Construction of in vitro neural circuits with complex higher-order functions would be achieved by converging development in diverse major disciplines including neuroscience, stem cell biology, tissue engineering, electrical engineering and computer science.Over the years, techniques have been developed to culture and assemble neurons, which brought us closer to creating neuronal circuits that functionally and structurally mimic parts of the brain. Starting with primary culture of neurons, preparations of neuronal culture have advanced substantially. Development of stem cell research and brain organoids has opened a new path for generating three-dimensional human neural circuits. Along with the progress in biology, engineering technologies advanced and paved the way for construction of neural circuit structures. In this article, we overview research progress and discuss perspective of  One of the highlights of embryogenesis is the formation of the brain, which is achieved by the most complex choreography and orchestration of cells. In addition to cellular processes that occur during formation of other organs , diverse types of neurons extend and connect their dendrites and axons, and go through maturation process, to generate complex networks of neural circuits. The functions of neurons as the key elements of the brain are to propagate action potential within the cells and to transmit and receive activity through synapses. Neurons can be cultured in vitro, go through maturation process, retain their elementary functions as neurons, and exhibit characteristics of diverse cell types. Therefore, it would be natural to hope and be optimistic for the formation of functional neural circuits in vitro. However, construction of functional neural circuits in vitro could be considered as one of the most difficult tasks that have been sought after for a long time.During development in vitro neural circuits have demonstrated their diverse mechanistic functions including complex oscillation activity and network plasticity. It is fair to assume that more unique and higher-order functions could be achieved by constructing in vitro neural circuits with certain structures that are organized to acquire signal processing capability. Ultimately, neural circuits with desired functions might be generated by connecting neurons just as a computer assembled with semiconductor and other elements.Functions of neural circuits could be appreciated from two perspectives. Considering neurons as elements for neural circuits, abilities or characteristics of neurons emerged by forming networks could be considered as functions of the circuits. Considering neural circuits as sub-systems of the brains, activity or response that provide mechanistic processes for the brain performance could be functions of the circuits as well. With these criteria, in vitro neural circuit that precisely mimics the functional in vivo circuit structure. The neural circuit structures in the brain have been explored in detail by classical anatomical approaches and recent connectome studies [in vivo have been revealed rapidly by various approaches including electrophysiology, fMRI, EEG, electrode array, calcium imaging and optogenetics [in vitro neural circuit models that can investigate the circuits’ fundamental mechanisms increased correspondingly.Attempts to construct neural circuits with certain functions directly link with efforts to understand minimal structural requirements of the circuits which execute mechanistic functions within the brain. If a particular function of the brain is executed by a specific neural circuit, in theory, the same function would be reproduced by an  studies , 2. The genetics –6. As stin vitro disease models that precisely recapitulate the symptomatic regions or circuits of the brain to understand their pathophysiological mechanisms. Correspondingly, in vitro disease modeling provide important insights into functionality of circuits by understanding their deregulation phenotypes in relation to the disease symptoms.Neurological diseases indicated that disruption of various circuits triggers diverse functional defects of the brain. It is essential to create in vitro to provide structural basis for functionalization of neurons. Various engineering techniques have been developed to control positioning, connection, and assembly of living neural cells that are fragile, unstable and hard to control. In parallel, stem cell research enabled formation of three-dimensional brain-like tissues (brain organoids) from human stem cells, which mimic brain structures including layers of subtypes of neurons. Brain organoids showed great potentials of creating neural circuit models that resembles the brain structure and functionality more than two-dimensional cell cultures. Research of neural circuit formation is currently dynamically changing and advancing through convergence of engineering and biology and incorporation of other diverse research fields. Could higher-order functions emerge and be recognized in the in vitro neural circuits by extending the current efforts to control and assemble neural cells? We review the progress and perspectives toward building functional neural circuits in this review.Conventional two-dimensional culture of neurons generate limited functionality due to random orientation and interaction of neurons. Efforts have been made to mimic neural circuits with neurons cultured Individual neurons exhibit their “elementary functions” including generation of action potentials (left). The neurons form circuits and gain “mechanistic functions” including generation of complex network activity (middle). The brain exhibit “higher-order functions” by coordinating activity of the neural circuits (right).in vitro relies on the culture of neurons capable of performing their elementary functions  have facilitated understanding of their asymmetric/symmetric division and differentiation in vitro . Culturein vitro , 20, illin vitro –23. Whilin vitro , 25, difin vitro for cellular and molecular neuroscience and other fields of study.Embryonic stem (ES) cells isolated from the inner cell mass of fertilized eggs can be differentiated into diverse cell types including neurons –32. In 1in vivo neural circuits are complex yet well organized. To extract and obtain the functional neural circuits, acute slices of the brain from animals are prepared. Acute slices of rodent hippocampus are the most studied ex vivo neural circuits and have significantly advanced the study of synaptic plasticity [in vivo [ex vivo for a couple of weeks, which allows researchers to observe temporal  changes of the cells. However, it is challenging to keep the circuit structure perfect for an extended period of culture, since the cells tend to migrate within and out from the tissue. Furthermore, a major limitation of brain slice methods is that it is not applicable to humans, which restricts the potential for drug development and human brain studies.While the above-mentioned neurons are randomly oriented in the culture, asticity –47. Braiasticity –50. They[in vivo , indicatThe cultured neurons have the property of spontaneously extending neurites and forming synapses with other neurons. If no physical restriction is applied to the neurites, they will extend in random directions, form synapses between accidentally encountered neurons, and create circuits without architectural plans. Synapses can even be formed between the dendrites and axons extended from the same cell .Towards establishing organized neural circuits in two-dimensional culture, spatial control of neurons and their processes is one of the most critical techniques. Since the Campenot chamber was first introduced in 1977, various microfluidic culture chips have been developed and utilized to control neurites. Typically, these devices allow separation between cell bodies and axons through control of neurite outgrowth by size. Microslits in a wall separating multiple culture chambers are designed to be wide enough for axons (approximately 0.5 μm in width) to pass through, whereas cell bodies  cannot pass through. Thus, neurons plated into one chamber stay inside of the chamber, but axons grown from the neurons pass through the microslits and extend into another chamber. While porous membranes can be also used for separation of axons from cell bodies, microslit chips are superior for more elaborate designs aimed at circuit formation. By using the devices, neurons can be connected via axons within the microgroove to create circuit structures. Furthermore, more advanced microfluidic devices have been applied not only for connecting two types of neurons but also for connecting more than three types of neurons to achieve co-culture of multiple cell types . TechniqIt is also possible to direct neuron outgrowth by micropatterning and microstructures on the culture surface –61. Applin vitro, but improvements are still required to generate more complicated activities and acquire higher-order functions.Over the years, various technologies have been developed one after another in two-dimensional neuron cultures, and related ongoing research is steadily progressing towards the establishment of neural circuits with organized structures. The development of these circuits has made it possible to study neural plasticity and generate simple oscillatory activities Two-dimensional cultures of neurons contributed tremendously to the understanding of neuronal physiology, taking advantage of their accessibility for observation and ease of manipulation. Neuron culture systems and engineering techniques in three dimensions could advance the modeling of more complex neural circuits Fig. .in vivo environment and promote cellular maturation. More importantly, neurons could form synapses and generate unorganized circuits within the tissues, although the internal structure of neurospheres do not necessarily mimic the brain.As mentioned earlier, primary NSCs are often cultured in three-dimensional as neurospheres. The three-dimensional environment facilitates cell-cell interactions within the tissues and exposure to extracellular matrix produced by themselves, which mimic the in vitro culture. The physical segregation between axons and cell bodies is naturally achieved in vivo, but often cells migrate out of tissues in vitro, impeding the creation of neural circuits. This indicates that three-dimensional assembly of cells within the neurospheres provides strong adhesion between cells, serving as a robust structural foundation to build circuit-like structures.Inter-regional connections are critical for coordination and information processing of the brain. To achieve this, methods to integrate the neurospheres as building blocks have been developed . Neural in vivo cortical embryogenesis, which was not typically observed in the neurospheres that are mentioned in the previous section. This marked an important turning point for in vitro neuroscience research by demonstrating that stem cells can self-organize to produce complex and organized three-dimensional tissues.Stem cell research independently propelled three-dimensional tissue culture technologies. Dr. Yoshiki Sasai’s group first demonstrated that cultured three-dimensional embryoid bodies of mouse ES cells can spontaneously form neural tube-like neuroepithelial structures with inner lumens . The neuin vitro. In addition, brain organoids derived from patient iPS cells or genetically engineered disease-model human stem cells can enhance our understanding of disease pathogenesis and facilitate the search for new drugs.Similarly, human iPS cells and ES cells in three-dimensional aggregates can undergo differentiation and self-organization that result in tissues with brain-like structures ; such brin vivo human brain development. This could be extremely beneficial in the establishment of complex neural circuit constructs. Also, a population of neural stem cells named outer radial glia (oRG) cells that are present in human brains has been identified in the organoids, indicating that organoids are good models of the human brain.What distinguishes brain organoids from previous human neuron models is the structures of cellular assemblies e.g. spontaneously organized layers, similar to the Various regions of our brain are mostly originated from a neural tube, in which each region gradually segments and acquires its identity. Formation of the brain regions are controlled by morphogens that are differently expressed along the anterior-posterior and dorso-ventral axes of the neural tube and the body. Organoids resembling