Nocturia and frailty in older adults: a scoping review.

BACKGROUND: More than one in two older people wake up several times a night to urinate. Far from being a minor inconvenience, nocturia is associated with poor health outcomes. Given the importance of sleep as a foundation for resilience and healthy ageing, nocturia may promote the development of frailty, which is inextricably linked to physical decline, disability, and mortality. The aim of this scoping review was to collate published evidence on the relationship between nocturia and frailty, using the methodological framework of Arksey and O'Malley, together with the Joanna Briggs Institute methodology as guidance (OSF registration: osf.io/d5ct7). METHODS: Relevant publications were retrieved via PubMed, Embase, the Cochrane Library and Google Scholar. The Rayyan tool was used to facilitate the screening process. Data were extracted by two independent reviewers. 250 publications were initially identified, of which 87 met the eligibility criteria. RESULTS: Most of the evidence came from cross-sectional studies, most of which had been published within the last 5 years. The researchers were diverse, with 27% having a geriatric background. Only few publications established a clear association between nocturia and frailty. Other topics included: the association between nocturia and poor sleep quality and duration; the association between sleep and frailty; the association between frailty, multimorbidity, and age-related changes in the lower urinary tract. CONCLUSION: The findings emphasize the increasing interest and interdisciplinary nature of research into the relationship between frailty, nocturia, lower urinary tract symptoms, and sleep disturbances. Further research is required to enhance understanding, establish causality, and identify potential therapeutic approaches.

Relationship between Hypoxia and Hypercapnia Tolerance and Life Expectancy.

The review discusses the potential relationship between hypoxia resistance and longevity, the influence of carbon dioxide on the mechanisms of aging of the mammalian organism, and intermittent hypercapnic-hypoxic effects on the signaling pathways of aging mechanisms. In the article, we focused on the potential mechanisms of the gero-protective efficacy of carbon dioxide when combined with hypoxia. The review summarizes the possible influence of intermittent hypoxia and hypercapnia on aging processes in the nervous system. We considered the perspective variants of the application of hypercapnic-hypoxic influences for achieving active longevity and the prospects for the possibilities of developing hypercapnic-hypoxic training methods.

Impact of NLRP3 Depletion on Aging-Related Metaflammation, Cognitive Function, and Social Behavior in Mice.

Immunosenescence and chronic inflammation associated with old age accompany brain aging and the loss of complex behaviors. Neuroinflammation in the hippocampus plays a pivotal role in the development of cognitive impairment and anxiety. However, the underlying mechanisms have not been fully explained. In this study, we aimed to investigate the disruption of insulin signaling and the mechanisms underlying metabolic inflammation ("metaflammation") in the brains of wild-type (WT) and NLRP3 knockout (KO) mice of different ages. We found a significant upregulation of the NLRP3 inflammasome in the hippocampus during aging, leading to an increase in the expression of phosphorylated metaflammation proteinases and inflammatory markers, along with an increase in the number of senescent cells. Additionally, metaflammation causes anxiety and impairs social preference behavior in aged mice. On the other hand, deletion of NLRP3 improves some behavioral and biochemical characteristics associated with aging, such as signal memory, neuroinflammation, and metabolic inflammation, but not anxious behavior. These results are associated with reduced IL-18 signaling and the PKR/IKKß/IRS1 pathway as well as the SASP phenotype. In NLRP3 gene deletion conditions, PKR is down-regulated. Therefore, it is likely that slowing aging through various NLRP3 inhibition mechanisms will lessen the corresponding cognitive decline with aging. Thus, the genetic knockout of the NLRP3 inflammasome can be seen as a new therapeutic strategy for slowing down central nervous system (CNS) aging.

Expression of NLRP3 Inflammasomes in Neurogenic Niche Contributes to the Effect of Spatial Learning in Physiological Conditions but Not in Alzheimer's Type Neurodegeneration.

