Advances in tissue engineering continue at a rapid pace and have provided novel methodologies and insights into normal cell and tissue homeostasis, disease pathogenesis, and new potential therapeutic strategies. The evolution of new techniques has particularly invigorated the field and span a range from novel organ and organoid technologies to increasingly sophisticated imaging modalities. This is particularly relevant for the field of lung biology and diseases as many lung diseases, including chronic obstructive pulmonary disease (COPD) and idiopathic fibrosis (IPF), among others, remain incurable with significant morbidity and mortality. Advances in lung regenerative medicine and engineering also offer new potential avenues for critical illnesses such as the acute respiratory distress syndrome (ARDS) which also continue to have significant morbidity and mortality. In this review, an overview of lung regenerative medicine with focus on current status of both structural and functional repair will be presented. This will serve as a platform for surveying innovative models and techniques for study, highlighting the need and timeliness for these approaches. As lung diseases and critical illnesses have diverse pathophysiologies and manifestations, multiple potential regenerative medicine-based therapeutic approaches are being investigated. For functional repair, ARDS and sepsis/septic shock offer paradigms for disease entities marked by acute high intensity inflammation and associated injuries. A range of pharmacologic agents including monoclonal antibodies, corticosteroids, non-steroidal anti-inflammatory agents have only modestly improved morbidity and mortality if at all and current strategies are primarily focused on supportive care . AcknowlOne new promising approach centers on cell-based therapies, predominantly utilizing mesenchymal stromal cells (MSCs) . MSCs arThere have been a large number of investigations of both systemic and direct airway administration of MSCs in a wide range of pre-clinical models of lung diseases and critical illnesses. These have been predominantly in rodents but also include large animal models such as sheep and also in explanted human lungs . In virtAspergillus in bronchoalveolar lavage samples from cystic fibrosis (CF) patients resulted in rapid MSC death, through actions of fungal gliotoxin on mitochondrial function . Increasing data demonstrates that these cells can structurally and potentially functional engraft in pre-clinical models. One important caveat for these studies to date is that injury to the airway or alveolar epithelium is a predicate for engraftment. One simplistic explanation is that the damage provides “room” and allows the engrafting cells to integrate into previously intact cell monolayers. How this might translate into clinical practice is unclear at present but this is an exciting area of research.Regenerative medicine and engineering also incorporates developing devices that can augment or replace failing organs. This is well evidenced by examples of hemodialysis for chronic renal failure and left ventricular assist devices for heart failure. Such devices can also be utilized as bridges until organ transplantation or to improve quality of life in patients who do not qualify for organ transplants.This is an area of necessary growth in respiratory medicine. Mechanical ventilators and other non-invasive ventilation approaches are powerful and well-established tools. However, for intractable respiratory failure, there are limited options. Extra-corporeal membrane oxygenation (ECMO) is an established tool for use in both neonatal and adult patients with intractable respiratory failure and has had increasing utilization in adult patients, particularly in the setting of the COVID-19 pandemic. However, ECMO is a complex approach requiring specialized centers and highly trained support personnel and is currently limited to relatively few tertiary care centers. Further ECMO can be complicated by clotting and bleeding in the circuitry as well as by infection. ECMO is also limited to short term use in intensive care settings; there are no current available device options for long term use in ambulatory patients on lung transplant waiting lists or who will not qualify for transplantation .There are several devices undergoing development and investigation by both academic and commercial entities . This inWhile lung transplantation is available and increasingly successful for patients with end-stage lung diseases, there remains a significant shortage of suitable donor organs. Lung transplantation is also complicated by still unacceptably high rates of both acute and chronic rejection. Accordingly, extensive effort has taken place over the past approximate 15 years to develop functional gas exchange tissue that can be implanted to replace defective lungs. This has led to development of a number of novel technical approaches that will be overviewed here.ex vivo  tissue culture, 3D culturing proves a platform for 3D tissue formation and growth, in many cases resulting in miniature versions of the target organ or tissue . These oin situ human lung environment (Organoid culture approaches also offer a powerful opportunity to study the influence of the surrounding ECM composition on cell behaviors. However, to date, most organoid culture work, including those utilizing lung cells, have primarily utilized Matrigel™, a non-relevant ECM derived from cancerous mouse tissue typically utilized for stem and cancer cell proliferation, and thus, an insufficient ECM to replicate the ironment . Howeverironment . As suchironment . Used inironment , this prironment . These a3D bioprinting has an increasingly important role in replacing damaged or diseased structural tissues with successful clinical applications in fields such as orthopedics and cosmetic repair. With respect to respiratory tissues, 3D-bioprinted tracheas and large airways have a growing potential clinical role . HoweverOther considerations include the bioink utilized in the printing process . Will itin vivo use, these have provided platforms that underlie other attempts at structural and functional repair.A number of powerful tools have developed and are still in evolution. While not geared towards eventual direct Organ-on-a-chip model systems have developed for a number of organs and tissues. The first descriptions of lung-on-a-chip models highlights the general approach: cells are grown on either one or both sides of a biomimetic semi-permeable membrane in a small enclosed chamber. A general approach is to have epithelial cells on one side and vascular endothelial cells on the other . MicroflCulturing thin sections of lung tissue has been an approach used for many years, primarily to assess actions of pharmacologic agents on airway smooth muscle contraction and relaxation [reviewed in ]. Howevein situ utilizing conventional fluoresence or confocal microscopy [reviewed in (Powerful advances in imaging and visualization techniques have provided further insights into processes involved in lung repair and regeneration [reviewed in ]. This iiewed in ]. In pariewed in . As indiLight sheet fluorescence microscopy is a technique that utilizes a plane of light to optically section and view tissues with subcellular resolution, essentially functioning as a non-destructive microtome [reviewed in ]. As tisin situ genomics and proteomics, along with advances in machine learning, single cell RNA sequencing and development of multi-omics atlases including gene and protein lung maps, are all tools that will help advance lung regenerative medicine and engineering (In parallel with imaging, increasing advances in “omics” technologies, including ineering .Techniques and approaches developed to study lung regenerative medicine and engineering offer promise to both better understand normal homeostasis and disease pathophysiologies as well as to devise new therapeutics. However, there remain multiple challenges and thus opportunities for up-and-coming lung biologists, biomedical engineers, and pulmonary and critical care physicians. The dictum that “today’s science fiction is tomorrow’s science” is particularly appropriate in this rapidly moving field.