different brain regions can be created by mimicking and reproducing the corresponding developmental processes through treatment with extrinsic factors . Brain oStrikingly, organoids cultured for long periods of time  have been reported to exhibit complex oscillatory activities, with the activity patterns similar to those of premature infants’ brainwaves . Brain oConventional two-dimensional culture of neurons form random networks in a dish. Bioengineering techniques to generate organized networks mimicking local circuit structures have been developed. This could be further incorporated into a network of networks that mimic macroscopic circuit structures. Neurons could be also cultured in a three-dimensional aggregate known as a neurosphere. The brain organoids bearing advanced three-dimensional internal structures can be generated by spontaneous differentiation and maturation of stem cells in a spheroid. The brain organoids could be further used to develop advanced structures including fused and connected organoids.In the past, disease pathogenesis of the human brain was largely explored through post-mortem patient biopsy, which provides only snapshots of the disease. Among various types of disease models that allow researchers to actively investigate disease pathogenesis and develop treatment, brain organoids demonstrate their unique characteristics of mimicking the human brain.In 2013, Lancaster et al. reported the differentiation of cerebral organoids from iPS cells derived from microcephaly patients, marking the first time in which brain organoids were used to model disease phenotypes . The patin vitro. As our brain’s higher-order capacity depends on the functionality of neural circuits and their coordination, most neurological diseases are not limited to structural and cellular alterations of the brain, but also functional changes at a network level. As emerging evidence indicates that brain organoids contain intrinsically complex networks, the potential of brain organoids to study network disruptions in disease states has come under the spotlight. Sun et al. modeled Angelman Syndrome, a neurodevelopmental disease characterized by developmental delays and seizures, with CRISPR-mediated UBE3A knock-out (KO) human ES cells [The unique ability of brain organoids to self-organize and mature to form layered structures led early studies using patient-derived brain organoids to reveal critical physical phenotypes ES cells . The KO ES cells . CorrespES cells . AnotherES cells . Gamma oES cells .Other than neurological diseases that happen early in development, brain organoids have also shown promise in the study of late-onset, neurodegenerative diseases (NDDs). These diseases uniquely affect specific regions of the nervous system, but have the common hallmark of aggregated protein accumulation, linked to neuronal damage and their loss. NDDs cause dysfunctions of certain neural circuits that underly their distinct symptoms, making it appropriate to investigate functionality of representative neural circuits. In fact, correspondingly, recent studies suggest that aggregated proteins propagate along specific neural networks, which further suggest importance of neural circuits as disease models for understanding their pathological mechanisms. To date, various neurodegenerative disease models have been established, including the use of cerebral organoids for Alzheimer’s disease (AD) , 82, midIn addition to investigation on protein aggregation and cellular death, the functionality of NDD organoid models is gradually coming to light. For instance, APP mutant and PSEN1 mutant cerebral organoids were studied as AD models . The autin vitro are increasing, and further development is expected to uncover pathogenesis mechanisms of NDDs and to serve as platforms for drug development.A study by Foliaki et al. investigated the electrophysiological properties of cerebral organoid models generated with cells from donors representing Creutzfeldt Jakob disease and PD along with Down syndrome. In early stages (3–4 months), the control and the disease model organoids exhibited no difference in activity. Interestingly, by 6–10 months, the disease model organoids demonstrated slower burst rates and longer periods between network events, as well as neural oscillation changes . Since PBesides the diseases with genetic causes, models have also been developed for diseases caused by damages induced by external triggers including traumatic brain injury, viral infection and malaria –97. SubsIn vivo studies of patients revealed neural circuitry alterations that often span multiple brain regions. A deeper understanding of developmental progression and early network changes associated with psychiatric diseases can possibly result in earlier intervention and more treatment options. Due to the complexity of psychiatric diseases, it is extremely difficult to model the disorders in vitro. Cellular or molecular analyses have not been very effective to decipher the higher-order dysfunctions and it is now clear that analyses at a network level are necessary to study psychiatric disease. Fortunately, recent progress in technology and cultures has shown promises toward understanding psychiatric diseases in vitro.Psychiatric diseases exhibit diverse symptoms that affect higher-order brain functions. Our understanding of the etiology and pathophysiology underlying these disorders remain limited. With no definite biomarkers or anatomical changes, diagnosis of psychiatric diseases largely relies on clinical symptoms such as behavioral and cognitive impairments. Three-dimensional brain organoids have opened new opportunities in studying the developmental progression of psychiatric diseases. Several papers reported abnormalities in neurogenesis and neurodevelopment using organoid models of schizophrenia and bipolar disorder, including reduced neural progenitor cell proliferation, disrupted neural differentiation, and altered excitatory-inhibitory balance –104.Recent studies have attempted to explore the functional alterations in organoids modelling psychiatric disorders. Schizophrenia patient-derived organoids and bipolar disorder patient-derived organoids exhibited diminished response to stimuli, although the baseline electrical activity did not differ from the control , 106. ThIn this last decade, we have seen major advancements in brain organoids and disease models using them. Their complexity has offered unprecedented opportunities in uncovering the functional aspects of neurological diseases at a cellular and network level. More advanced models combined with diverse technologies would provide a deeper understanding of not only the diseases but also our normal brain functions and mechanisms.Inter-regional circuit models are crucial in understanding brain functions and mechanisms. Beyond the models of local and regional circuitry, researchers have developed more advanced strategies for modeling neural circuits structured at a relatively larger scale. Importantly, developmental interaction between brain regions is essential to form proper structures of the brain, and the interaction facilitate formation of neural networks. To model the inter-regional interaction, multiple approaches have been proposed with their effectiveness demonstrated.in vitro, a method of locally inducing ventral differentiation within an organoid has been proposed [The first approach is to induce developmental body axes for generating multiple regions within an organoid along the axes. During brain development, the dorsal forebrain produces excitatory neurons and becomes the cerebral cortex. Meanwhile, the ventral forebrain becomes ganglion eminence and produces inhibitory neurons which migrate tangentially to the dorsal forebrain and are incorporated into the cerebral cortex. To reproduce this process proposed . By incoproposed . Toward proposed , 110.Theproposed .Another type of approach is direct fusion of multiple organoids that models different regions of the brain. Various types of brain organoids are fused to model interaction between different pairs of regions. Such fused organoids are widely recognized as “assembloids” –115. ThiThough the above mentioned approaches have shown feasibility in modeling the interactions between multiple regions and creating circuit structures, none of them generate an axonal tract that appears to be the signature structure in the brain and serves as a critical hub for the inter-regional connections. In the brain, axonal tracts in and between cerebral hemispheres form the white matter. While distal cortical regions are connected through the white matter (extrinsic pathway) in the human brains, the corresponding axons in the mouse brains are either fewer or located inside the grey matter (intrinsic pathway) . This stin vitro axon tract model that contains self-assembled bundles of axons. With this in mind, we have previously developed a method to fabricate motor nerve organoids in microfluidic devices [The first step towards reconstructing a brain circuit that mimics the distal connections of different brain regions is the establishment of an  devices . In this devices . Human i devices , 121.in vitro.The difference between assembloids and connectoids lies in the axon bundles which can be useful for understanding how axonal tracts assemble and form. In addition to the importance of the axon bundle in structural and developmental studies, functionality of the tissue could be dramatically changed by the structure. Using multi-electrode array (MEA), the activities of connectoids were observed to be significantly more complex and active than those of single cerebral organoid and cortico-cortical assembloids , suggestin vitro neural cultures as a result of fruitful collaboration between stem cell biology and tissue engineering  and electrical engineering, which will be described below. The merging interest and participation from various fields of study on in vitro neural circuits create a unique opportunity for the researchers to explore new ways to code and decode neural activity patterns along with developing the circuits at the same time. This could construct a new platform and scientific community to probe fundamental mechanisms of neural circuits underlying higher-order functions of the brain.As described, organoid models of neural circuits are quickly advancing through the application of bioengineering technologies on biologically generated tissues, in parallel to the advances in two-dimensional neural circuits, where recent improvements largely depend on bioengineering techniques Fig. . The neuing Fig. . This isin vitro models.Stem cell biology and tissue engineering technologies converge into organoid engineering technologies. By incorporating other technologies including genome editing, patient-derived cells, artificial intelligence, and electrical engineering would generate advanced functional in vitro circuits of three snail neurons that generate oscillation is one of the simplest and most sophisticated examples of in vitro network executing clear functions [A demonstration of unctions . Since tin vitro often generate simple periodic burst activity, which by itself could be considered as a functional signature of the neural circuits. The periodic bursts could be generated by strong recruitment of individual neurons to fire together with other neurons to form synchronous burst activity, due to their tight and strong synaptic connections and uniformness of the circuits. This could be also considered as a hyperactive (possibly epileptic) pattern in which too many neurons fire together and hence its information coding capability is hindered, which indicate that too strong and too uniform connections limit functionality of neural circuits. Adequate amount of inhibitory neurons and astrocytes are also critical to maintain complex activity patterns of the networks. In contrast to the random network, regional circuits in the brain are connected with other regions with their axons projected between the regions, allowing information to flow from one region to another, and the whole brain to function coordinately. Additionally, the axonal projections could serve as structural constraint for the circuit to protect them from excessive connection and thus epileptic seizures. The axonal connections also could provide time delay as important factor for functionality of computation and oscillation of neural circuits. One could speculate that the structural constraint can provide convergence points for information and neural activity, whereas the cortical regional networks as divergence