Rapid Tissue Perfusion Using Sacrificial Percolation of Anisotropic Networks.

Tissue engineering has long sought to rapidly generate perfusable vascularized tissues with vessel sizes spanning those seen in humans. Current techniques such as biological 3D printing (top-down) and cellular self-assembly (bottom-up) are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. We present a flexible and scalable technique termed SPAN - Sacrificial Percolation of Anisotropic Networks, where a network of perfusable channels is created throughout a tissue in minutes, irrespective of its size. Conduits with length scales spanning arterioles to capillaries are generated using pipettable alginate fibers that interconnect above a percolation density threshold and are then degraded within constructs of arbitrary size and shape. SPAN is readily used within common tissue engineering processes, can be used to generate endothelial cell-lined vasculature in a multi-cell type construct, and paves the way for rapid assembly of perfusable tissues.

Geometry and length control of 3D engineered heart tissues using direct laser writing.

Geometry and mechanical characteristics of the environment surrounding the Engineered Heart Tissues (EHT) affect their structure and function. Here, we employed a 3D tissue culture platform fabricated using two-photon direct laser writing with a high degree of accuracy to control parameters that are relevant to EHT maturation. Using this platform, we first explore the effects of geometry based on two distinct shapes: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites that are placed symmetrically along hemicylindrical membranes. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal fiber alignment. We systematically increase the length of the seeding wells for both configurations and observe a positive correlation between fiber alignment at the center of the EHTs and tissue length. With increasing length, an undesirable thinning and "necking" also emerge, leading to the failure of longer tissues over time. In the second step, we optimize the stiffness of the seeding wells and modify some of the attachment sites of the platform and the seeding parameters to achieve tissue stability for each length and geometry. Furthermore, we use the platform for electrical pacing and calcium imaging to evaluate the functional dynamics of EHTs as a function of frequency.

High throughput screening system for engineered cardiac tissues.

Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging. Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol. Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats.

Dynamic Control of Contractile Force in Engineered Heart Tissue.

Three-dimensional engineered heart tissues (EHTs) derived from human induced pluripotent stem cells (iPSCs) have become an important resource for both drug toxicity screening and research on heart disease. A key metric of EHT phenotype is the contractile (twitch) force with which the tissue spontaneously beats. It is well-known that cardiac muscle contractility - its ability to do mechanical work - depends on tissue prestrain (preload) and external resistance (afterload). OBJECTIVES: Here, we demonstrate a technique to control afterload while monitoring contractile force exerted by EHTs. METHODS: We developed an apparatus that can regulate EHT boundary conditions using real-time feedback control. The system is comprised of a pair of piezoelectric actuators that can strain the scaffold and a microscope that can measure EHT force and length. Closed loop control allows dynamic regulation of effective EHT boundary stiffness. RESULTS: When controlled to switch instantaneously from auxotonic to isometric boundary conditions, EHT twitch force immediately doubled. Changes in EHT twitch force as a function of effective boundary stiffness were characterized and compared to twitch force in auxotonic conditions. CONCLUSION: EHT contractility can be regulated dynamically through feedback control of effective boundary stiffness. SIGNIFICANCE: The capacity to alter the mechanical boundary conditions of an engineered tissue dynamically offers a new way to probe tissue mechanics. This could be used to mimic afterload changes that occur naturally in disease, or to improve mechanical techniques for EHT maturation.

Comparative genomics of Trueperella pyogenes available in the genome database reveals multidrug resistance genomic islands.

OBJECTIVE: An opportunistic pathogen, Trueperella pyogenes can infect cattle, buffalo, pig, goat, cat, dog, forest musk deer, etc., affecting various organs. The aim of this study was to identify the multidrug resistance genomic islands of T. pyogenes genomes available in NCBI database and also in the recently isolated strain TN_CUL_2020. METHODS: The strain TN_CUL_2020 isolated from swine lung abscess was sequenced by Illumina platform, and all the available T. pyogenes genome in NCBI database was retrieved for the comparative analysis. The ABRicate searches was used to identify antimicrobial resistance genes, and genomic islands (GIs) were predicted using IslandViewer 4. RESULTS: The strains SH01, SH02, and TP1 were predicted with maximum number of drug resistance genes. Genomic islands identified had multidrug resistance genes along with the class I integron and/or IS6100 elements in SH01, SH02, TP1. Composite transposons of IS6100 were noted in T2849, T4479, and TP3 intercalating tet(33) resistance genes. Several strains were predicted with phage elements, type IV secretion system, the toxin-antitoxin system in the GIs. CONCLUSION: Swine strains SH01, SH02 were predicted with multidrug resistance genes along with class I integrons. The presence of class I integrons, insertional elements, type IV secretion system, toxin-antitoxin system, and phage elements may aid in the horizontal transfer of antimicrobial resistance genes.