However, primary hepatocytes rapidly dedifferentiate in conventional 2D in vitro platforms. Physiologically relevant or physiomimetic microsystems, are increasingly popular alternatives to traditional two-dimensional (2D) monocultures for in vitro studies. Physiomimetic systems reconstruct and incorporate elements of the native cellular microenvironment to improve biologic functionality in vitro. Multiple elements contribute to these models including ancillary tissue architecture, cell co-cultures, matrix proteins, chemical gradients and mechanical forces that contribute to increased viability, longevity and physiologic function for the tissue of interest. These microsystems are used in a wide variety of applications to study biological phenomena. Here, we explore the use of physiomimetic microsystems as tools for studying viral hepatitis infection in the liver and how the design of these platforms is tailored for enhanced investigation of the viral lifecycle when compared to conventional 2D cell culture models. Although liver-based physiomimetic microsystems are typically applied in the context of drug studies, the platforms developed for drug discovery purposes offer a solid foundation to support studies on viral hepatitis. Physiomimetic platforms may help prolong hepatocyte functionality in order to sustain chronic viral hepatitis infection in vitro for studying virus-host interactions for prolonged periods.Viral hepatitis is a leading cause of liver disease and mortality. Infection can occur acutely or chronically, but the mechanisms that govern the clearance of virus or lack thereof are poorly understood and merit further investigation. Though cures for viral hepatitis have been developed, they are expensive, not readily accessible in vulnerable populations and some patients may remain at an increased risk of developing hepatocellular carcinoma (HCC) even after viral clearance. To sustain infection  While most are quickly cleared after acute infection, hepatitis viruses B (HBV), C (HCV), D (HDV), and E (HEV) can all manifest as chronic infections . The mecin vitro models. Conventional two-dimensional (2D) in vitro models are excellent for generating high throughput data, but fail to provide cells with surroundings that mimics their native physiologic microenvironment. Because of this disconnect between in vitro culture and the in vivo microenvironment, primary hepatocytes, the gold standard of in vitro hepatocyte models, suffer from a loss of functionality after extended culture periods in vitro. This loss of functionality inhibits accurate long-term study of host responses to infection, which are instrumental for providing insight into how the transition from acute to chronic infection occurs. Furthermore, hepatoma cell lines and iPSC-derived hepatocyte-like cells (HLCs) do not provide a fully differentiated hepatocyte phenotype and it is difficult to draw relevant biologic conclusions from the observed cellular responses to infection using these models.One reason the transition from acute to chronic viral infection has not been properly investigated is a lack of sufficient in vitro models cannot replicate a systemic response to infection, but these animal models must be supplemented by human-specific in vitro models.Animal models are indeed capable of sustaining chronic viral infections and have been critical to our understanding of the viral hepatitis and its progression to liver disease. However, in the context of viral hepatitis, humans and chimpanzees are the only two known naturally hosts for HBV and HCV, which makes studying the virus in animal models incredibly challenging given the ethical concerns of non-human primate models. Non-human analogs of hepatitis viruses exist, but these are fundamentally different from HBV and HCV, and their naturals hosts are challenged with replicating a human’s immunological response . Even gein vitro models. The purpose of these microsystems is to capture elements of the native cellular microenvironment in vitro, in an effort to improve experimental results as compared to a conventional 2D plate format. In the liver specifically, much of the work in the development of physiologically relevant microsystems has been directed toward pharmacokinetic modeling and drug-induced liver injury. Nonetheless, many of the same principles used in these systems to improve cell functionality can be effectively utilized in modeling virus-host responses. In this review we investigate how physiologically relevant microsystems recapitulate liver physiology and also how this has led to advances in the study of viral hepatitis infection in vitro.In an effort to better supplement animal models, physiologically relevant microsystems are a potential solution to insufficient in vitro. We then examine different cell models for studies on viral hepatitis in vitro. Next, we provide an overview of a wide variety of practical designs of physiologically relevant microsystems that model the liver and discuss the applications of these models as tools to study virus-host interactions. Finally, we discuss some of the elements of physiologically relevant microsystems that enhance their ability to study viral hepatitis biology specifically, and how these systems are working toward better informing clinical predictions.We first review the physiology of the liver and the necessary components required for relevant studies of viral hepatitis infection in vitro. Overall, the hepatic lobule is the functional unit of the liver  each with several different genotypes. These viruses vary greatly; however, in this review, we focus on HBV and HCV due to their worldwide prevalence and strong link to significant morbidity and mortality.In vitro models to further characterize the progression of HBV infection from acute to chronic infection are desperately needed and also models that can be utilized to further develop effective curative regimens are highly sought after.HBV is a partially double-stranded DNA virus that produces high levels of viral antigens which enabled the development of a highly effective vaccines against the virus . AlthougHCV is a single-stranded positive-sense RNA virus that manifests chronically in 75–85% of those infected . HCV entin vitro. Primary hepatocytes, hepatoma cell lines and iPSC-derived hepatocytes have significant differences in various experimental characteristics; however, each model may be desirable for use depending on the specific experimental application.Choice of hepatocyte source has a substantial impact on the biologic validity of experimental results as well as on the efficiency of supporting infection in vitro liver models in general. They display a notable differentiated phenotype at the time of plating. In addition, they support both HBV and HCV infection; unfortunately, their potent innate antiviral response is difficult to circumvent rendering this model unsuitable for the production of high titer virus stocks. In concordance with this, it is more difficult to establish long-term infections in these cells without artificially downregulating the antiviral response. Additionally, PHHs are a relatively rare cell source given that they come directly from patients or fetal tissue and they do not naturally divide once cultured in vitro. Inherent interpatient variability also drives the need to source cells from multiple donors to achieve more reproducible results (in vivo which has rendered these cells somewhat easier to source (Primary human hepatocytes (PHHs) are the gold standard for  results . Howevero source .in vitro at the cost of being unable to provide normal physiologic antiviral responses. Here we describe three different cell lines frequently used to model viral hepatitis infection in vitro.Hepatoma cell lines provide a high degree of accessibility for modeling viral infection The Huh7 cell line and its genetically modified successors are very receptive to HCV infection. These cells are excellent at propagating infectious HCV virions and they divide rapidly. Huh7 cell are extremely useful for replicating viral infection, and this cell line also supports infection with other important pathogenic viruses including SARS-CoV-2 . HoweverHepG2 cells have been used for studies of the HBV lifecycle. HepG2 cells are polarized, can be manipulated to express the HBV genome and can generate a cell-intrinsic innate antiviral response that makes them useful for the studies on host defense pathways. However, HepG2 cells must be genetically modified in order to fully support HBV infection as they lack significant expression of the NTCP HBV viral entry receptor, and even when this receptor is expressed, HepgG2 cells are still less susceptible to HBV infection than fully differentiated HepaRG cells .The HepaRG cell line is derived from a bipotent progenitor that can differentiate into either the hepatocyte or cholangiocyte lineage. If differentiated toward the hepatocyte lineage, HepaRGs confer a phenotype that mirrors that of PHHs in terms of its ability to support both HBV and HCV infection while demonstrating contact inhibition . HoweveriPSC-derived hepatocyte-like cell (HLCs) are similar to HepaRGs in the sense that they can be differentiated in order to confer a phenotype that approaches that of PHHs; however, they have yet to prove capable of exhibiting the same level of differentiation as PHHs that includes minimal to no expression of alpha-fetoprotein. Unlike PHHs however, iPSCs offer a limitless supply of cells while minimizing donor variability. HLCs have demonstrated the capacity of supporting both HBV and HCV infection .in vitro liver models are typically designed with the goal of promoting and sustaining hepatocyte differentiation and viability and are then applied to study relevant functional biological mechanisms. This preliminary step of promoting the maintenance of viable and differentiated hepatocytes is critical for the establishment of a microenvironment that is conducive to support long-term viral infection. Here, we describe how different technologies recapitulate the hepatic microenvironment to best support and maintain physiologically relevant hepatocyte function. The advantages and disadvantages of each type of physiomimetic platform are listed in Physiomimetic Static models established the foundation for the subsequent development of dynamic