segments, which could serve as an important principle for information processing. Thus, neural circuits that contains regional circuits that are inter-connected with axons could be useful for assessing the importance of the convergence/divergence structure motif. Due to structural and functional complexity, the connected organoids could be an ideal platform to assess these hypotheses and functional importance of the inter-regional connections and to formulate a fundamental structural requirement for maximizing functionality of neural circuits. Two-dimensional cultures with the macroscopic separation and connections between segmented neural circuits have demonstrated promising structural contribution to their network activity [in vitro neural circuits, more unique brain region- or input- specific functions should be modeled by incorporating more organoids in numbers and types into the circuits.Random neural circuits activity , 125. OnIn vitro neural circuits have been demonstrated to exhibit functionality including synchronized bursts, oscillation, network propagation, and neural avalanches. These activity patterns generated as neural circuits could be considered as mechanistic functions that provide foundation for higher-order functions of the brain. One of the important questions is whether higher-order functions could emerge in the neural circuits that exhibit mechanistic functionality. This question leads to another question of whether higher-order functionality could be appreciated by observers, even if the neural circuits have the capability of executing higher-order functions in itself. To answer these questions, it is essential to provide a system to relate input to the neural circuits and output from the circuits  or a “reservoir” to achieve tasks, suggesting that employing the mechanistic structure for artificial intelligence could be a powerful approach for understanding higher-order functionality of neural circuits [in vitro neural circuits have been investigated to achieve responsive game task [in vitro neural circuits are capable of processing information and executing higher-order functions. These highlights that in vitro neural circuits could be ideal for testing theories and methods to unlock the ability of neural circuits and execute higher-order functions. Higher-order functionality of the biological in vitro neural circuits are still limited at the current stage but combining closed loop systems with circuit models equipped with more sophisticated structures would pave the way to unleash advanced higher-order functions, and lead to better understanding on the basic principles of neural network mechanisms of the brain. In vitro neural circuits could be ideal for both exploring methods to unlock higher-order functions and inventing new methods to effectively process information inspired by the neural circuits.its Fig. . Since mcircuits . Anotherame task , in whicDo neurons have higher-order functions? We could investigate this by real-time interaction with them through a system with continuous input and output that provides virtual environments.Human stem cell-derived brain organoids provide numerous advantages to model brain functions and structure. However, currently there are a few major limitations. One of the potential concerns is the variability between organoids. Organoids often differ in their structures and activity patterns even within the same batch of organoids from the same iPS cell line, resulting in difficulty to control their quality. Cell-line-to-cell-line variability also exists. Relatively long time required for maturation and production of some of the cell types could also a limitation. Human brain organoids need a few hundreds of days for their maturation, which is critical for producing some of the late-emerging cell types including upper layer neurons and astrocytes. Also, the brain organoids usually lack major cell types and structures, including those for the blood and immune systems. By resolving these issues, the brain organoids would have better ability to model the human brain.Considering the functions of brain organoids, one of the most critical limitations of the current organoids is the lack of input and output as stated above. This is critical not only for understanding their hidden functional capability, but also for creating functional circuits within or with the organoids through activity-dependent synapse formation and organization mechanisms. Another limitation is the recording techniques. Although high density electrode arrays and wide-field calcium imaging techniques enables recording of neuronal activity, it is still difficult to record from all neurons in the complex tissue with substantial three-dimensional size.Engineering techniques are rapidly developing for brain organoids, but there are many technical limitations to overcome. For example, it is currently difficult to build complex three-dimensional structures that are compatible with long-term culture and observation of the organoids. It is also difficult to establish stable long-term gradient of morphogens to establish body axes within organoids. While numerous technical challenges exist, we believe that new technologies in biology and engineering will continue to be invented to advance the construction and modeling of functional neural circuits.in vitro neural circuits and their ability and potential to acquire functions. The field is quickly advancing and changing as exciting discoveries and inventions emerge almost every week. We apologize that many important and interesting studies are not mentioned in this review due to the space limitation. We hope that this review helps the readers to know current advances in the field and to feel the potential for future developments.In this review, we overviewed research progress and discussed perspective of


Animal models are essential for investigating the pathogenesis and developing the treatment of human diseases. Identification of genetic mutations responsible for neurodegenerative diseases has enabled the creation of a large number of small animal models that mimic genetic defects found in the affected individuals. Of the current animal models, rodents with genetic modifications are the most commonly used animal models and provided important insights into pathogenesis. However, most of genetically modified rodent models lack overt neurodegeneration, imposing challenges and obstacles in utilizing them to rigorously test the therapeutic effects on neurodegeneration. Recent studies that used CRISPR/Cas9-targeted large animal (pigs and monkeys) have uncovered important pathological events that resemble neurodegeneration in the patient’s brain but could not be produced in small animal models. Here we highlight the unique nature of large animals to model neurodegenerative diseases as well as the limitations and challenges in establishing large animal models of neurodegenerative diseases, with focus on Huntington disease, Amyotrophic lateral sclerosis, and Parkinson diseases. We also discuss how to use the important pathogenic insights from large animal models to make rodent models more capable of recapitulating important pathological features of neurodegenerative diseases. The prevalence of age-dependent neurodegenerative diseases (NDs), such as Alzheimer’s disease (AD), Parkinson’s diseases (PD), Amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), has increased worldwide in parallel with the rise in life expectancy. The common features of these diseases include selective neurodegeneration of specific populations of neurons that occurs in an age-dependent manner in the human brain  , superoxide dismutase 1 (SOD1), fused in sarcoma (FUS), and C9ORF72  that is featured by neurodegeneration in association with mitochondria dysfunction , however, the nuclear TDP-43 is redistributed in the cytoplasm and forms cytoplasmic inclusions  as well as a relatively short gestation period (about 114 days) and are able to produce multiple offspring (about 10–12 piglets per liter). In addition, the pig endogenous genes can be edited via somatic cell nuclear transfer (SCNT) to produce knock-in or knockout models. All these advantages make pigs a promising and alternative large animal model for investigating human diseases  of monkeys at different ages  were generated via SCNT, but most of the transgenic HD piglets died postnatally  in the endogenous HTT gene , it is important to examine the protein level of the targeted gene in different brain regions. On the other hand, injection of AAV into the monkey substantia nigra to co-edit the PINK1 and DJ-1 genes could mediate severe nigra dopaminergic cell loss and motor function deficits  transgenic pigs via the SCNT method  of the primate SQSTM1 transcript  in the cytoplasm in the brains of rhesus monkeys and pigs , the gestation period lasts for about 165 days, and each female animal often produces a single offspring each year. In addition, generation of embryonic genome edited non-human primate models is technically challenging and labor intensive, as multiple processes including hormonal superovulation, embryo transfer with surgical implantation, and experienced animal care, would require the well-equipped animal facility, enough animal resources, and a highly trained team.Additionally, CRISPR/Cas9 system creates the mosaic mutations that may result in various types of mutations and different cellular phenotypes. The mosaicism in offspring can be reduced by outcrossing the mosaic founders with wild type animals. However, it will take years to eliminate mosaicism in large animal models, especially for the non-human primates that require 4–5 years of sexual maturity to produce next generation. In pig models, however, the gene editing tools combined with SCNT have been successfully to establish the pig models that carry the same single genetic mutation as in human patients. SCNT was also applied to the non-human primate, and the results demonstrate the feasibility to generate knock-in non-human primate models (Liu et al., Several other technical hurdles also need to be resolved. Although the mosaic mutations in different animals may not yield the same phenotypes, one can analyze the correlation of genetic mutations and phenotypes at the cellular level, which can be achieved by imaging and RNAseq analysis at single cell resolution (Zhang et al., Alternatively, genetic modification can be performed in somatic cells to mirror neuropathology in the specific brain regions in large animals. The direct administration of AAV expressing gRNA/Cas9 or transgenes into specific brain regions of large animals at multiple developmental stages or different ages could more rapidly recapitulate brain region-dependent or age-related neuropathology. Treatment of gene-targeted animals with environmental stress or chemicals that induce oxidative stress could also facilitate disease progression, as oxidative stress has been implicated in the progression of a number of NDs. Moreover, the continued improvement of viral vectors that can readily penetrate the blood–brain barrier may greatly facilitate the delivery of the gRNA/Cas9 system or transgenes in the brains of large animals (Chan et al., This review updates the large animal models of neurodegenerative diseases, including HD, PD and ALS. Although investigation of large animal models led to new discoveries of important pathological events or mechanism that have not been found in small animals, there are challenges and limitations that are largely stemmed from the costly and time-consuming investigation. However, the important information gained from the large animal models would be highly valuable for understanding the pathogenic mechanisms and identifying new therapeutic targets. Given the lack of obvious neurodegeneration phenotypes in most of genetically modified rodent models, the demand for establishing large animal models to study neurodegenerative diseases is well-appreciated, and the publications of large animal models with gene editing are steadily increased recently. It is expected that more of large animal models would be embraced as the preferred model for human diseases and translational medicine research. Moreover, the important information gained from large animal models would be highly valuable for generating more humanized mouse models. Specifically, important differences in pathological phenotypes between small and large animals would guide us to create humanized mouse models that can more closely emulate the important pathological features seen in large animal models and patients.