A common feature of neurodegenerative disorders, in particular Alzheimer's disease (AD), is a chronic neuroinflammation associated with aberrant neuroplasticity. Development of neuroinflammation affects efficacy of stem and progenitor cells proliferation, differentiation, migration, and integration of newborn cells into neural circuitry. However, precise mechanisms of neurogenesis alterations in neuroinflammation are not clear yet. It is well established that expression of NLRP3 inflammasomes in glial cells marks neuroinflammatory events, but less is known about contribution of NLRP3 to deregulation of neurogenesis within neurogenic niches and whether neural stem cells (NSCs), neural progenitor cells (NPCs) or immature neuroblasts may express inflammasomes in (patho)physiological conditions. Thus, we studied alterations of neurogenesis in rats with the AD model (intra-hippocampal injection of Aß1-42). We found that in Aß-affected brain, number of CD133+ cells was elevated after spatial training in the Morris water maze. The number of PSA-NCAM+ neuroblasts diminished by Aß injection was completely restored by subsequent spatial learning. Spatial training leads to elevated expression of NLRP3 inflammasomes in the SGZ (subgranular zones): CD133+ and PSA-NCAM+ cells started to express NLRP3 in sham-operated, but not AD rats. Taken together, our data suggest that expression of NLRP3 inflammasomes in CD133+ and PSA-NCAM+ cells may contribute to stimulation of adult neurogenesis in physiological conditions, whereas Alzheimer's type neurodegeneration abolishes stimuli-induced overexpression of NLRP3 within the SGZ neurogenic niche.

NLRP3 Inflammasome Blocking as a Potential Treatment of Central Insulin Resistance in Early-Stage Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder. In recent years, attention of researchers has increasingly been focused on studying the role of brain insulin resistance (BIR) in the AD pathogenesis. Neuroinflammation makes a significant contribution to the BIR due to the activation of NLRP3 inflammasome. This study was devoted to the understanding of the potential therapeutic roles of the NLRP3 inflammasome in neurodegeneration occurring concomitant with BIR and its contribution to the progression of emotional disorders. METHODS: To test the impact of innate immune signaling on the changes induced by Aß1-42 injection, we analyzed animals carrying a genetic deletion of the Nlrp3 gene. Thus, we studied the role of NLRP3 inflammasomes in health and neurodegeneration in maintaining brain insulin signaling using behavioral, electrophysiological approaches, immunohistochemistry, ELISA and real-time PCR. RESULTS: We revealed that NLRP3 inflammasomes are required for insulin-dependent glucose transport in the brain and memory consolidation. Conclusions NLRP3 knockout protects mice against the development of BIR: Taken together, our data reveal the protective role of Nlrp3 deletion in the regulation of fear memory and the development of Aß-induced insulin resistance, providing a novel target for the clinical treatment of this disorder.

Blood-Brain Barrier and Neurovascular Unit In Vitro Models for Studying Mitochondria-Driven Molecular Mechanisms of Neurodegeneration.

Pathophysiology of chronic neurodegeneration is mainly based on complex mechanisms related to aberrant signal transduction, excitation/inhibition imbalance, excitotoxicity, synaptic dysfunction, oxidative stress, proteotoxicity and protein misfolding, local insulin resistance and metabolic dysfunction, excessive cell death, development of glia-supported neuroinflammation, and failure of neurogenesis. These mechanisms tightly associate with dramatic alterations in the structure and activity of the neurovascular unit (NVU) and the blood-brain barrier (BBB). NVU is an ensemble of brain cells (brain microvessel endothelial cells (BMECs), astrocytes, pericytes, neurons, and microglia) serving for the adjustment of cell-to-cell interactions, metabolic coupling, local microcirculation, and neuronal excitability to the actual needs of the brain. The part of the NVU known as a BBB controls selective access of endogenous and exogenous molecules to the brain tissue and efflux of metabolites to the blood, thereby providing maintenance of brain chemical homeostasis critical for efficient signal transduction and brain plasticity. In Alzheimer's disease, mitochondria are the target organelles for amyloid-induced neurodegeneration and alterations in NVU metabolic coupling or BBB breakdown. In this review we discuss understandings on mitochondria-driven NVU and BBB dysfunction, and how it might be studied in current and prospective NVU/BBB in vitro models for finding new approaches for the efficient pharmacotherapy of Alzheimer's disease.

Pericytes in Alzheimer's Disease: Novel Clues to Cerebral Amyloid Angiopathy Pathogenesis.