models and they are utilized for the development of tissue-like structures beyond traditional 2D cell monolayers. Any fresh media that the culture receives has to be administered manually and these cultures are characterized by the lack of any consistent media perfusion or flow through the model.Sandwich culture is a layered culture approach where cells are sandwiched between two matrix layers . Cells bin vitro  and cellulose where the porous structures support the transport of cell signaling molecules between hepatocytes  are deterministically patterned co-cultures, often achieved by selective matrix deposition or surface treatment . The orier cells . Like sa formats . This pa formats . Ploss eT3 cells . In addiin vitro  bioreactors are perfusion devices that typically feature two sets of hydrophilic media capillaries and one set of hydrophobic oxygen capillaries . The medex vivo . Gerlachex vivo . Though Rotational bioreactors, or rotating wall vessels (RWVs), are platforms for spheroid generation and/or maintenance that continuously suspend cells and allow them to aggregate into spheroids . The sphin vitro.Microfluidic platforms are the miniaturized successors to bioreactors that possess numerous advantages including easier multiplexing, imaging and sample collection. Furthermore, it leverages the use of fewer reagents and cells in its layout to better mimic the liver’s architecture. These microfluidic platforms are often designed as perfusion analogs to the static models delineated above. Liver microfluidic devices are most commonly fabricated from PDMS elastomer, PMMA (acrylic), or polystyrene. They can also incorporate numerous other materials in their design that can be manufactured with a wide variety of formfactors. Despite their differences in design and means of construction, these devices are ultimately unified in their aim to better capture the physiology of the liver in vivo. Without this barrier, hepatocytes are at risk of exposure to non-physiological shear that may affect the long-term maintenance of a differentiated phenotype. Emulate’s Liver-Chip features a membrane that divides dual parenchymal and non-parenchymal perfusion channels separated by a PDMS membrane with individual flow rate control that is amenable to imaging (As a perfusion analog to monolayers and sandwich culture, planar and layered microfluidic platforms expose a monolayer or multiples layers of cells to laminar flow . This la imaging . Kang et imaging . Several imaging . Zonatio3D microfluidic devices are acutely similar to planar microfluidic devices by design, but provide a difference in cell orientation. Like static 3D cultures, cells are cultured as scaffold-free spheroids/organoids, individual scaffold-encapsulated 3D cells, or scaffold-encapsulated spheroids/organoids. Scaffold-free cultures require physical barriers in the form of micropillars or protective membranes to immobilize and protect spheroids from being perturbed by direct exposure to flow . In Ma eLike bioprinted static culture, bioprinting offers additional spatial customizability for cell deposition. ® design to a 384 well plate with 40 individual culture chambers. Each chip has phaseguides that separate the matrix-encapsulated cells from rocking-induced gravity flow, but still permit nutrient exchange (While many of the platforms detailed above are not optimized for multiplexing and generating high-throughput data, several commercially available systems feature plate formats that are advantageous for adding biological replicates and testing a larger number of experimental conditions . The PREexchange . Lena Biexchange . High-thin vitro modeling, it is important to consider what these platforms may provide as clinically predictive models. For viral hepatitis specifically, physiologically relevant microsystems hold great potential as tools to make clinical predictions about disease progression because these platforms incorporate numerous microenvironmental factors that are implicated in viral pathogenesis. For example, Kupffer cells and stellate cells recruit T cells and contribute to the inflammatory microenvironment during infection (Although the scope of applications for physiologically relevant systems has thus far been limited to nfection . Severalnfection , and altnfection . Moreovein vitro results against clinical data in terms of drug toxicity response. Jang et al. showed species-dependent differences in toxicity response to a Janssen compound that was discontinued during animal trials because of hepatoxicity in rats. These results were confirmed from with rat hepatocytes on the in vitro platform, but when the same experiment was conducted on human hepatocytes, no such toxic effect was observed (Outside of the context of viral hepatitis, Emulate’s liver chip has already compared observed . This fiobserved . These fIn vitro studies are needed to aid in the ascertainment of new knowledge on the mechanisms that govern the transition of viral hepatitis from acute to chronic infection and related pathogenic processes. A lack of readily available in vivo models necessitates the use of in vitro systems that can attain the level of physiological relevance that is observed in vivo. Importantly, the capacity to study hepatotrophic viruses on human cells using models that can be interrogated through sophisticated and informative molecular assays is required. The developments of physiologically relevant platforms are in their early stages in terms of their use as models of viral hepatitis infection; however, the available studies thus far have demonstrated significant improvements in infection efficacy and longevity as compared to conventional cell culture methods. These preliminary steps in establishing more robust infection models will undoubtedly create beneficial tools to study virus-host interactions and uncover novel insights about chronic viral infection and related mechanisms that would not otherwise be possible through the use of more rudimentary platforms. Some of the systems mentioned above have yet to be specifically utilized for viral hepatitis modeling, but their shared goal of improved hepatocyte function in vitro translates across all applications. Nonetheless, physiomimetic platforms still have a variety of barriers to overcome before becoming reliable models of viral infection. For example, making these models higher throughput with easy access to cultures in order to introduce and assay infection is critical for these platforms to compete with conventional 2D culture where these two elements are commonplace. Fortunately, the plethora of tools and models that now exist can serve as a solid foundation for improving platforms to study virus-host interaction. Many of these platforms are now available for use, but need to be introduced to viral applications.DM, DB, AA, and ET: manuscript concept, design and drafting of the manuscript. All authors contributed to the article and approved the submitted version.


Aging is strongly associated with cognitive decline and degenerative diseases. Therefore, unravelling the basic mechanisms of aging is essential to improve the quality of life of older people. Among others, mitochondrial dysfunction and genomic instability have been considered key hallmarks of aging, and these deficiencies seem to play important roles in age associated cognitive decline . MitochoAlthough the role of the factors mentioned above has been extensively studied, it is still not known whether these aging related factors can serve as indicators of cognitive function in human studies. In a recent paper, we have addressed this question and performed molecular analyses on blood cells and plasma with the purpose of identifying key biomarkers related to cognitive abilities in very old individuals . We perfWe used Seahorse real-time cell metabolic analysis technology to measure different mitochondrial parameters in peripheral blood mononuclear cells (PBMCs). Centenarians were found to display well-preserved mitochondrial respiration in their PBMCs when compared to young individuals. Additionally, we observed that only centenarian males and not females showed a negative correlation between the rate of mitochondrial respiration and cognitive scores. Thus, our results suggest that lower rates of mitochondrial respiration may indicate better cognitive capacities in males. This agrees with a recent study performed in mice, which also associated a moderation of the mitochondrial activity with better health outcomes . We beliOur study revealed that sex differences must be considered as an important biological variable in research within the aging field. We have found negative correlations between cognitive abilities and mitochondrial respiration only in males, which could be due to the sexual dimorphism that has been observed for mitochondrial function, in the way females and males cope with oxidative stress . Althoug+/NADH levels in blood plasma, also correlated positively with cognitive capacity. Lastly, our results showed a high level of protein carbonylation, which is an indicator of oxidative stress, in individuals with lower cognitive scores. Based on these observations, we hypothesize that increased NAD+/NADH levels as well as BDNF could possibly slow down cognitive decline or aging related diseases through the stimulation of DNA repair pathways and the consequent lowered oxidative stress. This finding incites to look deeper into the network formed by DNA damage sensing proteins, DNA repair pathways and oxidative stress in relation to aging. Of note, NAD+ boosting strategies are already under investigation in order to prevent the loss of cognitive function in humans [Furthermore, we found that an increased amount and activity of the core BER enzyme, APE1 in PBMCs correlated with well-maintained cognitive health in both sexes. In the same way, the amount of circulating BDNF and NADn humans .+, BDNF, DNA repair and mitochondrial function in relation to aging and cognitive decline (In summary, we have presented several molecular markers that can be measured in blood and which correlate with the maintenance of cognitive capacities. However, in order to be able to design strategies to delay the aging associated cognitive impairment in humans, we still need a better understanding of the interplay between factors like NAD decline .