Next-generation engineered microsystems for cell biology: a systems-level roadmap.

Engineered microsystems for in vitro studies of cultured cells are evolving from simple 2D platforms to 3D architectures and organoid cultures. Despite advances in reproducing ever more sophisticated biology in these systems, there remain foundational challenges in re-creating key aspects of tissue composition, architecture, and mechanics that are critical to recapitulating in vivo processes. Against the backdrop of current progress in 3D fabrication methods, we evaluate the key requirements for the next generation of cellular platforms. We postulate that these future platforms - apart from building tissue-like structures - will need to have the ability to readily sense and autonomously modulate tissue responses over time, as occurs in natural microenvironments. Such interactive robotic platforms that report and guide cellular events will enable us to probe a previously inaccessible class of questions in cell biology.

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization.

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC­derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

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Coupled oscillation and spinning of photothermal particles in Marangoni optical traps.

Cyclic actuation is critical for driving motion and transport in living systems, ranging from oscillatory motion of bacterial flagella to the rhythmic gait of terrestrial animals. These processes often rely on dynamic and responsive networks of oscillators-a regulatory control system that is challenging to replicate in synthetic active matter. Here, we describe a versatile platform of light-driven active particles with interaction geometries that can be reconfigured on demand, enabling the construction of oscillator and spinner networks. We employ optically induced Marangoni trapping of particles confined to an air-water interface and subjected to patterned illumination. Thermal interactions among multiple particles give rise to complex coupled oscillatory and rotational motions, thus opening frontiers in the design of reconfigurable, multiparticle networks exhibiting collective behavior.

Transdermal oestradiol for androgen suppression in prostate cancer: long-term cardiovascular outcomes from the randomised Prostate Adenocarcinoma Transcutaneous Hormone (PATCH) trial programme.

BACKGROUND: Androgen suppression is a central component of prostate cancer management but causes substantial long-term toxicity. Transdermal administration of oestradiol (tE2) circumvents first-pass hepatic metabolism and, therefore, should avoid the cardiovascular toxicity seen with oral oestrogen and the oestrogen-depletion effects seen with luteinising hormone releasing hormone agonists (LHRHa). We present long-term cardiovascular follow-up data from the Prostate Adenocarcinoma Transcutaneous Hormone (PATCH) trial programme. METHODS: PATCH is a seamless phase 2/3, randomised, multicentre trial programme at 52 study sites in the UK. Men with locally advanced or metastatic prostate cancer were randomly allocated (1:2 from August, 2007 then 1:1 from February, 2011) to either LHRHa according to local practice or tE2 patches (four 100 µg patches per 24 h, changed twice weekly, reducing to three patches twice weekly if castrate at 4 weeks [defined as testosterone ≤1·7 nmol/L]). Randomisation was done using a computer-based minimisation algorithm and was stratified by several factors, including disease stage, age, smoking status, and family history of cardiac disease. The primary outcome of this analysis was cardiovascular morbidity and mortality. Cardiovascular events, including heart failure, acute coronary syndrome, thromboembolic stroke, and other thromboembolic events, were confirmed using predefined criteria and source data. Sudden or unexpected deaths were attributed to a cardiovascular category if a confirmatory post-mortem report was available and as other relevant events if no post-mortem report was available. PATCH is registered with the ISRCTN registry, ISRCTN70406718; the study is ongoing and adaptive. FINDINGS: Between Aug 14, 2007, and July 30, 2019, 1694 men were randomly allocated either LHRHa (n=790) or tE2 patches (n=904). Overall, median follow-up was 3·9 (IQR 2·4-7·0) years. Respective castration rates at 1 month and 3 months were 65% and 93% among patients assigned LHRHa and 83% and 93% among those allocated tE2. 157 events from 145 men met predefined cardiovascular criteria, with a further ten sudden deaths with no post-mortem report (total 167 events in 153 men). 26 (2%) of 1694 patients had fatal cardiovascular events, 15 (2%) of 790 assigned LHRHa and 11 (1%) of 904 allocated tE2. The time to first cardiovascular event did not differ between treatments (hazard ratio 1·11, 95% CI 0·80-1·53; p=0·54 [including sudden deaths without post-mortem report]; 1·20, 0·86-1·68; p=0·29 [confirmed group only]). 30 (34%) of 89 cardiovascular events in patients assigned tE2 occurred more than 3 months after tE2 was stopped or changed to LHRHa. The most frequent adverse events were gynaecomastia (all grades), with 279 (38%) events in 730 patients who received LHRHa versus 690 (86%) in 807 patients who received tE2 (p<0·0001) and hot flushes (all grades) in 628 (86%) of those who received LHRHa versus 280 (35%) who received tE2 (p<0·0001). INTERPRETATION: Long-term data comparing tE2 patches with LHRHa show no evidence of a difference between treatments in cardiovascular mortality or morbidity. Oestrogens administered transdermally should be reconsidered for androgen suppression in the management of prostate cancer. FUNDING: Cancer Research UK, and Medical Research Council Clinical Trials Unit at University College London.