Pericytes in the central nervous system attract growing attention of neurobiologists because of obvious opportunities to use them as target cells in numerous brain diseases. Functional activity of pericytes includes control of integrity of the endothelial cell layer, regeneration of vascular cells, and regulation of microcirculation. Pericytes are well integrated in the so-called neurovascular unit (NVU) serving as a platform for effective communications of neurons, astrocytes, endothelial cells, and pericytes. Contribution of pericytes to the establishment and maintaining the structural and functional integrity of blood-brain barrier is confirmed in numerous experimental and clinical studies. The review covers current understandings on the role of pericytes in molecular pathogenesis of NVU/BBB dysfunction in Alzheimer's disease with the special focus on the development of cerebral amyloid angiopathy, deregulation of cerebral angiogenesis, and progression of BBB breakdown seen in Alzheimer's type neurodegeneration.

Establishment of neurogenic microenvironment in the neurovascular unit: the connexin 43 story.

Connexins (Cx) play an important role in the coordination of intercellular communication, and autocrine and paracrine regulation of cells within the neurovascular unit (NVU). Gap junctional mechanisms control proliferation and differentiation processes underlying neurogenesis and angiogenesis in the brain. Cx43 possesses some unique properties [the ability to form either intercellular channels permeable for regulatory molecules and ions or hemichannels open to the extracellular space to provide release of cell metabolites; functional coupling with nicotinamide adenine dinucleotide (NAD+)-consuming and NAD+-dependent enzymatic processes] which may be of great importance for the fate of the stem cells. Dynamic changes in Cx43 expression are associated with different stages of brain cells development either at embryonic or adult periods of ontogenesis. This review summarizes recent data on Cx43-controlled neurogenesis in the context of NVU development and functioning. Understanding the molecular mechanisms of gap junctional intercellular communication will support translational studies focused on the development of regeneration-based approaches for the therapy of central nervous system pathology.

Frailty after COVID-19: The wave after?

The COVID-19 pandemic poses an ongoing public health challenge, with a focus on older adults. Given the large number of older persons who have recovered from COVID-19 and reports of long-lasting sequelae, there is reasonable concern that the COVID-19 pandemic may lead to a long-term deterioration in the health of older adults, i.e., a potential "wave of frailty." Therefore, it is critical to better understand the circumstances surrounding the development of frailty as a result of COVID-19, as well as the underlying mechanisms and factors contributing to this development. We conducted a narrative review of the most relevant articles published on the association between COVID-19 and frailty through January 2023. Although few studies to date have addressed the effects of COVID-19 on the onset and progression of frailty, the available data suggest that there is indeed an increase in frailty in the elderly as a result of COVID-19. Regarding the underlying mechanisms, a multicausal genesis can be assumed, involving both direct viral effects and indirect effects, particularly from the imposed lockdowns with devastating consequences for the elderly: decreased physical activity, altered diet, sarcopenia, fatigue, social isolation, neurological problems, inflammation, and cardiovascular morbidity are among the possible mediators. Since the COVID-19 pandemic is leading to an increase in frailty in the elderly, there is an urgent need to raise awareness of this still little-known problem of potentially great public health importance and to find appropriate prevention and treatment measures.

Early Life Stress and Metabolic Plasticity of Brain Cells: Impact on Neurogenesis and Angiogenesis.

Early life stress (ELS) causes long-lasting changes in brain plasticity induced by the exposure to stress factors acting prenatally or in the early postnatal ontogenesis due to hyperactivation of hypothalamic-pituitary-adrenal axis and sympathetic nervous system, development of neuroinflammation, aberrant neurogenesis and angiogenesis, and significant alterations in brain metabolism that lead to neurological deficits and higher susceptibility to development of brain disorders later in the life. As a key component of complex pathogenesis, ELS-mediated changes in brain metabolism associate with development of mitochondrial dysfunction, loss of appropriate mitochondria quality control and mitochondrial dynamics, deregulation of metabolic reprogramming. These mechanisms are particularly critical for maintaining the pool and development of brain cells within neurogenic and angiogenic niches. In this review, we focus on brain mitochondria and energy metabolism related to tightly coupled neurogenic and angiogenic events in healthy and ELS-affected brain, and new opportunities to develop efficient therapeutic strategies aimed to restore brain metabolism and reduce ELS-induced impairments of brain plasticity.

NLRP3 deficiency-induced hippocampal dysfunction and anxiety-like behavior in mice.