Bioelectromagnetism has contributed some of the most commonly used techniques to human neuroscience such as magnetoencephalography (MEG), electroencephalography (EEG), transcranial magnetic stimulation (TMS), and transcranial electric stimulation (TES). The considerable differences in their technical design and practical use give rise to the impression that these are quite different techniques altogether. Here, we review, discuss and illustrate the fundamental principle of Helmholtz reciprocity that provides a common ground for all four techniques. We show that, more than 150 years after its discovery by Helmholtz in 1853, reciprocity is important to appreciate the strengths and limitations of these four classical tools in neuroscience. We build this case by explaining the concept of Helmholtz reciprocity, presenting a methodological account of this principle for all four methods and, finally, by illustrating its application in practical clinical studies. To study the human brain, scientists often rely on non-invasive methods from bioelectromagnetism such as magnetoencephalography (MEG) and electroencephalography (EEG) as well as transcranial brain stimulation techniques. MEG is closely related to EEG and both afford noninvasive, multichannel measurements of neuronal activity at high temporal resolution in the order of milliseconds. They are therefore ideally suited to study brain dynamics. Excellent reviews provide comprehensive introductions to and overview of MEG and EEG and their applications e.g., . SimilarTheoretically, both sets of techniques are fundamentally linked by the Helmholtz reciprocity theorem that we will describe in more detail below . ThroughIn the following sections we will lay out the close relationship between MEG/EEG and TMS/TES from theoretical principles to their practical implications. We will start with a short introduction to these methods, then describe the importance of their close relationship for forward and inverse modeling  are more difficult to excite via transcranial direct current stimulation/transcranial alternating current stimulation (tDCS/tACS) than cortical structures which are located closer to the tDCS patches (red) and neural generators in deeper structures reciprocally evoke smaller potential differences between the electrodes. Although deeper structures are excitable by stronger currents only, this stimulation would in parallel excite superficial sources to a much stronger degree unless sophisticated interference techniques are used . SimilarThe concept of Helmholtz reciprocity is related to the concepts of forward and inverse problems that are equally relevant for the remainder of this manuscript. The forward problem in the context of EEG/MEG refers to the problem of estimating the potentials and/or magnetic fields at EEG electrode and/or MEG sensor locations on or/and outside the head for a dipole at a specific location in the brain with a given orientation. The inverse problem refers to the problem of estimating the current source distribution in the brain that gives rise to a given potential or/and magnetic field pattern on or/and outside the head. It was already noted by Helmholtz that the solution to the EEG/MEG inverse problem is not unique—that is, an infinite number of current distributions can lead to the same potential or/and magnetic field pattern. Therefore, EEG/MEG source localisation methods employ additional constraints to yield a unique solution.MEG/EEG sensors silently and non-invasively sample with millisecond temporal resolution the magnetic field or electric potential, respectively, that is caused by neuronal activity in the brain. The direct relationship between the recorded signals and the underlying neuronal currents is not affected by intermediate processes (such as neurovascular coupling) and thereby leads to an information-rich dynamic representation of large-scale neuronal activity. While MEG/EEG perform non-invasive recordings of signals generated by neuronal activity, the aim of TMS/TES is the controlled modulation of neuronal activity by means of injecting magnetic fields or electric currents into the brain .There are several advantages in combining MEG/EEG with neurostimulation techniques . First, MEG and EEG signals can even be simultaneously recorded during TMS/TES with the current exception of simultaneous TMS/MEG. While EEG recordings can momentarily be interrupted during phasic TMS, preventing EEG amplifier saturation , this opOptically pumped magnetometers (OPMs) have been developed recently and represent a promising alternative to traditional SQUID-based MEG systems . OPMs doAnother important consideration for EEG/MEG-TES studies is the optimization of the stimulation parameters, including TES electrode location . StimulaWhile simultaneous TMS-MEG is not currently possible, TMS has been used in conjunction with MEG by exploiting longer-lasting stimulation effects  or alternating current stimulation (tACS), is a noninvasive method to manipulate brain excitability via changes in membrane polarization and to induce long-lasting (minutes or even hours) changes in the brain, depending on polarity, duration, and intensity of the stimulation . In clasPlease note that the simple scenario where injecting currents parallel to target orientation leads to excitation and anti-parallel leads to inhibition holds for tDCS only. Other stimulation methods and protocols will engage other mechanisms of action. For example, tACS employs electric stimulation with rapidly alternating current direction. rTMS applies single pulses in rapid succession. Both methods are thought to lead to frequency-specific resonance effects that may be used to change the amplitude of neuronal rhythms .To improve performance, it can be vital to couple the aforementioned inverse and optimization approaches with modern forward modeling methods. The EEG and TES as well as MEG and TMS forward problems are closely related through Helmholtz’s reciprocity principle , which manisotropy refers to the property of a material (here white matter) to allow changes in different directions in contrast to isotropy, that is, the conductivity parallel to the white matter tracts is higher than in the two perpendicular directions. To illustrate white matter conductivity anisotropy volume conduction effects, the lower row of A tangentially oriented source located in the somatosensory cortex of a 5-compartment head model (A) built of skin, skull, cerebrospinal fluid CSF), gray matter, and anisotropic white matter (D) would generate EEG isopotential lines on a model surface as shown in (B) and (C). The only difference between B and C is that skull conductivity was modelled ten times higher in (B) compared to (C) leading to much higher surface potentials and a smaller distance between the positive potential peak and the negative potential trough in (B) compared to (C), as indicated by the black arrows. In the context of the EEG inverse problem, ignoring the variance of individual skull conductivities would thus predominantly lead to depth localization errors (as the distance between peak and trough increases with source depth). Apart from skull conductivity modeling, the importance of accurate CSF conductivity modeling has been shown not only in simulations  but also, gray maRealistic individualized head models can be constructed using (semi-)automatic processing pipelines based on T1-weighted- (T1w-), T2w-MRI and diffusion-weighted-MRI . This alIn the next section, we present four studies that combine MEG/EEG and TMS/TES and sample the wide spectrum of their synergistic use.The number of studies combining high-density EEG/MEG neuroimaging with targeted TMS/TES brain stimulations increased exponentially in the past decade. Accordingly, the spectrum of applications in cognitive and clinical neuroscience has expanded considerably. The following four examples were picked to provide a glimpse on the range of methods, method combinations, and applications. This specific choice in part reflects the clinical orientation of the author’s research interests and is in no way intended to represent any kind of superiority over the multitude of excellent studies in the field.Advances in functional neuroimaging continuously increase our understanding of pathological alterations in neural circuits that underlie brain disorders. This advanced knowledge leads to an increased interest in brain stimulation methods to modulate the identified aberrant neuronal activity patterns. Reciprocally, clinical treatment effects of brain stimulation can also inform us about underlying disease mechanisms and allow us to evaluate causal relations of stimulated brain areas in distributed networks going beyond purely correlational associations.EEG and MEG source analysis and connectivity investigations can, for instance, fundamentally contribute to the understanding of pathomechanisms underlying epileptogenesis, seizure generation, and seizure propagation . It may Individual targeting by optimized multichannel TES can improve therapeutic effects and decrease negative side effects. Swallowing relies on highly complex sensorimotor functions requiring widely distributed neural network activities . It is tIn a randomized control trial, Suntrup-Krueger and colleagues revealed that excitatory tDCS stimulation of the unaffected motor cortical swallowing network resulted in greater improvement of swallowing functions compared with a sham stimulation . In factThis example illustrates that even non-individualized brain stimulation can modulate highly automatized and complex brain functions such as swallowing, holding out the prospect of standardized use in clinical practice.The so-called ventromedial prefrontal cortex (vmPFC) is one of the most widely reported structures identified as dysfunctional in mood and anxiety disorders . In two Schizophrenia patients typically show deficits in evoked gamma band activity and gamma synchrony at rest and during various cognitive tasks e.g., . By takiThe above examples gave an impression on how combinations of TMS/TES with MEG/EEG can produce synergistic effects. They also illustrated how different brain stimulation methods can be informed by MEG/EEG and then, for instance, be applied in the attempt to test potential novel treatment strategies or to uncover or prove pathological neural mechanisms. However, despite the increasingly successful use of targeted brain stimulation, knowledge about the mechanisms of action of the different brain stimulation methods, about their dependence on stimulation parameters such as duration, repetition, strength, frequency, and orientation, and about the modulation of stimulation effects by physiological and psychological states and characteristics is still rather scarce.Here, we showed that due to Helmholtz’s reciprocity , EEG andWhile we here focus mostly on the issue of reciprocity, it should be noted that long-lasting effects of brain stimulation that are exploited for therapeutic purposes, might not solely be related to direct modulation of targeted brain areas, but could also be caused by more general mechanisms, such as modulations of more distant areas and effects mediated by changes in neurotransmitters and gene expression e.g., .Looking into the future, we expect that hardware developments and improved targeting procedures will increase the effectiveness of neurostimulation as scientists continue to strive to better control and understand the stimulation effects. It is now well established that modeling of stimulation effects  is important. However, stimulation effects depend on many factors that need to be considered together. In summary, the complementary use of MEG/EEG and TMS/TES holds great potential not only for improving our understanding of cognitive processes in the brain but also for developing new therapeutic approaches for the treatment of neurological and psychiatric disorders.