Vocal communication is used across extant vertebrates, is evolutionarily ancient, and been maintained, in many lineages. Here I review the neural circuit architectures that support intraspecific acoustic signaling in representative anuran, mammalian and avian species as well as two invertebrates, fruit flies and Hawaiian crickets. I focus on hindbrain motor control motifs and their ties to respiratory circuits, expression of receptors for gonadal steroids in motor, sensory, and limbic neurons as well as divergent modalities that evoke vocal responses. Hindbrain and limbic participants in acoustic communication are highly conserved, while forebrain participants have diverged between anurans and mammals, as well as songbirds and rodents. I discuss the roles of natural and sexual selection in driving speciation, as well as exaptation of circuit elements with ancestral roles in respiration, for producing sounds and driving rhythmic vocal features. Recent technical advances in whole brain fMRI across species will enable real time imaging of acoustic signaling partners, tying auditory perception to vocal production. Homo neanderthalis and sapiens cries were probably species-specific. Courtship songs in other tetrapods are also typically innate, with only a few exceptions, most notably songbirds  is essential for social communication  are among the most ancient acoustic communicators, appearing in the fossil record ~270 mya (Ranids) emerged from a world-wide extinction event and underwent massive radiations at the KT boundary  and vocalizations are produced by a larynx modified for underwater sound production to produce sounds without airflow  is our current research focus.Placental mammals are also ancient acoustic communicators  ~85 mya—include many vocal genera . Mice (Mus) have dominated investigations of rodent ultrasonic vocalizations (USVs), due in part to the genetic advantages of specific laboratory strains. The Chiroptera (bats)—another highly vocal group—diverged from other mammals ~75 mya  is recent  ~240 MYA and bird species radiated ~60 mya, again reflecting the worldwide extinction event at the K-T boundary. All extant birds communicate vocally  in response to stimulation of attached laryngeal nerves that mimics call patterns . Two forebrain nuclei  and the Bed Nucleus of the Stria Terminalis (BNST) project to their contralateral counterparts as well as to Ra and PB. The resulting pattern of connectivity .The behavioral effects of HCG on male calling are due to gonadotropin itself, to direct effects on neurons expressing gonadotropin receptors in the CeA  produce complex courtship songs directed toward the females in their harems at dawn and dusk, sandwiched between territorial songs  and rats (Rattus) are nocturnal while other genera , are diurnal (see below). Vocal communication is also prominent in subterranean genera such as naked mole rats , visual signals from conspecifics influence the activity of auditory neurons  and two rodents: Alston's singing mice (Scotinomys teguina) as well as mice (Mus musculis), vocal species with well-characterized repertoires and CNS vocal circuits. As for humans, features of acoustic communication in some species of birds are learned , used to locate prey, are also employed during social behaviors. Auditory cortex neurons tuned for echolocation contribute to recognition of USVs used to interact socially at the roost , the reticular formation, the PAG and the PB. Stimulating the CeA evokes agonistic vocalizations  but has not yet been described.CNS vocal circuitry: In Scotinomys teguina, are neotropical, diurnal Cricitine rodents, whose evolutionary divergence was more recent  comprise ~40% of extant mammalian species and diverged from a common ancestor with lagomorphs ~75 MYA  into vocal muscles and then following transneuronal (retrograde) spread at successive intervals. This PRV approach identifies CNS nuclei that participate in vocal production  and can Mice Arriaga and Jarvis  and retrograde . We used this approach , a brain region recently proposed as a key node for courtship displays across vertebrates  were first identified by Schmidt . In X. lorebrain .ex vivo brain evokes “fictive calling” in adult males  for the male advertisement call  consists of several interconnected sub-regions (L1-3) whose circuit architecture resembles processing in mammalian auditory cortex . The young bird “evaluates” the match between a learned song and his own match to that song, linking motor output to its acoustic consequences  is a CPG for the male advertrisement call [reviewed in Kelley et al.  while the homolog of PB contributes controls vocal patterning. If so, the origin of the PB as a vocal CPG could be evolutionarily ancient.As bird songs are coordinated with respiration, one approach to finding a vocal CPG in birds is to identify the respiratory CPG. Wild  describepattern, suggesting separate CPGs for cry production and minibreaths. Interneurons innervating TA and CT motor neurons form three groups: rv-iRF (glutamatergic neurons), Botzinger and preBotzinger nuclei (gabaergic) and Nucleus Retroambiguus (mixed). Interneurons innervating both tongue motor neurons and TA motor neurons were also found in rv-iRF. Inactivating rv-iRF disrupted the interval between cry bouts as well as intervals within a bout, but not basal breathing. Brief optogentic stimulation of the rv-iRF produced cry bouts throughout the longer breath. Comparing activity patterns in brain slices that included the rv-iRF and the pre-Botzinger nucleus revealed a faster oscillation (every 6s as compared to 23s) in the former. These experiments provide strong evidence that the rv-iRF generates the pattern of pup cries. This vocal CPG provides input to preBotzinger neurons to drive inspiration (triggering minibreaths) and coordinates activity in the laryngeal TA and CT muscles that control glottal opening.A recent study  are rhythmically active during inspiration, post-inspiration and expiration  in auditory and vocal neurons  emerged during the Jurassic ~170 mya and the genus Xenopus ~50 mya  suggest an ancient role in the coordination of vocal signaling during reproduction.The capacity for synthesizing estrogen arose before the evolution of the ancestral ER  controls male clasping  has been described as the “autonomic” amygdala because of its role in respiration and heart rate. Given the prominence of expiration for vocal expression across vertebrates, CeA involvement in vocal communication makes sense. In Xenopus, a species that uncoupled breathing from calling many millions of years ago, the CeA matches acoustic stimuli to vocal expression. Lesions of the CeA in Xenopus result in socially inappropriate responses of males to song playbacks  in the PB was identified electrophysiologically in both species . When syvice versa). In Xenopus, each sound pulse includes two dominant frequencies   and pulse preference  co-localize in the genome, raising the possibility that the linkage between male performance and female preference contributes to shared QTL.Crickets use acoustic communication at a distance (far field) during courtship. Interestingly, female preferences co-evolve with acoustic features of male courtship songs in Hawaiian crickets  ranging from ~5 (simulans) to ~13 mya  that is activated by similar social contexts, but drives different motor outputs . D. melanogaster can produce clack songs, suggesting that circuitry for this song type is shared between species  is a prominent circuit motif. In mouse pups, the output of the CeA to the PAG is also inhibitory and disinhibition gates production of their cries . The A group is the most speciose in the genus and the female release call  is not yet clear but if so, could be due to gonadal hormones.In other frogs, recognizing a heterospecific male is selected for in females because F1 hybrids are less fit. In Acoustic signaling is ancient and phylogenetically associated with extant species that are nocturnal or especially vocal at dawn or dusk . Birds aof the same species for reproduction. This is particularly for true females whose gametes are finite and provide resources for the embryo, unlike those of males.Because speciation reflects both sexual selection (success in attracting mates) and natural selection , sex and speciation are linked at many levels. In vertebrates, sexual differentiation is governed by pituitary and gonadal hormones. Patterns of AR and ER expression—from sensory receptors through to neural circuits for muscle effectors—are targets for evolutionary selection. Broder et al.  argue thRobert and the late Dorothy Cheyney argued , we do use biting language. We also devote considerable attention to decoding how people feel about us from cues in voice to construct a socially appropriate response. Areas of the human brain involved in language production and perception must (at the very least) access other areas that identify social context-driven voice cues regulated by the endocrine and neuromodulatory systems described in this review. Advances in fMRI now allow imaging of entire brains in response to conspecific and heterospecific vocal sounds in other animals (Van Ruijssevelt et al., The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.DBK wrote this manuscript and adapted from published work or created the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Modulating the epigenome has long been considered a potential opportunity for therapeutic intervention in numerous disease areas with several approved therapies marketed, primarily for cancer. Despite the overall promise of early approaches, however, these drugs have been plagued by poor pharmacokinetic and safety/tolerability profiles due in large part to off-target effects and a lack of specificity.Recently, there has been marked progress in the field on a new generation of epigenomic therapies which address these challenges directly by targeting defined loci with highly precise, durable, and tunable approaches. Here, we review the promise and pitfalls of epigenetic drug development to date and provide an outlook on recent advances and their promise for future therapeutic applications.Novel therapeutic modalities leveraging epigenetics and epigenomics with increased precision are well positioned to advance the field and treat patients across disease areas in the coming years. In the 70 years since the term “epigenetics” was first coined, the field has yet to fulfill its true therapeutic potential, but has nonetheless proven a boon to basic researchers, to understand how cells process genetic information, differentiate, and respond to external stimuli . At its Epigenetic effectors, enzymes capable of inducing changes in chromatin state, are varied and are often divided into three major categories—writers, erasers, and readers . At the The principal promise of epigenetic-based therapies is the