Neuroinflammation has been classified as a trigger of behavioral alterations and cognitive impairments in many neurological conditions, including Alzheimer's disease, major depression, anxiety and others. Regardless of the cause of neuroinflammation, key molecules, which sense neuropathological conditions, are intracellular multiprotein signaling inflammasomes. Increasing evidence shows that the inflammatory response, mediated by activated nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasomes, is associated with the onset and progression of a wide range of diseases of the CNS. However, whether the NLRP3 inflammasome in the CNS is involved in the learning, development of anxiety and adult neurogenesis remains elusive. Therefore, the present study was designed to assess NLRP3 inflammasome contribution in anxiety and reveal its potential involvement in the experimental acquisition of fear responses and hippocampal neurogenesis. Behavioral, immunohistochemical and electrophysiological alterations were measured to evaluate role of neuroinflammation in the limbic system of mice. In this study, we describe interrelated neurophysiological mechanisms, which culminate in absence of NLRP3 inflammasome in young 4 months mice. These include the following: anxious behavior and deterioration in learning and memory of fear conditioning; impairment of adult neurogenesis; reduction and altered morphology of astrocytes in the brain; hyperexcitability in basolateral amygdala (BLA); impaired activation in axons of pyramidal cells of CA1 hippocampal zone in NLRP3 KO mice particularly via the Schaffer collateral pathway; and impaired synaptic transduction in pyramidal cells mediated by an embarrassment of neurotransmitter release from presynaptic site in CA3 hippocampal zone. The present study has demonstrated the novel findings that basal level of NLRP3 inflammasome in the brain of young mice is required for conditioning-induced plasticity in the ventral hippocampus and the basolateral amygdala. The deletion of NLRP3 impair synaptic transduction and caused anxiety-like behavior and labored fear learning, suggesting that low grade inflammation, mediated by NLRP3 expression, play a key role in memory consolidation.

Inflamm-Aging and Brain Insulin Resistance: New Insights and Role of Life-style Strategies on Cognitive and Social Determinants in Aging and Neurodegeneration.

Over the past decades, the human life span has dramatically increased, and therefore, a steady increase in diseases associated with age (such as Alzheimer's disease and Parkinson's disease) is expected. In these neurodegenerative diseases, there is a cognitive decline and memory loss, which accompany increased systemic inflammation, the inflamm-aging, and the insulin resistance. Despite numerous studies of age-related pathologies, data on the contribution of brain insulin resistance and innate immunity components to aging are insufficient. Recently, much research has been focused on the consequences of nutrients and adiposity- and nutrient-related signals in brain aging and cognitive decline. Moreover, given the role of metainflammation in neurodegeneration, lifestyle interventions such as calorie restriction may be an effective way to break the vicious cycle of metainflammation and have a role in social behavior. The various effects of calorie restriction on metainflammation, insulin resistance, and neurodegeneration have been described. Less attention has been paid to the social determinants of aging and the possible mechanism by which calorie restriction might influence social behavior. The purpose of this review is to discuss current knowledge in the interdisciplinary field of geroscience-immunosenescence, inflamm-aging, and metainflammation-which makes a significant contribution to aging. A substantial part of the review is devoted to frontiers in the brain insulin resistance in relation to neuroinflammation. In addition, we summarize new data on potential mechanisms of calorie restriction that influence as a lifestyle intervention on the social brain. This knowledge can be used to initiate successful aging and slow the onset of neurodegenerative diseases.

CD157 and Brain Immune System in (Patho)physiological Conditions: Focus on Brain Plasticity.