The connection between international trade and food systems (un)sustainability is both contentious and critical for policy work supporting progress towards achieving the twin goals of hunger alleviation and dietary health while improving the overall sustainability of development. We characterize the food system using a set of metrics based upon the EAT-Lancet commission dietary guidelines for both over- and under-consumption of different foods to assess country-level dietary health and sustainability in tandem. Using a partial equilibrium model of agricultural production and trade, we then project the functioning of the global agricultural system to 2050 and calculate the metrics for that year. For most regions we find increased overconsumption above the expert-defined healthy and sustainable diet thresholds, with more limited progress towards closing dietary health and sustainability gaps where they currently exist. Trade influences this dynamic into the future under certain socioeconomic conditions, and we find that under a “business as usual” trade environment, future agricultural import profiles continue to be misaligned with dietary health and sustainability outcomes, suggesting the potential for early intervention in trade policy as a means to positively influence food system outcomes. The impact of global agricultural trade on the environment and human health is a contested space in agricultural policy. The importance of the relationship between trade and dietary health is well established in the literature, especially as it relates to the question of trade liberalization –5. SuppoOn the other side, proponents of global agricultural trade point to reduced hunger and increased opportunities for agricultural producers in the developing world. They highlight increased opportunities for food system actors to buy and sell goods via integration into global value chains and the expansion of robust agricultural markets to more and more communities . The autTrade is clearly a critical component of the global food economy, but the lack of consensus regarding the specific effects of trade indicates that there is much left to understand about the potential roles of trade in supporting food system sustainability. While there are broad explorations of the relationship between trade and dietary (un)sustainability –18 in thTo this end, we operationalize two quantitative measures of dietary quality using the recent EAT-Lancet commission on Food, Planet, and Health’s dietary guidelines and food availability data from the FAOSTAT database  to benchUnhealthy diets are one of the largest risk factors driving the global disease burden . As therThe transition from a food security focus to a food system emphasis in food policy includes the recognition of a need to develop and track quantitative indicators of multiple facets of global food systems functions. In order to ensure that progress is being made towards ambitious system-level goals, consistent and comparable measures are required to understand whether food systems are functioning in a healthy, equitable, and sustainable manner , 31. ConExtending this line of reasoning, we propose here to examine the role of trade in promoting healthy and sustainable diets on a country-level basis. The EAT-Lancet guidelines have been formulated so as to achieve tandem goals of providing the nutrition necessary for human health at the individual level, while avoiding transgressing an environmental safe operating space at the global level . This yields the To compute these metrics, data on production, imports, exports, and food availability was first obtained from FAOSTAT , coverinFor the foresight component of the analysis, we use annual projection data from the International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT) version 3.0 . IMPACT While the EAT-Lancet diet has been used for scenario analysis in the original EAT commission report , we focuWe use the Shared Socioeconomic Pathways framework to drive projection of dietary quality under four (SSPs 2–5) structurally different socioeconomic futures. These scenarios provide different trajectories of population and GDP growth which are exogenously imposed on the IMPACT modeling system to introduce demand-side changes. GDP and Population growth rate values are adapted from the SSP public database  and usedThese SSPs are compatible with climate change assumptions of a 1C-2C increase in global mean surface temperature over preindustrial levels by 2050 , North American (NAM), Former Soviet (FSU), and European regions (EUR) for example see dietary undershoot that is functionally the same or potentially worse than dietary undershoot in 2010, while progress in reducing dietary undershoot in Latin American and Caribbean regions (LAC) is expected to occur across a similarly narrow band of possible outcomes.The SSP5 scenario is projected to drive the most progress in reducing dietary undershoot across regions but is also projected to drive a significant increase in dietary overshoot—almost 60% higher on average than the next highest scenario (SSP2). This implies that the increase in average purchasing power may be effective in some regions  for reducing dietary undershoot, but at the cost of driving overconsumption of other food commodities. In other regions , increased purchasing power may drive overconsumption with regards to the EAT-Lancet diet, but with little to no reduction in underconsumption.The effect of trade on healthy and sustainable diets is in some sense bounded by the portion of the average diet that is derived or expected to be derived (for future years) from traded goods. 1) weak but significant negative relationships between change in imports per capita and dietary undershoot across all scenarios, suggesting that regions with larger change in import volume relative to 2010 are relatively more successful at reducing dietary undershoot regardless of global socioeconomic patterns; and 2) weak but significant positive associations between dietary overshoot and change in imports per capita across all scenarios except SSP3, suggesting that regions with larger change in imports per capita are relatively less successful at containing dietary overshoot except in a scenario where socioeconomic outcomes are fairly stagnant (SSP3).As agricultural trade is expected to increase in importance for the average diet out to mid-century, rather than decrease, it is prudent to try and understand whether countries with larger increases in trade , to assess the degree of benefit derived from agricultural trade. These results are displayed in If we expect agricultural trade to affect dietary quality in a mostly negative manner due to the potential for increased overshoot, we might also ask whether less trade would provide for better dietary outcomes. To answer this question we estimate a hypothetical “no-trade” diet , dietary undershoot conditions are slightly improved by consuming a “pre-trade” diet across most regions. In the North American region (NAM) for example, the dietary undershoot gap is closed nearly completely by eating only what countries in this region produce. This is reversed for the MEN region, and dietary undershoot in the Sub-Saharan African region (SSA) is roughly comparable both before and after trade. On the dietary overshoot side however, most countries experience a large benefit from global trade in terms of reduction of dietary overshoot. Latin American diets, for example, see a nearly 100 percentage point closure of the dietary overshoot gap achieved via agricultural trade in 2010. The MEN and South Asian (SAS) regions, however, see dietary overshoot increased through international food trade. South Asian states are the only region that see both dietary undershoot and dietary overshoot performance become worse after accounting for the effect of trade.We then project the effect of trade on diets to 2050 under the four SSPs used for this study (right panel). Here we observe that South Asian (SAS) states are still projected to see dietary health and sustainability reduced by international food trade across most scenarios (excluding SSPs 2–3). By contrast, Sub-Saharan Africa (SSA) is projected to benefit from international food trade overall (reducing both overshoot and undershoot). For most other regions, reductions in dietary undershoot are still expected to be at least partially offset by international food trade, with the dietary outcomes on the undershoot side remaining more or less the same as in 2010 across most scenarios. On the overshoot side however, the degree to which international food trade benefits dietary quality is expected to increase under most scenarios and most regions. Latin American countries (LAC), for instance, are expected to see their dietary overshoot decrease by over 200 percentage points after accounting for traded foods under a high growth—low population future (SSP5).While traded commodities are found to benefit most global regions (on average) from our dietary analysis—there are several countries for which trade is expected to continue to affect negatively dietary health and sustainability on both overshoot and undershoot sides. In order to understand why a certain country or region may or may not benefit in dietary terms from agricultural trade, we then looked into the food group categories that are contributing to dietary overshoot and undershoot at the regional level in In this study, we sought to untangle the expected future relationship between international food trade and healthy and sustainable diets for a globally-representative set of countries, leveraging established projection models and socioeconomic scenarios , 57. UndOur analysis shows that under projections of international trade environments that are fundamentally price-driven , dietary health and sustainability benefits/losses from trade are projected to accrue to different sets of countries in different ways. Problems of overconsumption are expected under this projection framework to continue to be a major issue for dietary health and sustainability for large parts of the world out to mid-century. Under a high-income per capita scenario (SSP5) dietary overshoot is projected to be significantly worse in several regions compared to other scenarios. Low-income countries, which tend to struggle relatively more with underconsumption in dietary quality terms, are projected to see positive returns from agricultural trade in reducing dietary undershoot. However, under either “business-as-usual” (SSP2) or high-growth (SSP5) scenarios, undershoot reduction is expected to occur alongside increased dietary overshoot for those low-income countries. This outcome nuances in the utility of increased purchasing power for enabling better food systems outcomes in low-income countries, where, under demand-driven trade environments absent substantial policy intervention, small advances in reduction of underconsumption are expected to be granted at the expense of large advances in overconsumption with respect to human health and environmental wellbeing.Because overshoot and undershoot of EAT-Lancet dietary guidelines are projected to ‘co-exist’ for most regions under future scenarios, a “double-burden” dynamic of over- and under consumption with respect to the EAT-Lancet dietary guidelines is expected. For upper-middle income countries especially, and low-income countries to a lesser degree, global agricultural trade may exacerbate a dietary double-burden dynamic under some scenarios  as well as to limit the oversupply of certain types of foods , so our results here are thus best interpreted as following from a “baseline” trade future, which is largely price-driven and tends toward country-level specialization on the production-side. Against this background, it is important to keep in mind that political economic considerations  will conThere are also some important considerations for these results stemming directly from the EAT-Lancet diet’s formulation. For instance, the significant dietary overshoot in the Sub-Saharan African region is mostly associated with a large portion of dietary energy coming from roots and tuberous foods—for which the EAT-Lancet dietary guidelines set a relatively low maximum threshold (100 g/cap/day) citing high glycemic load associated with the consumption of tubers and starchy vegetables , 74. ProThe world today faces challenges on two distinct fronts of dietary health and sustainability—with countries both undershooting  and overshooting (exceeding) many dietary health and sustainability goals and limits. The dietary metrics constructed in this paper present a way to track progress towards, or movement away from, “ideal” dietary and sustainability states, and the results make clear that managing the balance between improving dietary undershoot and limiting dietary overshoot will continue to be a major challenge across countries and regions globally in the future. The results further show that trade, though expected to provide benefits for dietary health and sustainability to many countries, is also projected to exacerbate dietary harms in others under business-as-usual trade conditions. We find this to be true to varying degrees when accounting for potential socioeconomic uncertainty under the SSP framework, evidencing that socioeconomic patterns of development will have implications for both the impact of trade on dietary outcomes, and dietary outcomes themselves. In general, we find that scenarios with higher growth conditions imply far greater dietary overshoot potential across a number of regions  while soIn sum, while there is much room for trade policy to be re-aligned and create healthy and sustainable dietary incentives, it cannot be relied upon as a tool to address dietary health in isolation. Rather, other policy interventions on both the production and demand side of diets will need to be undertaken in conjunction with trade policy in order to address global dietary challenges, especially those related to overconsumption of food groups that are expected to be widely over-available  or under-available .