Diffuse Large B-Cell Lymphoma: Clinical Presentation and Treatment Outcomes From the OncoCollect Lymphoma Registry.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the commonest subtype of lymphoma, standard CHOP was the treatment of choice, 42% of patients received rituximab, and 29% of patients were lost to follow-up during therapy, were reported in a study that collected retrospective data at 13 public and private hospitals for patients diagnosed with lymphoma between January 2005 and December 2009. The OncoCollect Registry was set up in 2017 to address the challenges in the collection of retrospective data through chart review, recording access to anthracycline and rituximab-based treatment, and to study outcomes and any improvement in the patient follow-up. METHODOLOGY: The OncoCollect Lymphoma group registry was set up at a national level with 9 participating centers. Lymphoma patients registered at these centers between 2011 and 2017 were included. The clinical features, prognostic stratification, associated comorbidities, response to first-line treatment, and 3-year outcomes of adult patients with DLBCL were analyzed. RESULTS: Of the 5,886 lymphoma patients registered in the OncoCollect registry, 2,581 (44%) had DLBCL. A total of 1,961 were evaluable for frontline therapy. The median age at presentation was 57 years. Gender ratio was 1.6:1. At presentation, 43% were early stage, 70% had low and low intermediate IPI, 53% had extranodal disease, and 30.9% had one or more comorbidities (data available for 1,136 patients). The commonest extra nodal site was gastro-intestinal (23.98%) followed by head and neck (19.24%). The overall response rate was 79.29%. Complete remission was seen in 61.75%, partial response in 17.5%, stable disease in 4.3%, and progression in 7.9%. Patients who received anthracycline-based therapy (86.7%) and rituximab-based therapy (83.7%) had a 3-year event-free survival (EFS) of 69.67% and 68.48%, respectively. With a median follow-up of 33 months, the 3-year overall Survival (OS) and EFS were 75.37% and 66.58%, respectively. CONCLUSIONS: DLBCL remains the commonest (44%) lymphoma subtype and is curable with standard anthracycline- and rituximab-based therapies. The availability of rituximab has increased the proportion of patients receiving standard chemoimmunotherapy.

Robots learn to identify objects by feeling.

Multimodal tactile sensors help robot hands accurately identify grasped objects by measuring thermal properties in addition to contact loads.

Timing of radiotherapy after radical prostatectomy (RADICALS-RT): a randomised, controlled phase 3 trial.