Ectoenzyme and receptor BST-1/CD157 has been considered as a key molecule involved in the regulation of functional activity of cells in various tissues and organs. It is commonly accepted that CD157 catalyzes NAD+ hydrolysis and acts as a component of integrin adhesion receptor complex. Such properties are important for the regulatory role of CD157 in neuronal and glial cells: in addition to recently discovered role in the regulation of emotions, motor functions, and social behavior, CD157 might serve as an important component of innate immune reactions in the central nervous system. Activation of innate immune system in the brain occurs in response to infectious agents as well as in brain injury and neurodegeneration. As an example, in microglial cells, association of CD157 with CD11b/CD18 complex drives reactive gliosis and neuroinflammation evident in brain ischemia, chronic neurodegeneration, and aging. There are various non-substrate ligands of CD157 belonging to the family of extracellular matrix proteins (fibronectin, collagen I, finbrinogen, and laminin) whose activity is required for controlling cell adhesion and migration. Therefore, CD157 could control structural and functional integrity of the blood-brain barrier and barriergenesis. On the other hand, contribution of CD157 to the regulation of brain development is rather possible since in the embryonic brain, CD157 expression is very high, whereas in the adult brain, CD157 is expressed on neural stem cells and, presumably, is involved in the neurogenesis. Besides, CD157 could mediate astrocytes' action on neural stem and progenitor cells within neurogenic niches. In this review we will summarize how CD157 may affect brain plasticity acting as a molecule at the crossroad of neurogenesis, cerebral angiogenesis, and immune regulation.

Excitation/inhibition imbalance and impaired neurogenesis in neurodevelopmental and neurodegenerative disorders.

The excitation/inhibition (E/I) balance controls the synaptic inputs to prevent the inappropriate responses of neurons to input strength, and is required to restore the initial pattern of network activity. Various neurotransmitters affect synaptic plasticity within neural networks via the modulation of neuronal E/I balance in the developing and adult brain. Less is known about the role of E/I balance in the control of the development of the neural stem and progenitor cells in the course of neurogenesis and gliogenesis. Recent findings suggest that neural stem and progenitor cells appear to be the target for the action of GABA within the neurogenic or oligovascular niches. The same might be true for the role of neuropeptides (i.e. oxytocin) in neurogenic niches. This review covers current understanding of the role of E/I balance in the regulation of neuroplasticity associated with social behavior in normal brain, and in neurodevelopmental and neurodegenerative diseases. Further studies are required to decipher the GABA-mediated regulation of postnatal neurogenesis and synaptic integration of newly-born neurons as a potential target for the treatment of brain diseases.

Vascular RAGE transports oxytocin into the brain to elicit its maternal bonding behaviour in mice.

Oxytocin sets the stage for childbirth by initiating uterine contractions, lactation and maternal bonding behaviours. Mice lacking secreted oxcytocin (Oxt -/-, Cd38 -/-) or its receptor (Oxtr -/-) fail to nurture. Normal maternal behaviour is restored by peripheral oxcytocin replacement in Oxt -/- and Cd38 -/-, but not Oxtr -/- mice, implying that circulating oxcytocin crosses the blood-brain barrier. Exogenous oxcytocin also has behavioural effects in humans. However, circulating polypeptides are typically excluded from the brain. We show that oxcytocin is transported into the brain by receptor for advanced glycation end-products (RAGE) on brain capillary endothelial cells. The increases in oxcytocin in the brain which follow exogenous administration are lost in Ager -/- male mice lacking RAGE, and behaviours characteristic to abnormalities in oxcytocin signalling are recapitulated in Ager -/- mice, including deficits in maternal bonding and hyperactivity. Our findings show that RAGE-mediated transport is critical to the behavioural actions of oxcytocin associated with parenting and social bonding.

Neurobiological Aspects of Face Recognition: The Role of Oxytocin.

Face recognition is an important index in the formation of social cognition and neurodevelopment in humans. Changes in face perception and memory are connected with altered sociability, which is a symptom of numerous brain conditions including autism spectrum disorder (ASD). Various brain regions and neuropeptides are implicated in face processing. The neuropeptide oxytocin (OT) plays an important role in various social behaviors, including face and emotion recognition. Nasal OT administration is a promising new therapy that can address social cognition deficits in individuals with ASD. New instrumental neurotechnologies enable the assessment of brain region activation during specific social tasks and therapies, and can characterize the involvement of genes and peptides in impaired neurodevelopment. The present review sought to discuss some of the mechanisms of the face distinguishing process, the ability of OT to modulate social cognition, as well as new perspectives and technologies for research and rehabilitation of face recognition.

Plasticity of Adipose Tissue-Derived Stem Cells and Regulation of Angiogenesis.