ability to control gene expression directly at the pre-transcriptional level and thus correct gene dysregulation at its source. Perhaps the greatest benefit of this approach lies in being able to turn gene expression up or down in a durable but  not permanent manner, without making any changes to the underlying genomic sequence. This capability aids in the study of cellular differentiation, lineage specification, and programming as well as enabling this understanding to be harnessed to treat disease.The ability to leverage the endogenous mechanisms by which cells control gene expression seemed like a new key to unlock therapeutic avenues for a variety of diseases. Cancer has been, and remains, an exemplar for the utility of epigenetic modulation as a therapeutic approach . In manyUnfortunately, translating these advances in our understanding of epigenetics into medicines has proven more challenging than initially anticipated. While there are eight FDA-approved and marketed epigenetic therapies with six to treat hematologic malignancies and two approved for use in solid tumors Table , trials There has also been limited success in applications of epigenetics outside of oncology. Although there is strong evidence that epigenetic dysregulation plays a role in other areas like autoimmunity and hemoglobinopathies, there has been minimal efficacy leveraging available compounds. Well-known HDAC inhibitors like butyrate have shown some proof-of-concept efficacy in certain settings, like sickle cell disease and beta-thalassemia , but notFollowing the elucidation of the DNA double helix structure, epigenetic markers, DNA methylation, and histone modifications were soon identified . One keyDNA methyltransferases are a class of cytosine methylases that play a key role in epigenetic regulation by depositing marks on the DNA itself. DNA methylation is important in the etiology of cancer as it epigenetically regulates the expression (or lack thereof) of cancer-related genes . In 1980Various other nucleoside analogs have also demonstrated DNA hypomethylation activity, but have stalled in development due to low biological activity and/or high levels of toxicity, impacting organs like the liver and heart , 24–26. Histone deacetylases are enzymes that remove acetyl marks from lysine residues on histones, allowing chromatin to be wound more tightly, reducing accessibility for transcription. The first epigenetic drugs approved in this class were vorinostat and romidepsin . These aCarboxylic acid is another zinc-binding motif that has been studied for its HDAC inhibiting properties . The sodAssays for DNMT and HDAC activity were available by the early 1990s . Given tWith the second-generation HDACs, applications have broadened to include non-hematological cancers . These mAnother successful structural class of compounds are the benzamides, which demonstrate selectivity toward Class I HDACs . One exaWith multiple DNMT and HDAC inhibitors approved for clinical use, the fundamental hypothesis that epigenetics can be harnessed for therapeutic use has been borne out . With imThe third wave of epigenetic drug discovery has identified three new targets: lysine histone methyltransferases (KMTs), lysine demethylases (KDMs), and bromodomain inhibitors . Agents Histone methyltransferases, either KMTs or protein arginine methyltransferases (PRMTs), post-translationally add between one and three methyl groups to lysine or arginine residues on histone proteins, which can have a range of important effects . DependiLysine demethylases reverse lysine methylation on either DNA or histone proteins (among other molecules) which can alter either the transcription of genes at the promoter or via changes in chromatin state. One family of KDMs, lysine-specific demethylases (LSDs) are homologous to monoamine oxidases in their mechanism; thus, MAOIs have been repurposed as epigenetic therapies . TranylcBromodomain proteins play a critical role in transcriptional regulation acting via histone acetylation, chromatin remodeling, and recruitment of other transcriptional machinery. Bromodomains are epigenetic readers with the BET  family garnering the most attention in drug discovery efforts . One of Although it is well accepted that epigenetic interventions should be feasible therapeutic options outside of oncology, previous small molecule approaches have yielded suboptimal results thus far due to toxicity and off-target effects. With more recent advances, however, we believe epigenetic therapies are poised to become an important component of patient care in the near future Table  1]..1].While monotherapy approaches have not produced results as strong as the field expected, there may be promise in combination studies, particularly in solid tumors. Given that tumors often accumulate multiple defects (both mutations and epigenetic dysregulations) as cancers grow and metastasize, combining therapies to target more than one at a time is an enticing hypothesis. As reviewed in Morel et al. , the epi+ER− breast cancer, HDAC inhibition plus EGFR inhibition in non-small cell lung cancer, and HDAC inhibitors with checkpoint inhibitor treatment in colorectal cancer. In some cases, like that of entinostat plus exemestane, promising Phase II results in breast cancer did not translate into approvable Phase III results [Some of the more promising combinations have been HDAC inhibition combined with aromatase inhibition in HR results ; howeverWhile there is promise to this multi-hit approach, both from preclinical and in early clinical studies, many of the limitations of current epigenetic monotherapies have still arisen. For example, toxicity profiles in combination settings have led to high rates of discontinuations and/or study terminations without a significant boost in survival or tumor shrinkage. Additionally, off-target effects of one epigenetic therapy may be compounded by epi/epi combos, which could offset the benefits. Other tactics may be required to make progress therapeutically harnessing epigenetics.While epigenetic therapies have had modest success in oncology, their overall impact has been significantly less than many had hoped and certainly far less than their full therapeutic potential across disease areas. One of the key limitations of these early epigenetic modulators has been specificity. By targeting key effectors in the epigenetic network, one can impact diseased cells, but without a constraint on the sites to which they are delivered , it is impossible to narrow the impact across all genes and prevent healthy cells from being similarly affected. As such, the levels of toxicity seen with existing classes of inhibitors have restricted their use.In order to deliver on the broader promise of epigenetics and epigenomics, this issue of molecular specificity must be resolved. The newest era of epigenomic therapies addresses this challenge head-on. Rather than targeting endogenous effector proteins directly, these approaches leverage sequence-specific DNA binding domains (DBD) to direct epigenetic changes to a precise genomic locus or loci. Effector proteins linked to these DBDs allow for exquisite targeting of activity, whether that be writing, modifying, or erasing marks. By leveraging this high degree of sequence specificity, this “epigenomic programming” approach aims to dramatically reduce off-target influences and increase the tolerability and applicability of epigenetic therapies.The first demonstration of site-specific targeting of epigenetic modifications was published in 2013 by Gilbert et al. . In thisThis proof-of-concept observation was subsequently expanded upon, with additional studies exploring the use of similar systems with alternative effector proteins , 73, 74 The past several years have seen a number of companies emerge that have taken up the mantle of developing epigenomic technologies into therapies that could represent truly meaningful therapeutic options for patients. Newer epigenetic medicines are targeted to work pre-transcriptionally with high specificity and control over the level of gene expression for a specified period.Precision and specificity can be conferred utilizing several different DBDs. One commonly used research tool for exploring the potential of these approaches rapidly has been dCas. Here, the enzymatic activity of the Cas protein, made famous by CRISPR gene editing discoveries, has been ablated, allowing this protein to act as a chaperone for an epigenetic effector, directed to the appropriate locus by a separate gRNA. Much of the early literature in the field leverages this technology for proof-of-concept gene activation or silencing.While this dCas system has been embraced as both a research tool and an emerging therapeutic option, other strategies for targeting use zinc finger proteins (ZFs) or transcription activator-like effectors (TALEs) that encode sequence-specific motifs that directly bind to the DNA without a need for Watson–Crick pairing or the use of separate guide RNA and can be directly linked to effector proteins. These single-component therapeutics, which may have advantages and efficiencies for dosing or multiplexing different effectors to discrete loci in a single therapeutic, are currently under development.Additional specificity of epigenetic programming relative to small molecule approaches comes from specialized delivery methodologies. Most of these therapeutics are delivered via cell- or tissue-specific vectors. Many drug developers are delivering DBD-effector fusions encoded as DNA and encapsulated in viral vectors like adeno-associated viruses (AAVs). AAVs have distinct tropisms for certain cells/tissues based on serotype, providing greater control compared to systemic delivery of small molecules, which generally have indiscriminate biodistribution. AAV-delivered approaches tend to generate long-lived or even permanent expression systems; however, in the context of epigenomic therapies, these suffer from concerns over redosability due to pre-existing or induced immunogenicity to the viral capsid and the inability to withdraw or negate the activity of the effectors if any untoward effects are observed clinically. Further, the payload capacity of the AAV genome is limited, making it challenging to encode large sequences like dCas and linked effector domains plus guide RNAs in a single viral vector.An alternative option to viral vectors being employed increasingly is lipid nanoparticles (LNPs) , 13. TheOne of the unique advantages to working with mRNA/LNPs is the ability to decouple the pharmacokinetics (PK) and pharmacodynamics (PD) of these novel therapeutics which is not possible with earlier generations of small molecule epigenetic modulators or viral delivery. mRNA-encoded approaches using LNPs are transient expression systems. While the expressed therapeutic proteins themselves typically persist in vivo on the