell types as key examples that can inform platelet research. Therapeutic translation of these modern approaches to understanding platelet regulatory mechanisms will enable the development of novel anti-thrombotic strategies.
Apoptosis and programmed necrosis balance each other as alternate first line host defense pathways against which viruses have evolved countermeasures. Intrinsic apoptosis, the critical programmed cell death pathway that removes excess cells during embryonic development and tissue homeostasis, follows a caspase cascade triggered at mitochondria and modulated by virus-encoded anti-apoptotic B cell leukemia (BCL)2-like suppressors. Extrinsic apoptosis controlled by caspase 8 arose during evolution to trigger executioner caspases directly, circumventing viral suppressors of intrinsic (mitochondrial) apoptosis and providing the selective pressure for viruses to acquire caspase 8 suppressors. Programmed necrosis likely evolved most recently as a trap door adaptation to extrinsic apoptosis. Receptor interacting protein (RIP)3 kinase (also called RIPK3) becomes active when either caspase 8 activity or polyubiquitylation of RIP1 is compromised. This evolutionary dialog implicates caspase 8 as a supersensor alternatively activating and suppressing cell death pathways.
Pathogens specifically target both the caspase 8-dependent apoptotic cell death pathway and the necrotic cell death pathway that is dependent on receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3). The fundamental co-regulation of these two cell death pathways emerged when the midgestational death of mice deficient in FAS-associated death domain protein (FADD) or caspase 8 was reversed by elimination of RIP1 or RIP3, indicating a far more entwined relationship than previously appreciated. Thus, mammals require caspase 8 activity during embryogenesis to suppress the kinases RIP1 and RIP3 as part of the dialogue between two distinct cell death processes that together fulfil reinforcing roles in the host defence against intracellular pathogens such as herpesviruses.
Advancement in wireless health sensor systems has triggered rapidly expanding research in continuous activity monitoring for chronic disease management or promotion and assessment of physical rehabilitation. Wireless motion sensing is increasingly important in treatments where remote collection of sensor measurements can provide an in-field objective evaluation of physical activity patterns. The well-known challenge of limited operating lifetime of energy-constrained wireless health sensor systems continues to present a primary limitation for these applications. This paper introduces CARER, a software system that supports a novel algorithm that exploits knowledge of context and dynamically schedules sensor measurement episodes within an energy consumption budget while ensuring classification accuracy. The sensor selection algorithm in the CARER system is based on Partially Observable Markov Decision Process (POMDP). The parameters for the POMDP algorithm can be obtained through standard maximum likelihood estimation. Sensor data are also collected from multiple locations of the subjects body, providing estimation of an individuals daily activity patterns.
Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.
Outcome measures of mobility for large stroke trials are limited to timed walks for short distances in a laboratory, step counters and ordinal scales of disability and quality of life. Continuous monitoring and outcome measurements of the type and quantity of activity in the community would provide direct data about daily performance, including compliance with exercise and skills practice during routine care and clinical trials.
Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.
Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNFalpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNFalpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance.
Energy efficiency presents a critical design challenge in wireless, wearable sensor technology, mainly because of the associated diagnostic objectives required in each monitoring application. In order to maximize the operating lifetime during real-life monitoring and maintain sufficient classification accuracy, the wearable sensors require hardware support that allows dynamic power control on the sensors and wireless interfaces as well as monitoring algorithms to control these components intelligently. This paper introduces a context-aware sensing technique known as episodic sampling - a method of performing context classification only at specific time instances. Based on Additive-Increase/Multiplicative-Decrease (AIMD), episodic sampling demonstrates an energy reduction of 85 percent with a loss of only 5 percent in classification accuracy in our experiment.
In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.
The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co-ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin-stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS-PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT-ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip-55. HIP-55 is an SH3-binding protein important in T-cell receptor signalling. Further analysis of HIP-55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin beta3 upon platelet activation. Analysis of HIP-55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP-55 to be an important regulator of platelet function.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation.
Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.
Fas, a TNF family receptor, is activated by the membrane protein Fas ligand expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas-induced cytokines, the IL-1? family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1? cytokines via caspase-1. However, the mechanisms by which Fas regulates IL-1? activation remain unresolved. In this article, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells, leading to the maturation of IL-1? and IL-18 independently of inflammasomes or RIP3. Hence, Fas controls a novel noncanonical IL-1? activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance, and control of infectious diseases.
Large-scale activity monitoring is a core component of systems aiming to improve our ability to manage fitness, deliver care, and diagnose conditions. While much research has been devoted to the accurate classification of motion, the challenges arising from scaling to large communities have received little attention. This paper introduces the problem of scaling, and addresses two of the most important issues: enabling robust large-scale ground-truth acquisition and building a common database for systems comparison. This paper presents a voice powered mobile acquisition system with efficient annotation tools and an extendable online searchable activity database with 331 datasets totaling over 700 h with 8 sensing modalities and 15 activities.
Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair.
Energy efficiency has been a longstanding design challenge for wearable sensor systems. It is especially crucial in continuous subject state monitoring due to the ongoing need for compact sizes and better sensors. This paper presents an energy-efficient classification algorithm, based on partially observable Markov decision process (POMDP). In every time step, POMDP dynamically selects sensors for classification via a sensor selection policy. The sensor selection problem is formalized as an optimization problem, where the objective is to minimize misclassification cost given some energy budget. State transitions are modeled as a hidden Markov model (HMM), and the corresponding sensor selection policy is represented using a finite-state controller (FSC). To evaluate this framework, sensor data were collected from multiple subjects in their free-living conditions. Relative accuracies and energy reductions from the proposed method are compared against nai?ve Bayes (always-on) and simple random strategies to validate the relative performance of the algorithm. When the objective is to maintain the same classification accuracy, significant energy reduction is achieved.
Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense. Receptor-interacting protein kinase (RIP) 3 (also called RIPK3) mediates RIP homotypic interaction motif (RHIM)-dependent programmed necrosis induced by murine cytomegalovirus (MCMV) infection or death receptor activation and suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI, also known as ZBP1 or DLM-1) sensitizes cells to virus-induced necrosis and that DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3(-/-) mice, vIRA mutant MCMV pathogenesis is restored in DAI(-/-) mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.
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