This study is the first to examine the impact of FinTech firms on bank financial stability. Using a sample of 26 banks from an emerging market , over the period 2003–2018, we find that the development of FinTech firms over time increases bank financial stability. We uncover further evidence that FinTech firms’ impact on bank financial stability holds when we conduct sub-sample analyses by bank size, bank type , and level of corporate governance. The results are robust to alternative model specifications, measures of financial stability, and FinTech. Financial technology (FinTech) companies have become increasingly important sources of financial services in both developed and emerging markets , 18, ErnThe motivation for conducting this study was based on three important sources. First, no systematic empirical study has been conducted on this topic. This study fills this research gap. Second, we believe that this topic is important and worth empirically examining because the FinTech market is growing rapidly, and the banking industry is under market pressure to adopt sophisticated financial technologies in their transactions and services. This pressure has received significant attention in the banking industry, and consequently, FinTech-based financial services are being prioritized by banks , 36, 46.priori indeterminate. In this study, we empirically determine the nexus between FinTech firms and bank stability.The following question arises: How do FinTech firms affect bank financial stability? Besanko and Thakor  and Boydriba), uncertainty (gharar), gambling (mysir), and other business activities that are deemed detrimental to people’s well-being [Our contributions to the literature are threefold: First, this study is the first to examine FinTech firms’ impact on bank financial stability. Second, the present study is also the first to investigate this impact in the context of both Islamic and conventional banks. The former bank type follows Islamic law (Shariah) as a mode of operation, which prohibits dealing with interest  regression as the baseline research method. We examine both the contemporaneous and lagged effects of FinTech on banks’ financial stability. The use of lagged values helps reduce the concern of reverse causality and permits our variable of interest some time lag to affect bank financial stability. We further use the dynamic panel generalized method of moments (GMM) estimator, which allows us to account for unobserved heterogeneity, simultaneity, and dynamic endogeneity in our estimation. We use a sample of both Islamic and conventional banks from Malaysia for the 2003–2018 period. Our results show that the development of FinTech firms over time increases bank financial stability. We find robust evidence when we conduct subsample analyses by bank size, bank type , and level of corporate governance. Our results are consistent with alternative model specifications, measures of financial stability, and FinTech.This study has important implications for regulators, policymakers, and bank managers. It provides important insights for regulators to adopt strategies to promote FinTech development to ensure the banking industry’s financial stability. Our research can guide policy makers to promulgate FinTech related policies and ensure good governance, as FinTech development promotes banking stability. This study’s findings may also guide bank managers in both Islamic and conventional banks to augment FinTech-based financial services.The remainder of this paper is organized as follows: Sect. FinTech firms have received increasing attention in recent years owing to their rapid development and expansion across economies. The rise of FinTech firms has been welcomed by numerous observers who believe that new technological innovations in the financial industry have great potential to transform financial services by offering less expensive transactions, thus making these services more convenient and secure . We defAs suggested by Thakor , we use Finally, we use control variables following the banking literature on the determinants of financial stability. Specifically, we use bank size measured as the logarithm of total assets. Further, bank age represents the number of years from the year of establishment to the end of 2018; the non-performing loans (NPL) ratio is the ratio of NPL to gross loans; equity capital ratio is the ratio of equity capital to total assets; assets growth is the annual growth rate of total assets; income diversity is the ratio of non-interest income to total operating income; ROA is the return on assets; bank concentration ratio is the assets of the three largest banks as a percentage of total banking industry assets; GDP growth rate is the annual growth rate of per capita GDP in percentage and bank-level corporate governance score; the governance score is the average of eight bank-level board of directors’ attributes—namely, board size, board independence, CEO duality, board busyness, board members’ financial expertise, audit committee size, audit committee chairman independence, and risk management committee size. The board governance score is constructed following the governance literature . We follow this approach, as in Arellano and Bover  and BlunWe begin our preliminary investigation by presenting descriptive statistics for the variables in Table A pairwise correlation matrix is presented in Appendix A1. The correlation matrix shows a positive correlation between financial stability (Z-score) and FinTech, indicating that the development of FinTech firms contributes to higher bank financial stability. The highest correlation between ROA and financial stability is 0.28. Therefore, multicollinearity is not a problem in this estimation. Finally, for the control variables, income diversity and bank-level governance exhibit a statistically significant positive correlation with financial stability, while bank concentration exhibits a significant negative correlation with financial stability. These significant associations between financial stability and the other control variables suggest the need to control for these variables in our regression model. Notably, none of the pairwise correlations among independent variables exceed 0.44 , and all variance inflation factor values remain below 10 (untabulated). Thus, multicollinearity is unlikely to be a major concern in our study context.The empirical results are presented in Table In the next step of our investigation, we classified our sample banks into large and small groups. Table We further conduct a sub-sample analysis based on bank-level corporate governance. Specifically, the sample banks in Malaysia are divided into high and low corporate governance groups, and the results are displayed in Table We further perform a sub-sample analysis by bank type. Table We conduct further investigation using alternative measures of financial stability (a relative measure using the stochastic frontier approach) and FinTech . The use of a stochastic frontier-based financial stability measure accommodates the relative financial stability measure and ranks a bank’s financial stability relative to the optimal financially stable banks in the entire industry. Table We further examine FinTech firms’ impact on bank financial stability using an alternative measure of FinTech. In the previous sections, we used FinTech firms closely related to the banking industry as our primary FinTech measure. In this section, we expand our measure by accounting for all FinTech firms to understand their overall impact, the results of which are reported in Table Finally, using alternative estimation techniques such as the dynamic panel GMM, we estimate the models and report the results in Table To account for the rapid changes in FinTech development in the post-2010 period, we examine FinTech firms’ impact on bank financial stability in this period. The empirical results are presented in Table Overall, our empirical results show that Fintech companies have a significant positive impact on the financial stability of banks in Malaysia. Our study further demonstrates that Fintech firms exhibit a more positive influence on the financial stability of small banks, low corporate governance banks, and Islamic banks. Given these findings, we offer several policy implications that are crucial for further strengthening the financial stability of banks in Malaysia. Specifically, we argue that large banks are not as quick as small banks in implementing appropriate actions to improve their financial stability. Hence, we suggest that large banks continue to protect their customer base by offering competitive and innovative services to meet the expectations of customers in the market. This recommendation is consistent with the findings of Chen et al. , who argFurther, we suggest that Malaysian banks’ financial stability, in general, has increased with the presence of FinTech companies; however, it varies considerably across banks. FinTech firms continue to grow due to the changing circumstances in the economy , technological innovations, and convenient and low-cost financial services. Thus, banks must be ready to protect their customer base and market power by providing competitive and attractive services to the customers. This study’s main takeaway is that FinTech companies’ presence does not negatively influence banks’ financial stability, creates healthy market competition, and may improve banking services for unbanked customers. Finally, we suggest that future studies may try investigating COVID-19’s impact on banks’ financial stability as well as FinTech firms’ growth. Future studies may also examine FinTech firms’ impact on both banking and non-banking firms, thereby further enhancing our understanding of FinTech and its wider impact.