Adipose tissue is recognized as an important organ with metabolic, regulatory, and plastic roles. Adipose tissue-derived stem cells (ASCs) with self-renewal properties localize in the stromal vascular fraction (SVF) being present in a vascular niche, thereby, contributing to local regulation of angiogenesis and vessel remodeling. In the past decades, ASCs have attracted much attention from biologists and bioengineers, particularly, because of their multilineage differentiation potential, strong proliferation, and migration abilities in vitro and high resistance to oxidative stress and senescence. Current data suggest that the SVF serves as an important source of endothelial progenitors, endothelial cells, and pericytes, thereby, contributing to vessel remodeling and growth. In addition, ASCs demonstrate intriguing metabolic and interlineage plasticity, which makes them good candidates for creating regenerative therapeutic protocols, in vitro tissue models and microphysiological systems, and tissue-on-chip devices for diagnostic and regeneration-supporting purposes. This review covers recent achievements in understanding the metabolic activity within the SVF niches (lactate and NAD+ metabolism), which is critical for maintaining the pool of ASCs, and discloses their pro-angiogenic potential, particularly, in the complex therapy of cardiovascular and cerebrovascular diseases.

Oxytocin and excitation/inhibition balance in social recognition.

Social recognition is the sensitive domains of complex behavior critical for identification, interpretation and storage of socially meaningful information. Social recognition develops throughout childhood and adolescent, and is affected in a wide variety of psychiatric disorders. Recently, new data appeared on the molecular mechanisms of these processes, particularly, the excitatory-inhibitory (E/I) ratio which is modified during development, and then E/I balance is established in the adult brain. While E/I imbalance has been proposed as a mechanism for schizophrenia, it also seems to be the common mechanism in autism spectrum disorder (ASD). In addition, there is a strong suggestion that the oxytocinergic system is related to GABA-mediated E/I control in the context of brain socialization. In this review, we attempt to summarize the underpinning molecular mechanisms of E/I balance and its imbalance, and related biomarkers in the brain in healthiness and pathology. In addition, because there are increasing interest on oxytocin in the social neuroscience field, we will pay intensive attention to the role of oxytocin in maintaining E/I balance from the viewpoint of its effects on improving social impairment in psychiatric diseases, especially in ASD.

Designing in vitro Blood-Brain Barrier Models Reproducing Alterations in Brain Aging.

Blood-brain barrier (BBB) modeling in vitro is a huge area of research covering study of intercellular communications and development of BBB, establishment of specific properties that provide controlled permeability of the barrier. Current approaches in designing new BBB models include development of new (bio) scaffolds supporting barriergenesis/angiogenesis and BBB integrity; use of methods enabling modulation of BBB permeability; application of modern analytical techniques for screening the transfer of metabolites, bio-macromolecules, selected drug candidates and drug delivery systems; establishment of 3D models; application of microfluidic technologies; reconstruction of microphysiological systems with the barrier constituents. Acceptance of idea that BBB in vitro models should resemble real functional activity of the barrier in different periods of ontogenesis and in different (patho) physiological conditions leads to proposal that establishment of BBB in vitro model with alterations specific for aging brain is one of current challenges in neurosciences and bioengineering. Vascular dysfunction in the aging brain often associates with leaky BBB, alterations in perivascular microenvironment, neuroinflammation, perturbed neuronal and astroglial activity within the neurovascular unit, impairments in neurogenic niches where microvascular scaffold plays a key regulatory role. The review article is focused on aging-related alterations in BBB and current approaches to development of "aging" BBB models in vitro.

Blood-brain barrier-supported neurogenesis in healthy and diseased brain.

Adult neurogenesis is one of the most important mechanisms contributing to brain development, learning, and memory. Alterations in neurogenesis underlie a wide spectrum of brain diseases. Neurogenesis takes place in highly specialized neurogenic niches. The concept of neurogenic niches is becoming widely accepted due to growing evidence of the important role of the microenvironment established in the close vicinity to stem cells in order to provide adequate control of cell proliferation, differentiation, and apoptosis. Neurogenic niches represent the platform for tight integration of neurogenesis and angiogenesis supported by specific properties of cerebral microvessel endothelial cells contributing to establishment of partially compromised blood-brain barrier (BBB) for the adjustment of local conditions to the current metabolic needs of stem and progenitor cells. Here, we review up-to-date data on microvascular dynamics in activity-dependent neurogenesis, specific properties of BBB in neurogenic niches, endothelial-driven mechanisms of clonogenic activity, and future perspectives for reconstructing the neurogenic niches in vitro.