order of days, there is robust evidence that the epigenetic changes imparted can be heritable and the phenotypic effects on expression can last on the order of weeks or months; the durability can be specified depending on the nature of the intervention  and the requirements of the specific indication and gene target. As such, the epigenomic medicines delivered in this way can provide major advantages, including meaningful reduction in dosing frequency and a potential significant improvement in safety profile, which can help expand the clinical utility of these drugs into a wider array of diseases.In addition to increased specificity and durability, novel epigenomic therapies have the ability to tune gene expression rather than induce a binary on or off result. Certain genes, including many notable oncogenes, are required for normal function of cells but can be pathogenic if overexpressed. By intervening pre-transcriptionally with the appropriate epigenetic effectors, it is possible to modulate gene expression to a physiologically normal level without completely ablating expression, which could have other negative effects. The same holds true for targets requiring upregulation; restoring homeostatic expression rather than supraphysiological levels may be safer or more tolerable in the long term.A final feature that differentiates precision approaches from other genetic medicine strategies and earlier generations of epigenetic therapies is the ability to precisely multiplex targets to enhance efficacy. Multiplexing can take different forms, including the use of more than one effector targeted to a given region to synergistically drive multiple epigenetic changes or the epigenetic modulation of multiple genes that contribute to pathology in the same disease state to obtain improved efficacy. In both cases, this multiplexing utilizing, for example, multi-cistronic mRNA encoding multiple therapeutic proteins, is distinct from the off-target effects of small molecule epigenetic therapies which had broadly detrimental impacts. Being able to address genes in the context of their natural DNA structure, for example in IGDs, allows for epigenetic regulation of not only promoters but other regulatory elements like enhancers, repressors, and CTCF sites or long non-coding RNAs, which can have profound, tunable, and durable effects. Directing epigenetic effectors to multiple genes via one therapeutic can also overcome some of the challenges to treating complex diseases where it is not feasible to utilize multiple small molecules or biologics simultaneously due to safety or tolerability issues. The therapeutic potential unlocked by these advances in precision epigenomic programming is truly remarkable.HTT as a strategy to combat Huntington’s disease. By targeting a zinc finger protein-KRAB fusion to the amplified CAG repeats, the effector was able to repress transcription selectively with therapeutic benefit in both behavioral and histological measures in a murine model. In a 2021 presentation, both in vitro and in vivo data also provided evidence to support therapeutic downregulation of a-synuclein in a murine model utilizing a zinc finger protein linked to a KRAB repressor delivered in an AAV vector.The application of epigenomic technologies spans a wide range of therapeutic areas, in agreement with the broad reach of epigenetics. Development is underway in oncology, cellular regeneration, autoimmune disease, ex vivo cell therapy, rare genetic disorders, and neuroscience including neurodegenerative diseases. A 2019 publication by Zeitler et al. [, MYC-targeting via epigenomic programming led to a decrease in tumor size in multiple murine xenograft models. Together, these early publications and presentations are evidence of substantial progress in translational research for epigenomic programming and speak to the longer-term promise in clinical development. The first clinical trial for an epigenomic programming approach is currently ongoing with the first patient dosed in October 2022.In oncology, preclinical work in epigenomic programming has demonstrated significant progress on targeting cMYC, a key oncogene historically considered undruggable. Leveraging IGD biology, epigenomic controllers targeted the IGD containing MYC were able to robustly downregulate expression of the gene across both hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) cell lines. This decrease in MYC expression led to a concomitant decrease in tumor cell viability in vitro, with the associated epigenetic marks detectable at the sites of interest 2 weeks post-administration. Importantly, normal primary cells from both the liver and lung did not show the same increase in cell death, supporting the more targeted effect of targeted epigenomic therapies over other epigenetic drugs. In vivoWhile the evolution of the epigenetic therapeutics field has been marked by modest success alongside notable disappointments, the rise of new precision approaches is expected to be a transformational catalyst to unlock its full potential. Similarly, great strides have been made in recent years in furthering our understanding of the causal links between dysregulation of the epigenome, disease processes, and negative outcomes, contributing to greater clarity on the most relevant targets to pursue. We believe that this potent combination of enhanced mechanistic understanding coupled with new and highly refined tools with unprecedented specificity portends a revolution in the ability of epigenomic medicine to deliver on the full breadth of its promise of transformational therapies for patients living with cancer and other chronic life-threatening conditions.Although clinical development in this space is just beginning, we anticipate rapid progress over the next several years. Success in unlocking targets previously considered undruggable, like MYC, could yield orthogonal strategies for treating patients, either as monotherapies or in conjunction with existing approaches. The ability to integrate new targets into the therapeutic landscape in oncology could be a major advance for patients, particularly those who are not amenable to existing targeted therapies, although it may take some time to understand the ideal patient characteristics and associated biomarkers to best apply these technologies. Similarly, the regenerative potential of the epigenomic programming approach has the potential to address debilitating neurodegenerative diseases as well as other diseases of aging in a way that has not been achievable to date. Targets that are known to be critical in disease biology but have eluded existing technologies represent a meaningful opportunity to improve patient outcomes. Harnessing epigenomic programming could yield practice-changing medicines in many areas if current technologies can be translated into clinical results.Building on the lessons learned from the earliest clinical efforts and maximizing the new levels of control conferred by improvements in targeting, protein engineering, and delivery of genetic medicines, epigenetics has the potential to deliver on its promise in a significant way for a wide variety of patients in need.


Recently, two research traditions that bring together evolutionary biology and medicine, that is to say, Darwinian medicine and evolutionary medicine, have been identified. In this paper, I analyse these two research traditions with respect to explanatory and interdisciplinary integration. My analysis shows that Darwinian medicine does not integrate medicine and evolutionary biology in any strong sense but does incorporate evolutionary concepts into medicine. I also show that backward-looking explanations in Darwinian medicine are not integrated proximate-and-ultimate explanations but functional explanations that include reference to evolutionary concepts. Nevertheless, explanations in Darwinian medicine have heuristic roles as they potentially contribute to conceptual change and tie pieces of knowledge from different fields of medical research together. I argue that Darwinian medicine is an “interfield” that fosters cross-disciplinary exchange between evolutionary biologists and medical researchers and practitioners based on division of labour and separation, rather than unity. Research in evolutionary medicine, on the other hand, happens at the intersection of evolutionary biology and medicine where the two disciplines are already integrated and is designed to produce entangled proximate-evolutionary explanations. My analysis thus adds another important aspect to the philosophical discussion on the distinction between Darwinian medicine and evolutionary medicine. In the past three decades, calls for the integration of evolutionary biology and medicine have become increasingly pronounced. Darwinian medicine is one of the research traditions that aims at achieving this goal. Rephrasing Dobzhansky’s  famous dBased on Ernst Mayr  and NikoIn what follows, I analyse Darwinian medicine and evolutionary medicine with respect to interdisciplinary and explanatory integration. As an exemplary explanation in Darwinian medicine, I discuss the so-called “old-friends hypothesis”. My analysis shows that Darwinian medicine does not produce integrated proximate-and-ultimate explanations, but functional explanations with reference to evolutionary concepts. Nevertheless, these explanations are useful tools that tie together results from different fields of medical research, and potentially contribute to conceptual change, opening up new avenues of research. I argue that Darwinian medicine can still play an important role in promoting cross-disciplinary exchange despite being based on separation and division of labour rather than blending or unification. Evolutionary medicine, on the other hand, generates entangled proximate-evolutionary explanations. The example of an experimental evolution study of antibiotic resistance shows that in evolutionary medicine, interdisciplinary integration precedes explanatory integration.To date, explanations and explanatory practices in Darwinian medicine and evolutionary medicine have not received much philosophical scrutiny. In this section, I summarize the two main accounts by Pierre-Olivier Méthot  and MichMéthot , p. 76 iMéthot emphasises the different types of explanation generated by the two research traditions . He argua forward looking explanation tries to predict the effects of ongoing evolutionary processes on human health and disease in contemporary environments . In contrast, a backward looking explanation typically applies evolutionary principles from the vantage point of the evolutionary past of humans  in order to assess present states of health and disease among populations. , p. 76Cournoyea , p. 478 In the literature, the terms ‘evolutionary medicine’ and ‘Darwinian medicine’ are sometimes used interchangeably, and in