SM data to promulgate stereotypes about AIAN individuals and, second, in partnership with collaborators from Cherokee Nation, we provide five recommendations for utilizing data from publicly available datasets to advance health research in AIAN populations. Specifically, we argue for the consideration of (1) the heterogeneity of the communities represented, (2) the importance of focusing on AIAN health and well-being, (3) engagement of relevant communities and AIAN community leaders, (4) consideration of historical and ongoing injustices, and (5) engagement with AIAN regulatory agencies or review boards. These recommendations are founded on principles from broader indigenous research efforts emphasizing community-engaged research and principles of Indigenous Data Sovereignty and Governance.American Indian and Alaska Native (AIAN) populations have suffered a history of exploitation and abuse within the context of mental health research and related fields. This history is rooted in assimilation policies, historical trauma, and cultural loss, and is promulgated through discrimination and disregard for traditional culture and community knowledge. In recognition of this history, it is imperative for researchers to utilize culturally sensitive approaches that consider the context of tribal communities to better address mental health issues for AIAN individuals. The public availability of data from large-scale studies creates both opportunities and challenges when studying mental health within AIAN populations. This manuscript has two goals; first, showcase an example of problematic use of Adolescent Brain Cognitive Development (ABCD) Study Health and mental health research conducted among American Indian and Alaska Native (AIAN)Potential research harms to AIAN communities are both tangible  and dignitary  and require thoughtful consideration of researchers through community engagement . By not As AIAN communities experience a disproportionate burden of mental health concerns, research has focused primarily on understanding symptom development, maintenance, and treatment. However, certain risk factors for poor mental health,  are of particular relevance for AIAN communities and underscore the need for tailored research methodologies placed in appropriate theoretical and empirically-validated frameworks –15. ImplSM, launched by the National Institute on Drug Abuse (NIDA) in partnership with other intramural and extramural research programs [® is the largest study of human brain development in the United States, aiming to understand neurocognitive development and associated factors with a focus on development of substance misuse and related conditions. The study includes multi-method  longitudinal data to track biological and behavioral development through adolescence into early adulthood [n = 11,880 at baseline) and intent to reflect the sociodemographic characteristics of the USA [The increasing number of large-scale studies with publicly available datasets requires that the National Institutes of Health (NIH) emphasize data sharing policies that enforce appropriate and culturally informed research practices, such as the draft policy on responsible management and sharing of AIAN participant data . A promiprograms . The ABCdulthood . Given t the USA , AIAN yo® data could serve as a substantial resource for AIAN communities to leverage in efforts to promote mental health and wellness of their youth. However, substantial AIAN participation in the ABCD Study® also raises the possibility for researchers to conduct inappropriate research with adverse effects for AIAN communities. If problematic research practices are employed, these data represent a large source of threat to AIAN communities. Thus, it is imperative that researchers using ABCD data (or other publicly available datasets) to examine questions relevant to AIAN communities conduct work that ensures (1) high-level scientific rigor expected when working with large-scale studies [®) have a responsibility to acknowledge and critique improperly conducted research to mitigate irresponsible and harmful research practices. This responsibility also is carried by independent research reviewers, editors, and journal publishers who evaluate and disseminate such works [With thoughtful community collaboration, the ABCD Study studies  and (2)  studies . Moreovech works .® can be used improperly in such a way as to potentially do harm. Second, in partnership with expert behavioral health researchers from the Cherokee Nation (CN), we offer recommendations for engaging in research with the data from large-scale publicly available data including the ABCD Study®. The Cherokee Nation provides IRB oversight for the ABCD Study® site at the Laureate Institute for Brain Research (LIBR) in Tulsa, Oklahoma and representatives from CN sit on the community advisory board for the LIBR site. This partnership imparts a unique responsibility and opportunity to provide the commentary and recommendations contained herein. Although the recommendations are focused on the ABCD Study®, they are also relevant for other emerging population-based datasets available (or soon to be available) for analysis such as “All of Us” Study [The goals of the current paper are two-fold. First, we provide a critical response to Assari , as an es” Study  and the s” Study .® data, suggesting key protective factors against high body mass index (BMI) in childhood were not protective among AIAN populations. The paper [Recently Assari , publishhe paper  serves a® was designed with specific retention efforts in mind [® was not initially designed to address questions relevant only to AIAN populations, the published design elements of the ABCD study facilitate an articulation of how these considerations may impact conclusions drawn from analysis of the data. Such considerations are absent or minimally alluded to in the limitations reported in the Assari paper [The first and foremost concern is the dubious validity of the presented rationale. Specifically, the foundational theoretical model – previously developed by the author – suggests that minoritized communities experience reduced health benefits from protective factors. This model was not developed with AIAN communities, nor has the conceptual framework been validated with these populations. Moreover, the study did not take into account the large body of extant research on obesity in AIANs . It has  in mind  and samp in mind . Althougri paper .Another important construct missing from the presented rationale  is food Considering the broad health disparities among AIAN populations, properly conducted research is critical to supporting better health outcomes in these communities. Indeed, many efforts are underway which employ multi-level, community-engaged methodologies to advance health in AIAN populations  . Researc®  [n = 4 (post-graduate degree) and n = 8 (family income ≥ $100 K) compared to n = 3930 and n = 4842 in the NHW group, respectively. Mean estimates derived from such small samples are very likely to be unreliable and not representative of the broader AIAN population. Furthermore, reporting such small cell sizes increases the potential risks to participant confidentiality, particularly in populations that are more readily identifiable from study variables [The work in question also employs statistical analyses of questionable validity, thus precluding the ability to draw meaningful conclusions. Specifically, the analysis sample is extremely imbalanced across comparison groups, the non-Hispanic White (NHW) group having 8517 participants compared to 63 in the AIAN, Native Hawaiian, and Pacific Islander (NHPI) group. It is unclear why the AIAN/NHPI group is so small relative to what would be expected given baseline demographics reported for the ABCD Studyviduals)  as the rviduals)  lacks neariables .n = 63) to be representative of this collection of culturally, geographically, politically, and linguistically distinct populations. Additionally, the interpretation of statistical associations as “causal effects” is inappropriate for cross-sectional observational data [There is substantial cultural, historical, and ethnic heterogeneity within the AIAN/NHPI group that is ignored in the work in question. There are 574 federally recognized AIAN nations in the United States without accounting for heterogeneity among NHPI peoples. Similarities and dis-similarities across these populations will depend on the research question and relevant policies for population(s) of interest. Collapsing AIAN/NHPI participants into one collective group has been referred to as “ethnic gloss”, and creates substantial problems for external validity and replicability of findings , 37. It nal data . The effnal data . In shornal data  are unwaRecommendations for conducting research with AIANs call for substantial involvement of community members in the research process due to rich diversity of AIAN populations as well as prior research endeavors that have harmed these communities , 38–41. Research conducted to address health disparities in AIAN communities – including those concerning metabolic diseases – are laudable. However, such work should be appropriately culturally sensitive and consider the perspectives of the communities involved to increase the validity of the findings. The potential for promulgating harmful stereotypes and public policy decisions based on dubious research underscores the general imperative of rigorous research practices in large-scale data projects. Best practice in research among AIAN communities calls for significant community involvement  even in the case of otherwise scientifically and quantitatively rigorous methodologies. The paper in question  suffers ® engaged with CN-IRB to provide oversight on recruitment and study protocols when initiating the study. Representatives from CN sit on the advisory board for the study and CN-IRB provide ongoing oversight of the protocol, modifications, and dissemination  from LIBR researchers. In collaboration with CN partners, we provide a set of brief recommendations for conducting analysis on large-scale, publicly available datasets  to facilitate research that prioritizes well-being of AIAN communities. Additionally, these recommendations are informed by on-going work of researchers conducting community engaged research with AIAN communities in many different settings [generators  and users  of large-scale publicly available AIAN participant data carry a responsibility to apply the following recommendations to their work.Consider heterogeneity of large-scale AIAN samples: A key consideration in examining AIAN samples from large-scale publicly available data is the heterogeneity across these communities. The AIAN population comprises 574 federally recognized sovereign nations in the United States, each representing distinct cultural groups with unique and overlapping history, language, traditions, spiritual practices, and foods. This complexity is compounded by modern distributions of these populations across rural and urban settings as well as living on or off reservations. Understanding the potential influence of such heterogeneity on research findings is important to avoid overgeneralization and ethnic gloss [users should carefully consider in their analysis plans and transparently discuss within their publications the impact of such heterogeneity on their research questions. Notably, many large-scale studies do not collect tribal affiliation information to respect tribal autonomy as collecting tribal affiliation requires additional responsibilities . The strength of large-scale data can be leveraged only insofar as researchers consider the degree to which variables of interest are shared  or distinct  across these heterogeneous communities. Thus, generators of these datasets can ensure adequate data collection to characterize the potential heterogeneity with respect to such variables without the need to collect tribal affiliation.ic gloss . With rePrioritize advancement of health and wellbeing in AIAN communities: The foundational motivation for research should be a beneficial impact on communities the research is meant to serve. Often the nature of science is aimed at potential long-term benefits through incremental research endeavors. Although this is laudable and a necessary feature of much scientific work, research can be designed and executed with an emphasis on immediate direct and indirect benefits to communities. Many AIAN communities that partner in research are overburdened and under resourced with respect to mental health care and related services. Carelessly