Synapses do not facilitate prion-like transfer of alpha-synuclein: a quantitative study in reconstructed unidirectional neural networks.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Sensorization of microfluidic brain-on-a-chip devices: Towards a new generation of integrated drug screening systems.

Brain-on-a-chip (BoC) devices show typical characteristics of brain complexity, including the presence of different cell types, separation in different compartments, tissue-like three-dimensionality, and inclusion of the extracellular matrix components. Moreover, the incorporation of a vascular system mimicking the blood-brain barrier (BBB) makes BoC particularly attractive, since they can be exploited to test the brain delivery of different drugs and nanoformulations. In this review, we introduce the main innovations in BoC and BBB-on-a-chip models, especially focusing sensorization: electrical, electrochemical, and optical biosensors permit the real-time monitoring of different biological phenomena and markers, such as the release of growth factors, the expression of specific receptors/biomarkers, the activation of immune cells, cell viability, cell-cell interactions, and BBB crossing of drugs and nanoparticles. The recent improvements in signal amplification, miniaturization, and multiplication of the sensors are discussed in an effort to highlight their benefits versus limitations and delineate future challenges in this field.

[Recent advances in emerging three-dimensional in vitro models for sport-related traumatic brain injury].

Sports-related traumatic brain injury (srTBI) is a traumatic brain injury (TBI) caused by sports, which can result in cognitive and motor dysfunction. Currently, research on the molecular mechanism of srTBI and related drug development mainly relies on monolayer culture models and animal models. However, many differences exist in cell populations and inflammatory responses between these models and human pathophysiological processes. Most of the researches derived from the models can't effectively conducted translational research. Emerging three-dimensional (3D) in vitro models bridge the limitations of traditional models in simulating the pathophysiological processes of human srTBI and provide new means to understand srTBI. A literature has reported the research progress of emerging 3D in vitro models in neurological diseases, but there is a lack of systematic summary of the mentioned models in srTBI studies. Here, we review the research progress of emerging 3D in vitro models of srTBI, discuss the advantages and limitations of existing models, and further prospect the future trend of srTBI models. This paper aims to provide a new research perspective for researchers in tissue engineering and sports medicine to study the molecular mechanisms of srTBI and develop neuroprotective drugs.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip.

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features.NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections.

Unraveling brain diseases: The promise of brain-on-a-chip models.

Brain disorders, encompassing a wide spectrum of neurological and psychiatric conditions, present a formidable challenge in modern medicine. Despite decades of research, the intricate complexity of the human brain still eludes comprehensive understanding, impeding the development of effective treatments. Recent advancements in microfluidics and tissue engineering have led to the development of innovative platforms known as "Brain-on-a-Chip" (BoC) i.e., advanced in vitro systems that aim to replicate the microenvironment of the brain with the highest possible fidelity. This technology offers a promising test-bed for studying brain disorders at the cellular and network levels, providing insights into disease mechanisms, drug screening, and, in perspective, the development of personalized therapeutic strategies. In this review, we provide an overview of the BoC models developed over the years to model and understand the onset and progression of some of the most severe neurological disorders in terms of incidence and debilitation (stroke, Parkinson's, Alzheimer's, and epilepsy). We also report some of the cutting-edge therapeutic approaches whose effects were evaluated by means of these technologies. Finally, we discuss potential challenges, and future perspectives of the BoC models.

Engineered modular neuronal networks-on-chip represent structure-function relationship.