some cases ‘evolutionary medicine’ is used as an umbrella term that includes both research traditions. However, I agree with the distinctions discussed by Méthot and Cournoyea and add a new aspect to the discussion with my analysis of explanatory and interdisciplinary integration.Integration is seen as one of the central characteristics of interdisciplinary research –20, but An important question concerns the level at which integration takes place. Science studies scholars have shown that two fields can be bridged at the level of theory , or Proponents of Darwinian medicine frequently express the need and desire to achieve interdisciplinary integration of medicine and (evolutionary) biology, and argue that Darwinian medicine provides the conceptual framework for this “synthesis” , p. 1993In the context of Darwinian medicine, integration of evolutionary biology and medicine is usually understood as integration on the level of explanation or integration of evolutionary biology into medical education. There are many publications that address the question of how knowledge from evolutionary biology can be integrated into medical school curricula e.g., , 37, 38 Most proponents of Darwinian medicine understand interdisciplinary integration as a synthesis of ultimate and proximate explanations of human diseases , 4, 39. I have already mentioned that evolutionary medicine is a less cohesive research tradition than Darwinian medicine and that scientists are not necessarily aware that their research belongs to the research tradition of evolutionary medicine. For this reason, there is no unified theoretical framework that addresses the question of how explanatory or interdisciplinary integration can or should be achieved. However, since practitioners of evolutionary medicine apply methods, practices, and concepts from evolutionary biology to address medical problems, it is worthwhile investigating whether and how evolutionary medicine generates integrated explanations and how explanatory integration relates to interdisciplinary integration within this research tradition.In the following sections, I analyse Darwinian medicine and evolutionary medicine with respect to explanatory and interdisciplinary integration. In the next section, I take a closer look at the structure of explanations in Darwinian medicine and address the questions of whether research under the framework of Darwinian medicine produces integrated proximate-and-ultimate explanations and whether unification or blending of evolutionary biology and medicine is achieved by means of explanatory integration. I further discuss whether Darwinian medicine helps to establish common ground between researchers in evolutionary biology and medicine and fosters interdisciplinary research that is coordinated around a set of problems. In the following section, I discuss interdisciplinarity in evolutionary medicine and address the question of whether and how research in evolutionary medicine generates integrated proximate-evolutionary explanations.Since advocates of Darwinian medicine aim at achieving integration on the level of explanation, I use the so-called old-friends hypothesis as an example that illustrates the structure and roles that explanations generated under the framework of Darwinian medicine play in medical and biological research. I will show that Darwinian medicine does not generate truly integrated ultimate-and-proximate explanations, but proximate explanations complemented with evolutionary concepts. I further argue that Darwinian medicine does not facilitate strong interdisciplinary integration in the sense of creating unity or blending but works as an interfield that enables cross-disciplinary exchange.One of the main supporters of the “old-friends hypothesis” is Graham Rook. The old-friends hypothesis is an attempt to reformulate the hygiene hypothesis “to bring it in line with Darwinian Medicine, and with the latest epidemiological and experimental evidence” , p. 5. TWith the old-friends hypothesis, Rook  proposesTo support his hypothesis, Rook , p. 11 rStudies of animal models and clinical trials suggest that helminths or helminth-derived drugs can prevent autoimmune diseases like multiple sclerosis or decrease disease severity.The old-friends hypothesis is an exemplary case of evolutionary reasoning within the framework of Darwinian medicine. As I have argued, explanations of this kind are functional explanations of health or disease that also reference evolution. The case of the old-friends hypothesis shows that the functional explanation of the prevalence and increase of chronic inflammatory disorders in countries of the Global North is already a full-fledged explanation even without reference to evolution. The answer to the question why chronic inflammatory diseases are prevalent in countries of the Global North does not necessarily require reference to evolutionary processes see , 39, 63.In the previous section, I have already mentioned that there could be another reason why Rook and other researchers include evolutionary reasoning, namely, to have a framing for their argument that ties pieces of knowledge from several fields of medical research together. This role of evolutionary theory is probably the reason why evolution is often referenced but not properly discussed in articles that summarize results from different (bio)medical studies. Another heuristic role of the old-friends hypothesis is the promotion of a new perspective on host-parasite interaction, particularly human-helminth interaction. While most research programs conceptualize helminths as pathogens that harm the host, the old-friends hypothesis emphasizes the health benefits that might arise from interactions of the human immune system with helminths or helminth-derived substances. Conceptualizing helminths as friends instead of enemies could spur new research questions that might eventually lead to the development of new drugs against chronic inflammatory disorders. This suggests that hypotheses generated under the framework of Darwinian medicine have heuristic value for medical research, as they have the potential to open up new research avenues, promote new treatments of diseases or help identifying targets for clinical intervention see , 27. To addition of an evolutionary perspective” to medical research. Antolin et al. [borrowing knowledge from germ line evolution” could produce better diagnostic and prognostic strategies for cancer treatment. Rather than creating a synthesis of ultimate and proximate explanations, Darwinian medicine incorporates pieces of evolutionary theory into medicine. The potential of Darwinian medicine to integrate evolutionary biology and medicine in any strong sense is rather low because it does not promote a blending or unification of disciplines. In fact, it fosters only partial integration of evolutionary biology into medicine on a conceptual level, but there is no mutual integration of methods, practices, or data and the two disciplines remain more or less separate. One of the main reasons why explanations like the old-friends hypothesis do not foster strong interdisciplinary collaboration is the somewhat atemporal or static account of evolution provided by the framework of Darwinian medicine that does not include any details of evolutionary mechanisms or processes. In fact, mismatch hypotheses are accounts of changing environments, not of evolving species. These explanations ignore ongoing evolutionary processes and focus solely on the current state that presumably arose as an adaptation to the environment of the Pleistocene savannah.Explanations like the old-friends hypothesis do not require collaboration or communication with evolutionary biologists or expert knowledge of evolutionary theory and methods because they rely on a few evolutionary concepts rather than empirical evidence, data, or complex theorizing. Knowledge of the theoretical framework of Darwinian medicine is sufficient to be able to construct a functional explanation with reference to evolution.At best, Darwinian medicine enables what Margaret Boden , p. 5 caInstead of promoting integrated accounts, proponents of Darwinian medicine have created a new research tradition separate from evolutionary biology , p. 2. MProper backward-looking reasoning can be compared to detective work where the investigator relies on clues to reconstruct past events or processes see , p. 276.However, maybe strong interdisciplinary integration of evolutionary biology and medicine may not be necessary to foster a fruitful exchange between the two disciplines. Maria Kronfeldner  identifiThis type of cross-disciplinary exchange does not require a common goal or collaborative research but might eventually foster “piecemeal integration” , p. 207 The finding that Darwinian medicine does not yield truly integrated proximate-and-ultimate explanations does not imply that it is impossible to produce integrated explanations of health or disease. In this section, I discuss research in evolutionary medicine that generates integrated explanations. These explanations, however, are not produced by explanatory integration, meaning that they don’t result from synthesizing explanations from different disciplines. Instead, they are a result of interdisciplinary research at the intersection of evolutionary biology and medicine that is designed to generate entangled proximate-evolutionary explanations.Since many pathogens procreate and evolve rather fast, it is relatively easy to study their evolution. While studies of human evolution usually require some kind of backward-looking approach, microbial evolution can be studied in real-time, for example by means of experimental evolution see , 75. The14Escherichia coli (E. coli) bacteria to study the evolution of antibiotic resistance. Samples from a starting population of E. coli were used to start experimental lines that were exposed to 95 different stressors  to analyse whether and how they evolved resistance. The researchers created one line for each of the 95 stressors plus a control without any stressor times six replicates . They let the populations evolve over a period of 27 days which corresponds to approximately 250–280 generations. The researchers then produced resistance profiles for 192 of the evolved strains to study how common cross-resistance and collateral sensitivity occurred, meaning that they wanted to find out how many of the strains that evolved under a certain kind of stress also exhibit increased resistance or susceptibility to another kind of stress. They also performed genome sequencing analysis of the evolved strains to examine the genetic changes (mutations). After performing transcriptome analysis, the scientists were able to identify modular classes of gene expression profiles and analyse the relationships between genome, transcriptome and resistance profiles.Here, I