designed research may exacerbate such difficulties [generators, this could include dedicated funding opportunities, trainings, and data analytic infrastructure made available to AIAN tribal entities. Moreover, users analyzing extant publicly available data also should prioritize advancement of health and well-being of AIAN communities. Specifically, it is important that users employing publicly available AIAN participant data consider contextual factors, such as community resource burden, how to provide benefit to community partners  through the research process surrounding their research questions. Furthermore, users should conduct analyses with an understanding of how analyses may advance or hinder public policy and public perceptions relevant for AIAN communities. This concern is underscored by the potential policy impact of large-scale open access datasets [iculties . In concdatasets  and neurdatasets . Prior rdatasets –50. Thusdatasets , 51, 52 datasets , 54.Facilitate community engagement at each stage of the research process: CBPR [ss: CBPR  approachgenerators and users of the responsibility to make good faith efforts to establish appropriate research protocols including design, recruitment, as well as data collection, storage and sharing agreements. Such efforts of the generators may include (1) requests for information (RFI) from funding agencies sent directly to tribal institutions, (2) public comment periods, and (3) convening national work groups of AIAN community representatives and their research collaborators. Results of such approaches can then be made publicly available in study descriptions. Generators can also consider creating a protected access repository of relevant research findings, requiring users to submit lay summaries of their findings in addition to preprints and relevant analytic scripts or methods associated with their work that AIAN communities and agencies would have access to. Furthermore, generators could require that data use agreement applications from users specify plans for community engagement and/or include a letter of support from community partners to access publicly available AIAN participant data. Thus, users should engage with community partners to collaborate on projects that involve analysis of AIAN participant data from largescale publicly available data at the earliest stage of the process that the user is involved in and find avenues for dissemination of the research back to the community. As a first step in this direction, researchers should seek out collaborators who have expertise in community engaged research with AIAN populations.The scope and scale of national, publicly available studies may complicate and even preclude deep engagement with all potential AIAN communities of interest. However, this does not absolve Consider the impact of social injustices on study variables: Colonization has led to widespread historical trauma and loss for AIAN people [users should examine the influence of such variables on findings of interest. If unable to model such influences, it is incumbent on users to contextualize findings with this limitation. This is especially necessary in the examination of biological data within any particular racial group, as race is not a biological variable [generators of population level studies should employ sampling practices that reflect relative disease burden across demographic categories rather than census level population data. Generators of such studies should engage directly with AIAN communities to ensure the study addresses meaningful questions for these populations, assesses all relevant constructs, and uses appropriate measurements.N people –57. MoreN people  and discN people , 60. Thevariable , nor is variable . This isvariable  principlEngage with tribal research regulatory infrastructure: Although important, community engagement is insufficient to ensure ethical research practices [Users should engage with tribal regulatory bodies for consultation and or oversight of analysis focused on AIAN participant data from large publicly available datasets. Prior work has put forth a framework and principles of ethical conduct of research with Indigenous communities [generators  to establish regulatory bodies comprising tribal representatives and experts in community engaged research in AIAN populations. These entities may be tasked with the oversight of design, implementation, data storage, and data sharing agreements of publicly available data. Such efforts should be informed by recommendations for Indigenous Data Governance (IDG) and Indigenous Data Sovereignty (IDS) [ractices . Researcractices  thus, remunities  which maty (IDS)  consistety (IDS) . Consistty (IDS) , 69.In addition to the recommendations for researchers, self-determination of AIAN communities within publicly accessible datasets would be greatly enhanced by community lead research calls published by tribes themselves. Specifically, tribal entities interested in leveraging publicly available data could solicit proposals from potential collaborators and organizations with research infrastructures that may enhance the ability of communities to conduct such work. Lastly, funding agencies could partner with tribes to produce funding announcements commensurate with research priorities derived from the community directly.Recommendations from CBPR include engaging with AIAN community members in the generation of research questions at the early stage of study design , 9. Thissettings , 43. ImpResearch conducted among AIAN populations has often resulted in harm or lack of benefit to communities in which the research was conducted. The potential for harm or lack of benefit is amplified in large-scale studies with publicly available data. Recent work is illustrative of this risk . Thus, i


Migraine is a prevalent disorder and a cause of high disability, influenced by modifiable and non-modifiable risk factors. Comorbid and psychiatric illnesses are prevalent in migraine patients and should be considered when choosing preventive drugs. There have been unforeseen problems with the use of preventive treatment of migraine with oral drugs, mainly due to side-effects that cannot be tolerated and lack of efficacy, leading to high discontinuation rates. Anti-CGRP monoclonal antibodies (mAbs) have shown better tolerance profiles, based on the low dropout rates in clinical trials due to adverse events. First-line therapy is a term most expressed in some medical specialties that adopt standardized protocol treatments and may not be suitable for treating migraine. Regarding efficacy, mAbs don’t seem to perform much better than the current prophylactic oral drugs in reduction of monthly migraine days compared to placebo. Monoclonal antibodies against CGRP pathway have been prescribed recently, which raises some concern about their safety in the long term. Only side effects observation will confirm whether CGRP blockade causes susceptibility to severe side-effects, at least to specific subpopulations. CGRP may play a role in regulating uteroplacental blood flow and myometrial and uterine relaxation, as well as blood pressure control, raising the suspicion that its blockade could cause complications during pregnancy. Recent guidelines retain the recommendation of starting preventive treatment of migraine with oral drugs. Both the fact that it is new and costs are the reason why guidelines recommend the prescription of mAbs only after failure of at least two oral drugs. Migraine is a common neurologic disorder and the second cause of disability worldwide, leading to more disability than all other neurologic diseases,Migraine manifests as a continuum, from episodic to chronic migraine“First line therapy” is a current definition of the best treatment for a given disease and clearly implies better outcomes and lower adverse event rates when compared to other available treatments-Regarding efficacy, mAbs don’t seem to perform much better than the current prophylactic oral drugs  in reduction of monthly migraine days compared to placebo. Long-term safety is also relevant when selecting some first line therapy. One of the greatest advantages of mAbs is the low adverse effect rates, (most of them of mild intensity), leading to better adherence to treatmentThe function of CGRP in both peripheral and enteric nervous systems is well established - it is involved not only in migraine, but also other physiological processes and in homeostatic responses during pathophysiological conditions. CGRP is present in nerve fibers that innervate blood vessels and the heart, acts in the regulation of blood pressure and may have a role in maintenance of cardiovascular homeostasis during ischemic events and tissue remodeling in pulmonary hypertension. CGRP is also involved in inflammatory processes and facilitation of wound healing. In addition, it is found in the anterior pituitary, possibly influencing the regulation of hypothalamus-pituitary tract functions. To date, there are no reports on safety issues related to these functions, and only observation of side effects will confirm whether or not CGRP blockade causes susceptibility to severe side-effects, at least to specific subpopulationsMigraine mostly affects women during their fertile years. There are no controlled studies of mAbs involving pregnant or lactating women. A limited number of safety reports have indicated no specific maternal toxicities, major birth defects or increased reporting of spontaneous abortions when mAbs is administered during pregnancy, exposure shortly prior to pregnancy or breast-feedingFinally, recent guidelines retain the recommendation for starting the preventive treatment of migraine with oral drugs. The American Headache Society Consensus Statement criteria for initiating mAbs arePrescribed by a licensed clinician;Patient is at least 18 years of age;Diagnosis of ICHD-3 migraine with or without aura (4-7 monthly migraine days) orDiagnosis of ICHD-3 migraine with or without aura (8-14 monthly migraine days) orDiagnosis of ICHD-3 chronic migraine .And inability to tolerate (due to side effects) or inadequate response to an 8-week trial of two or more of the following:TopiramateDivalproex sodium/valproate sodiumBeta-blocker: metoprolol, propranolol, timolol, atenolol, nadololTricyclic antidepressant: amitriptyline, nortriptylineSerotonin-norepinephrine reuptake inhibitor: venlafaxine, duloxetineOther Level A or B treatments (established efficacy or probably effective) according to AAN scheme for classification of evidence.Inability to tolerate or inadequate response to a minimum of two quarterly injections (6 months) of onabotulinumtoxinA (only for chronic migraine).The recommendations of the European Headache Federation published in 2019 about the use of mAbs in subjects with migraine areWhen should treatment with anti-CGRP monoclonal antibodies be offered to patients with migraine?In patients with episodic or chronic migraine who have failed at least two of the available medical treatments or who cannot use other preventive treatments because of comorbidities, side effects or poor compliance, we suggest the use of erenumab, fremanezumab, or galcanezumab.The Consensus of the Brazilian Headache Society on the treatment of chronic migraine has not incorporated guidelines on CGRP mAbs, because they were not yet available in Brazil in 2019Costs of CGRP mAbs are not entirely known but, presumably, these are higher than oral treatments that are available. Further studies could provide information about the economic impact of mAbs, taking into account direct and indirect costs related to migraineIn conclusion, undoubtedly CGRP monoclonal antibodies are a breakthrough in migraine preventive treatment; their specific mode of action, rapid response and safety profile to date show they are innovative and could soon be the first line therapy for episodic and chronic headache. Nevertheless, the concept of “first line treatment” is not suitable for migraine, since doctors should practice clinical examination skills to make a proper diagnosis and “a tailor-made” treatment, taking into account age, comorbidities, pregnancy risk, previous treatments, etc. Medical and psychiatric comorbid conditions are addressed by current oral preventive drugs, but not by mAbs. As with all new therapies, mAbs have not been subject to the test of time regarding long-term side effects. Subpopulations could still be at risk while pregnant or breast-feeding women, alcohol or drug abusers, people with cardio and cerebrovascular diseases as well as severe mental disorders are the most relevant of these