Brain function is substantially linked to the highly organized modular structure of neuronal networks. However, the structure of in vitro assembled neuronal circuits often exhibits variability, complicating the consistent recording of network functional output and its correlation to network structure. Therefore, engineering neuronal structures with predefined geometry and reproducible functional features is essential to precisely model in vivo neuronal circuits. Here, we engineered microchannel devices to assemble 2D and 3D modular networks. The microchannel devices were coupled with a multi-electrode array (MEA) electrophysiology system to enable recordings from circuits. Each network consisted of 64 modules connected to their adjacent modules by micron-sized channels. Modular circuits within microchannel devices showed enhanced activity and functional connectivity traits. This includes metrics such as connection weights, clustering coefficient, global efficiency, and the number of hub neurons with higher betweenness centrality. In addition, modular networks demonstrated an increased functional modularity score compared to the randomly formed circuits. Neurons within individual modules displayed uniform network characteristics and predominantly participated in their respective functional communities within the same or neighboring physical modules. These observations highlight that the modular network structure promotes the development of segregated functional connectivity traits while simultaneously enhancing the efficiency of overall network connectivity. Our findings emphasize the significant impact of physical constraints on the activity patterns and functional organization within engineered modular networks. These circuits, characterized by stable modular architecture and intricate functional dynamics-key features of the brain networks-offer a robust in vitro model for advancing neuroscience research.

Exploring Kainic Acid-Induced Alterations in Circular Tripartite Networks with Advanced Analysis Tools.

Brain activity implies the orchestrated functioning of interconnected brain regions. Typical in vitro models aim to mimic the brain using single human pluripotent stem cell-derived neuronal networks. However, the field is constantly evolving to model brain functions more accurately through the use of new paradigms, e.g., brain-on-a-chip models with compartmentalized structures and integrated sensors. These methods create novel data requiring more complex analysis approaches. The previously introduced circular tripartite network concept models the connectivity between spatially diverse neuronal structures. The model consists of a microfluidic device allowing axonal connectivity between separated neuronal networks with an embedded microelectrode array to record both local and global electrophysiological activity patterns in the closed circuitry. The existing tools are suboptimal for the analysis of the data produced with this model. Here, we introduce advanced tools for synchronization and functional connectivity assessment. We used our custom-designed analysis to assess the interrelations between the kainic acid (KA)-exposed proximal compartment and its nonexposed distal neighbors before and after KA. Novel multilevel circuitry bursting patterns were detected and analyzed in parallel with the inter- and intracompartmental functional connectivity. The effect of KA on the proximal compartment was captured, and the spread of this effect to the nonexposed distal compartments was revealed. KA induced divergent changes in bursting behaviors, which may be explained by distinct baseline activity and varied intra- and intercompartmental connectivity strengths. The circular tripartite network concept combined with our developed analysis advances importantly both face and construct validity in modeling human epilepsy in vitro.

Long-term organotypic brain slices cultured on collagen-based microcontact prints: A perspective for a brain-on-a-chip.

Organotypic brain slices are three-dimensional 150 µm-thick sections of a postnatal day 10 mouse and can be cultured for several weeks in vitro. In such brain slices the complex cellular connections are preserved with a high viability. These brain slices can be connected to collagen-loaded microcontact prints to develop a simple brain-on-a-chip model. Using the microcontact printing technique, many peptides or proteins can be printed onto a semipermeable membrane and linked to brain slices. On these microcontact prints, brain-derived nerve fibers grow out, or microglia can get activated and migrate out, or also new brain vessels can be formed. Such a brain-on-a-chip model may allow to develop new drugs or a diagnostic method for neurodegenerative diseases.

Recent advances in sensor-integrated brain-on-a-chip devices for real-time brain monitoring.

Brain science has remained in the global spotlight as an important field of scientific and technological discovery. Numerous in vitro and in vivo animal studies have been performed to understand the pathological processes involved in brain diseases and develop strategies for their diagnosis and treatment. However, owing to species differences between animals and humans, several drugs have shown high rates of treatment failure in clinical settings, hindering the development of diagnostic and treatment modalities for brain diseases. In this scenario, microfluidic brain-on-a-chip (BOC) devices, which allow the direct use of human tissues for experiments, have emerged as novel tools for effectively avoiding species differences and performing screening for new drugs. Although microfluidic BOC technology has achieved significant progress in recent years, monitoring slight changes in neurochemicals, neurotransmitters, and environmental states in the brain has remained challenging owing to the brain's complex environment. Hence, the integration of BOC with new sensors that have high sensitivity and high selectivity is urgently required for the real-time dynamic monitoring of BOC parameters. As sensor-based technologies for BOC have not been summarized, here, we review the principle, fabrication process, and application-based classification of sensor-integrated BOC, and then summarize the opportunities and challenges for their development. Generally, sensor-integrated BOC enables real-time monitoring and dynamic analysis, accurately measuring minute changes in the brain and thus enabling the realization of in vivo brain analysis and drug development.

Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.

Methods for studying brain function and disease heavily rely onin vivoanimal models,ex-vivotissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects ofin vivohuman brain are needed as additional preclinical tools. In this context, 3D hydrogel-basedin vitromodels of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-ß-glycerophosphate (CHITO-ß-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-ß-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-ß-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

Biosensor integrated brain-on-a-chip platforms: Progress and prospects in clinical translation.

Because of the brain's complexity, developing effective treatments for neurological disorders is a formidable challenge. Research efforts to this end are advancing as in vitro systems have reached the point that they can imitate critical components of the brain's structure and function. Brain-on-a-chip (BoC) was first used for microfluidics-based systems with small synthetic tissues but has expanded recently to include in vitro simulation of the central nervous system (CNS). Defining the system's qualifying parameters may improve the BoC for the next generation of in vitro platforms. These parameters show how well a given platform solves the problems unique to in vitro CNS modeling (like recreating the brain's microenvironment and including essential parts like the blood-brain barrier (BBB)) and how much more value it offers than traditional cell culture systems. This review provides an overview of the practical concerns of creating and deploying BoC systems and elaborates on how these technologies might be used. Not only how advanced biosensing technologies could be integrated with BoC system but also how novel approaches will automate assays and improve point-of-care (PoC) diagnostics and accurate quantitative analyses are discussed. Key challenges providing opportunities for clinical translation of BoC in neurodegenerative disorders are also addressed.

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

Propagation of Distinct α-Synuclein Strains Within Human Reconstructed Neuronal Network and Associated Neuronal Dysfunctions.

Aggregated alpha-synuclein (α-Syn) in neurons is a hallmark of Parkinson's disease (PD) and other synucleinopathies. Recent advances (1) in the production and purification of synthetic assemblies of α-Syn, (2) in the design and production of microfluidic devices allowing the construction of oriented and compartmentalized neuronal network on a chip, and (3) in the differentiation of human pluripotent stem cells (hPSCs) into specific neuronal subtypes now allow the study of cellular and molecular determinants of the prion-like properties of α-Syn in vitro. Here, we described the methods we used to reconstruct a cortico-cortical human neuronal network in microfluidic devices and how to take advantage of this cellular model to characterize (1) the prion-like properties of different α-Syn strains and (2) the neuronal dysfunctions and the alterations associated with the exposure to α-Syn strains or the nucleation of endogenous α-Syn protein in vitro.

Brain physiome: A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain.

In the last few decades, adverse reactions to pharmaceuticals have been evaluated using 2D in vitro models and animal models. However, with increasing computational power, and as the key drivers of cellular behavior have been identified, in silico models have emerged. These models are time-efficient and cost-effective, but the prediction of adverse reactions to unknown drugs using these models requires relevant experimental input. Accordingly, the physiome concept has emerged to bridge experimental datasets with in silico models. The brain physiome describes the systemic interactions of its components, which are organized into a multilevel hierarchy. Because of the limitations in obtaining experimental data corresponding to each physiome component from 2D in vitro models and animal models, 3D in vitro brain models, including brain organoids and brain-on-a-chip, have been developed. In this review, we present the concept of the brain physiome and its hierarchical organization, including cell- and tissue-level organizations. We also summarize recently developed 3D in vitro brain models and link them with the elements of the brain physiome as a guideline for dataset collection. The connection between in vitro 3D brain models and in silico modeling will lead to the establishment of cost-effective and time-efficient in silico models for the prediction of the safety of unknown drugs.