BACKGROUND: The optimal timing of radiotherapy after radical prostatectomy for prostate cancer is uncertain. We aimed to compare the efficacy and safety of adjuvant radiotherapy versus an observation policy with salvage radiotherapy for prostate-specific antigen (PSA) biochemical progression. METHODS: We did a randomised controlled trial enrolling patients with at least one risk factor (pathological T-stage 3 or 4, Gleason score of 7-10, positive margins, or preoperative PSA ≥10 ng/mL) for biochemical progression after radical prostatectomy (RADICALS-RT). The study took place in trial-accredited centres in Canada, Denmark, Ireland, and the UK. Patients were randomly assigned in a 1:1 ratio to adjuvant radiotherapy or an observation policy with salvage radiotherapy for PSA biochemical progression (PSA ≥0·1 ng/mL or three consecutive rises). Masking was not deemed feasible. Stratification factors were Gleason score, margin status, planned radiotherapy schedule (52·5 Gy in 20 fractions or 66 Gy in 33 fractions), and centre. The primary outcome measure was freedom from distant metastases, designed with 80% power to detect an improvement from 90% with salvage radiotherapy (control) to 95% at 10 years with adjuvant radiotherapy. We report on biochemical progression-free survival, freedom from non-protocol hormone therapy, safety, and patient-reported outcomes. Standard survival analysis methods were used. A hazard ratio (HR) of less than 1 favoured adjuvant radiotherapy. This study is registered with ClinicalTrials.gov, NCT00541047. FINDINGS: Between Nov 22, 2007, and Dec 30, 2016, 1396 patients were randomly assigned, 699 (50%) to salvage radiotherapy and 697 (50%) to adjuvant radiotherapy. Allocated groups were balanced with a median age of 65 years (IQR 60-68). Median follow-up was 4·9 years (IQR 3·0-6·1). 649 (93%) of 697 participants in the adjuvant radiotherapy group reported radiotherapy within 6 months; 228 (33%) of 699 in the salvage radiotherapy group reported radiotherapy within 8 years after randomisation. With 169 events, 5-year biochemical progression-free survival was 85% for those in the adjuvant radiotherapy group and 88% for those in the salvage radiotherapy group (HR 1·10, 95% CI 0·81-1·49; p=0·56). Freedom from non-protocol hormone therapy at 5 years was 93% for those in the adjuvant radiotherapy group versus 92% for those in the salvage radiotherapy group (HR 0·88, 95% CI 0·58-1·33; p=0·53). Self-reported urinary incontinence was worse at 1 year for those in the adjuvant radiotherapy group (mean score 4·8 vs 4·0; p=0·0023). Grade 3-4 urethral stricture within 2 years was reported in 6% of individuals in the adjuvant radiotherapy group versus 4% in the salvage radiotherapy group (p=0·020). INTERPRETATION: These initial results do not support routine administration of adjuvant radiotherapy after radical prostatectomy. Adjuvant radiotherapy increases the risk of urinary morbidity. An observation policy with salvage radiotherapy for PSA biochemical progression should be the current standard after radical prostatectomy. FUNDING: Cancer Research UK, MRC Clinical Trials Unit, and Canadian Cancer Society.

Effect of scapular stabilization exercise program in patients with subacromial impingement syndrome: a systematic review.

Scapular stabilization approaches have been a mainstay of therapeutic training programs in the recent past for patients with subacromial im-pingement syndrome, whereas its contributions solely in the clinical outcome of reducing shoulder pain and disability are largely unclear. This systematic review aims to evaluate and summarize the best evi-dence regarding the role of scapular stabilization interventions in allevi-ating shoulder dysfunction among subjects with subacromial impinge-ment syndrome. Six reviewers involved in this systematic review. Liter-ature was retrieved systematically through searching 5 electronic data-bases (PubMed, MEDLINE, CINAHL, Cochrane, and Google Scholar). Articles published from the year 2010 up to and including 2019 were in-cluded. The literature search included clinical trials those intervened subjects with scapular exercises or scapular stabilization exercises or scapular rehabilitation, as an intervention for subacromial impingement syndrome. Seven studies, totaling 228 participants were included in this systematic review. Articles included in this review were graded ac-cording to Lloyd-Smith hierarchy of evidence scale and critically ap-praised with a tool developed by National, Heart, Lung and Blood Insti-tute (United States), named as quality assessment of controlled inter-vention studies tool. There was a significant effect on the scapular sta-bilization exercise program on improving pain and disability among sub-jects with subacromial impingement syndrome. This systematic review provides sufficient evidence to suggest that scapular stabilization exer-cises offers effectiveness in reducing pain and disability among sub-jects with subacromial impingement syndrome. However, more trials with larger sample are needed to provide a more definitive evidence on the clinical outcomes of scapular stabilization exercises among pa-tients with impingement.

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

Topology optimization and 3D printing of multimaterial magnetic actuators and displays.