Effects of Ischemia on the Migratory Capacity of Microglia Along Collagen Microcontact Prints on Organotypic Mouse Cortex Brain Slices.

Ischemic stroke is a severe insult in the brain causing cell death, inflammation, and activation of microglia. Microglia are the immune cells of the brain and play a role in any inflammatory process during neurodegeneration. Microglia are round ameboid and migrate to the lesion site, where they differentiate into ramified forms and activated phagocytic microglia. On the other hand, microglia can also release growth factors to repair degeneration. The aim of the present study is to explore the migratory capacity of microglia after ischemic insults. Organotypic brain slices of the mouse cortex (300 µm) were prepared. In order to study migration, the slices were connected to collagen-loaded microcontact prints (with or without monocyte chemoattractant protein-1, MCP-1) on the membranes. Slices were stimulated with lipopolysaccharide (LPS) for maximal microglial activation. Ischemic insults were simulated with oxygen-glucose deprivation (OGD) and acidosis (pH 6.5) for 3 days. After 3 weeks in culture, slices were fixed and immunohistochemically stained for the microglial markers Iba1, CD11b and macrophage-like antigen. Our data show that Iba1+ microglia migrated along the microcontact prints, differentiate and phagocyte 1.0 µm fluorescent microbeads. LPS significantly enhanced the number of round ameboid migrating microglia, while OGD and acidosis enhanced the number of ramified activated microglia. The effect was not visible on slices without any µCP and was most potent in µCP with MCP-1. We conclude that OGD and acidosis activate ramification and exhibit a similar mechanism, while LPS only activates round ameboid microglia. Collagen-loaded microcontact prints connected to mouse brain slices are a potent method to study activation and migration of microglia ex vivo.

Nanoscale Patterning of In Vitro Neuronal Circuits.

Methods for patterning neurons in vitro have gradually improved and are used to investigate questions that are difficult to address in or ex vivo. Though these techniques guide axons between groups of neurons, multiscale control of neuronal connectivity, from circuits to synapses, is yet to be achieved in vitro. As studying neuronal circuits with synaptic resolution in vivo poses significant challenges, we present an in vitro alternative to validate biophysical and computational models. In this work we use a combination of electron beam lithography and photolithography to create polydimethylsiloxane (PDMS) structures with features ranging from 150 nm to a few millimeters. Leveraging the difference between average axon and dendritic spine diameters, we restrict axon growth while allowing spines to pass through nanochannels to guide synapse formation between small groups of neurons (i.e., nodes). We show this technique can be used to generate large numbers of isolated feed-forward circuits where connections between nodes are restricted to regions connected by nanochannels. Using a genetically encoded calcium indicator in combination with fluorescently tagged postsynaptic protein, PSD-95, we demonstrate functional synapses can form in this region.

A Three-Dimensional Brain-on-a-Chip Using Human iPSC-Derived GABAergic Neurons and Astrocytes.

Brain-on-a-chip is a miniaturized engineering platform to mimic the structural and functional aspects of brain tissue. We describe a method to construct a three-dimensional (3D) brain-on-a-chip in this chapter. We firstly portray the method of a brain-on-a-chip model with cocultured mice neurons, microglia, and astrocytes to mimic brain tissue and membrane-free perfusion with endothelial cells, in which we successfully build the blood-brain barrier to screen neurotoxicity. Then we describe a method to construct a brain-on-a-chip with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded GABAergic neurons and astrocytes and a perfusion channel with dynamic flow. We also include the broader applicability test of this model using an organophosphate (OP), malathion, to induce acute and chronic neurotoxicity, and then using butyrylcholinesterase (BuChE) as an exogenous bioscavenger of OP. Following the methods listed in this chapter, we are able to measure the neurotoxic effects on construct integrity, viability, and total AChE and BuChE activity.

Microcontact Printing of Cholinergic Neurons in Organotypic Brain Slices.

Alzheimer's disease is a severe neurodegenerative disorder of the brain, characterized by beta-amyloid plaques, tau pathology, and cell death of cholinergic neurons, resulting in loss of memory. The reasons for the damage of the cholinergic neurons are not clear, but the nerve growth factor (NGF) is the most potent trophic factor to support the survival of these neurons. In the present study we aim to microprint NGF onto semipermeable 0.4 µm pore membranes and couple them with organotypic brain slices of the basal nucleus of Meynert and to characterize neuronal survival and axonal growth. The brain slices were prepared from postnatal day 10 wildtype mice (C57BL6), cultured on membranes for 2-6 weeks, stained, and characterized for choline acetyltransferase (ChAT). The NGF was microcontact printed in 28 lines, each with 35 µm width, 35 µm space between them, and with a length of 8 mm. As NGF alone could not be printed on the membranes, NGF was embedded into collagen hydrogels and the brain slices were placed at the center of the microprints and the cholinergic neurons that survived. The ChAT+ processes were found to grow along with the NGF microcontact prints, but cells also migrated. Within the brain slices, some form of re-organization along the NGF microcontact prints occurred, especially the glial fibrillary acidic protein (GFAP)+ astrocytes. In conclusion, we provided a novel innovative microcontact printing technique on semipermeable membranes which can be coupled with brain slices. Collagen was used as a loading substance and allowed the microcontact printing of nearly any protein of interest.

Review of Design Considerations for Brain-on-a-Chip Models.

In recent years, the need for sophisticated human in vitro models for integrative biology has motivated the development of organ-on-a-chip platforms. Organ-on-a-chip devices are engineered to mimic the mechanical, biochemical and physiological properties of human organs; however, there are many important considerations when selecting or designing an appropriate device for investigating a specific scientific question. Building microfluidic Brain-on-a-Chip (BoC) models from the ground-up will allow for research questions to be answered more thoroughly in the brain research field, but the design of these devices requires several choices to be made throughout the design development phase. These considerations include the cell types, extracellular matrix (ECM) material(s), and perfusion/flow considerations. Choices made early in the design cycle will dictate the limitations of the device and influence the end-point results such as the permeability of the endothelial cell monolayer, and the expression of cell type-specific markers. To better understand why the engineering aspects of a microfluidic BoC need to be influenced by the desired biological environment, recent progress in microfluidic BoC technology is compared. This review focuses on perfusable blood-brain barrier (BBB) and neurovascular unit (NVU) models with discussions about the chip architecture, the ECM used, and how they relate to the in vivo human brain. With increased knowledge on how to make informed choices when selecting or designing BoC models, the scientific community will benefit from shorter development phases and platforms curated for their application.

On the road to the brain-on-a-chip: a review on strategies, methods, and applications.

The brain is the most complex organ of our body. Such a complexity spans from the single-cell morphology up to the intricate connections that hundreds of thousands of neurons establish to create dense neuronal networks. All these components are involved in the genesis of the rich patterns of electrophysiological activity that characterize the brain. Over the years, researchers coming from different disciplines developedin vitrosimplified experimental models to investigate in a more controllable and observable way how neuronal ensembles generate peculiar firing rhythms, code external stimulations, or respond to chemical drugs. Nowadays, suchin vitromodels are namedbrain-on-a-chippointing out the relevance of the technological counterpart as artificial tool to interact with the brain: multi-electrode arrays are well-used devices to record and stimulate large-scale developing neuronal networks originated from dissociated cultures, brain slices, up to brain organoids. In this review, we will discuss the state of the art of the brain-on-a-chip, highlighting which structural and biological features a realisticin vitrobrain should embed (and how to achieve them). In particular, we identified two topological features, namely modular and three-dimensional connectivity, and a biological one (heterogeneity) that takes into account the huge number of neuronal types existing in the brain. At the end of this travel, we will show how 'far' we are from the goal and how interconnected-brain-regions-on-a-chip is the most appropriate wording to indicate the current state of the art.