Upcoming actuation systems will be required to perform multiple tightly coupled functions analogous to their natural counterparts; e.g., the ability to control displacements and high-resolution appearance simultaneously is necessary for mimicking the camouflage seen in cuttlefish. Creating integrated actuation systems is challenging owing to the combined complexity of generating high-dimensional designs and developing multifunctional materials and their associated fabrication processes. Here, we present a complete toolkit consisting of multiobjective topology optimization (for design synthesis) and multimaterial drop-on-demand three-dimensional printing for fabricating complex actuators (>106 design dimensions). The actuators consist of soft and rigid polymers and a magnetic nanoparticle/polymer composite that responds to a magnetic field. The topology optimizer assigns materials for individual voxels (volume elements) while simultaneously optimizing for physical deflection and high-resolution appearance. Unifying a topology optimization-based design strategy with a multimaterial fabrication process enables the creation of complex actuators and provides a promising route toward automated, goal-driven fabrication.

Learning the signatures of the human grasp using a scalable tactile glove.

Humans can feel, weigh and grasp diverse objects, and simultaneously infer their material properties while applying the right amount of force-a challenging set of tasks for a modern robot1. Mechanoreceptor networks that provide sensory feedback and enable the dexterity of the human grasp2 remain difficult to replicate in robots. Whereas computer-vision-based robot grasping strategies3-5 have progressed substantially with the abundance of visual data and emerging machine-learning tools, there are as yet no equivalent sensing platforms and large-scale datasets with which to probe the use of the tactile information that humans rely on when grasping objects. Studying the mechanics of how humans grasp objects will complement vision-based robotic object handling. Importantly, the inability to record and analyse tactile signals currently limits our understanding of the role of tactile information in the human grasp itself-for example, how tactile maps are used to identify objects and infer their properties is unknown6. Here we use a scalable tactile glove and deep convolutional neural networks to show that sensors uniformly distributed over the hand can be used to identify individual objects, estimate their weight and explore the typical tactile patterns that emerge while grasping objects. The sensor array (548 sensors) is assembled on a knitted glove, and consists of a piezoresistive film connected by a network of conductive thread electrodes that are passively probed. Using a low-cost (about US$10) scalable tactile glove sensor array, we record a large-scale tactile dataset with 135,000 frames, each covering the full hand, while interacting with 26 different objects. This set of interactions with different objects reveals the key correspondences between different regions of a human hand while it is manipulating objects. Insights from the tactile signatures of the human grasp-through the lens of an artificial analogue of the natural mechanoreceptor network-can thus aid the future design of prosthetics7, robot grasping tools and human-robot interactions1,8-10.

Systematic Review on Effectiveness of shoulder taping in Hemiplegia.

BACKGROUND: Shoulder pain and subluxation are the commonly encountered problems among subjects with hemiplegia. Rehabilitating the shoulder following stroke is a challenging task among physiotherapists in rehabilitation set up. There is a need to validate the effectiveness of externally applied taping materials in hemiplegic shoulder. OBJECTIVE: This systematic review analyses the efficacy of taping on hemiplegic shoulder in terms of alleviating pain and managing subluxation. METHODS: Systematic review of randomized controlled trials (RCTs) was conducted to determine the effects of taping on hemiplegic shoulder. Articles were electronically searched from the year 2000 to 2017 in the 4 databases, Google scholar, CINAHL, Pubmed, and Pedro. Reviewers graded the papers according to Lloyd-Smith's hierarchy of evidence scale. Papers were quality appraised using a systematic review of RCT tool developed by National Heart, Lung and Blood Institute (United States), named as quality assessment of controlled intervention studies tool. RESULTS: Eight papers were included, totaling 132 participants. All the RCT's included in this review were good quality. There was a significant effect on taping method for reduction of pain and subluxation among subjects with stroke. CONCLUSIONS: This systematic review provides sufficient evidence to suggest taping is a beneficial method for reducing pain and shoulder subluxation among stroke subjects.

Photothermocapillary Oscillators.

We present a new class of tunable light-driven oscillators based on mm-scale objects adsorbed at fluid interfaces. A fixed light source induces photothermal surface tension gradients (Marangoni stresses) that drive nanocomposite hydrogel discs away from a stable apex position atop a drop of water. The capillary forces on the disc increase with surface curvature; thus, they act to restore the disc to its original position. As the disc reenters the light source it again experiences Marangoni propulsion, leading to sustained oscillation for appropriate conditions. Propulsive forces can be modulated with incident light intensity, while the restoring force can be tuned via surface curvature-i.e., drop volume-providing highly tunable oscillatory behaviors. To our knowledge, this is the first example where Marangoni and capillary forces combine to incite sustained motion. As such, a model was developed that describes this behavior and provides key insights into the underlying control parameters. We expect that this simple approach will enable the study of more complex and coupled oscillatory systems.