present an example of an experimental evolution study of antibiotic resistance that illustrates how research in evolutionary medicine generates entangled proximate-evolutionary explanations. In experimental evolution studies, populations are studied across multiple generations to investigate “evolutionary changes occurring in experimental populations as a consequence of conditions  imposed by the experimenter” , p. 547.E. coli’s evolutionary dynamics is attributable to a relatively small number of intracellular states, indicating that it is likely equipped with only a limited number of strategies for antibiotic resistance” [As a result of their analyses, the scientists were able to identify molecular mechanisms associated with resistance acquisition and find a connection between changes in gene expression and stress resistance. They argue that their results suggest the existence of evolutionary constraints on accessible phenotypes, meaning that “istance” . The resistance” . Thus, uistance”  have cliStudies like these have a long tradition that predate the emergence of Darwinian medicine , p. 77. The example shows that there are studies in biomedical research that do not only produce proximate explanations but entangled proximate-evolutionary explanations. Maeda and collaborators  conducteE. coli populations to novel environments in a controlled setting, they also know which genetic changes have evolved in which environment. The difference between the two kinds of explanation is related to the time span that the respective explanations encompass. Backward-looking explanations encompass very large time spans and include events of the distant past while forward-looking explanations encompass relatively short time spans. It is much easier to generate integrated proximate-evolutionary explanations when the evolutionary pathways are known  than to generate integrated explanations of long-term evolution. As I have shown in the previous section, evolutionary events of the distant past can be inferred and reconstructed from traces, but this can take a lot of time and effort and requires expertise in relevant areas of research.There is an important difference between forward-looking explanations in evolutionary medicine and backward-looking explanations in Darwinian medicine, namely that backward-looking explanations rely on speculative hypotheses about evolution, whereas in forward-looking explanations, the evolutionary pathways are known. Proponents of Darwinian medicine hypothesize that certain traits have evolved as adaptations to the Pleistocene environment. In the experimental evolution study by Maeda and collaborators , howeverForward-looking explanations like the one presented by Maeda and collaborators  answer tI have shown that there is a difference between Darwinian medicine and evolutionary medicine with respect to explanatory and interdisciplinary integration. Research under the framework of Darwinian medicine does not produce truly integrated proximate-and-ultimate explanations but proximate explanations that incorporate evolutionary concepts. Explanations in Darwinian medicine can be useful to integrate results from different fields of (bio)medical research and potentially promote conceptual change in medicine, but their construction does not foster interdisciplinary integration of evolutionary biology and medicine in any strong sense. However, Darwinian medicine can work as an interfield that loosely integrates evolutionary biology and medicine by enabling exchange between medical researchers and evolutionary biologists. This cross-disciplinary exchange does not require shared goals or collaborative research projects and is based on separation rather than unity. Thus, Darwinian medicine maintains the division of labour between evolutionary biologists and medical researchers. Under this framework, the focus lies on the products of medical and biological research and partial integration of proximate and evolutionary explanations happens on the conceptual level. Research activities in evolutionary medicine, on the other hand, produce genuinely integrated explanations because they are, from the very beginning, designed to produce entangled proximate-evolutionary explanations. This kind of research usually happens at the intersection of evolutionary biology and medicine where concepts, methods and practices from evolutionary biology and medicine are already integrated. Thus, in evolutionary medicine, interdisciplinary integration precedes explanatory integration. My analysis thus adds another important distinction to the philosophical discussion of the different research traditions at the intersection of evolutionary biology and medicine.


For some, the vestibular system is so poorly understood that it is thought of simply as an accessory of the auditory system. As we have gained better understanding of this sensory system, we have learned that the vestibular system goes far beyond maintenance of posture and balance, participating in orientation in space, navigation, motor coordination, and body perception.The mature inner ear consists of a labyrinth of diverse sensory organs set within a finely organized spatial configuration. Vestibular sensory organs detecting angular acceleration reside in the ampullae of three orthogonal semicircular canals, while changes in gravity and linear acceleration are detected primarily by the maculae utriculi and sacculi in the vestibule. All vertebrates maintain body equilibrium in space for quality of life and survival. These basic requirements may explain why the phylogenetically old vestibular system is highly conserved, with all vertebrates sharing a common blueprint for basic inner ear configuration and central vestibular pathways. An accurate control of posture, balance, gaze, and their perception requires integrating diverse sensorimotor signals within a single central nervous system framework. Spatially and temporally precise signals from the vestibular sensory organs are transmitted over widespread areas starting mainly with the brainstem vestibular nuclei where the first level of sensory integration occurs. Signaling then proceeds to other brainstem centers: the thalamus, cerebral cortex, cerebellum, and spinal cord.Despite the evolutionary importance of these labyrinths for survival, malformation is not rare during development. Epidemiological studies report that up to 8% of children experience vertigo or balance problems , with veDuring the intense developmental period of childhood, vestibular disorders may produce similar deficits observed in adults, lesser problems due to more effective vestibular compensation in children, or more severe problems because some vestibular functions emerge before birth or during the 1st months of life, such as multisensory integration, basic postural reflexes, motor coordination, and gaze stability. The most obvious consequences of CVDs are hypotonia and delayed verticalization impacting sitting, standing, and the acquisition of walking. CVD children may naturally implement compensatory behaviors using visual or somesthetic cues, such as moving on the back to increase surface contact. Visual stability may be disturbed in CVD patients who experience decreased dynamic visual acuity and oscillopsia resulting in reading difficulties. These deficits induce fatigue in children and reduce their ability to concentrate that altogether hinder learning. Finally, vestibular deficits are associated with increased incidence of psychiatric comorbidities in CVD children .Peripheral Vestibular Dysfunction Is a Common Occurrence in Children With Non-syndromic and Syndromic Genetic Hearing Loss” by Wang et al. reports new data indicating that vestibular disorders are commonly found in children with syndromic and non-syndromic hearing loss. Two other studies focus on new approaches for clinical exploration in CVDs. An enlarged vestibular aqueduct is one of the first malformations in CVDs, an anomaly that may be explored using the video head impulse test (VHIT), a caloric test described in “Clinical Implication of Caloric and Video Head Impulse Tests for Patients With Enlarged Vestibular Aqueduct Presenting With Vertigo” by Li et al. Imaging is a recent major development in vestibular testing, especially for Meniere's disease. This topic is discussed in “Three-Dimensional Volumetric Measurement of Endolymphatic Hydrops in Meniere's Disease” by Noh et al., paving the way for new discoveries in CVDs where endolymphatic MRI may contribute to understanding the underlying mechanisms in children.The objective of this Research Topic is to describe clinical testing approaches and the results obtained in CVD children and CVD animal models presently available to better understand the pathology within central and peripheral vestibular system structures that produce behavioral deficits in CVD children. The study “Sustained Loss of Bdnf Affects Peripheral but Not Central Vestibular Targets” by Elliott et al. describes the effect of brain-derived neurotrophic factor (BDNF) on vestibular ganglion neurons and vestibular hair cells in a mouse model. A translational study “Vestibular Deficits in Deafness: Clinical Presentation, Animal Modeling, and Treatment Solutions” by Maudoux et al. describes non-syndromic, vestibular impairments and how animal models are leading to promising results in treatments. Lastly, the paper “Understanding the Pathophysiology of Congenital Vestibular Disorders: Current Challenges and Future Directions” by Peusner et al. reports on the challenges faced by children with syndromic CVDs and how animal models can be used to acquire more precise assessment of the vestibular pathophysiology underlying the disorders at the cellular level.Concerning animal models, “All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Viewing strategies are strategies used to support visual information processing. These strategies may differ between children with cerebral visual impairment (CVI), children with ocular visual impairment, and children with normal vision since visual impairment might have an impact on viewing behavior. In current visual rehabilitation practice a variety of strategies is used without consideration of the differences in etiology of the visual impairment or in the spontaneous viewing strategies used. This systematic scoping review focuses on viewing strategies used during near school-based tasks like reading and on possible interventions aimed at viewing strategies. The goal is threefold: (1) creating a clear concept of viewing strategies, (2) mapping differences in viewing strategies between children with ocular visual impairment, children with CVI and children with normal vision, and (3) identifying interventions that can improve visual processing by targeting viewing strategies. Four databases were used to conduct the literature search: PubMed, Embase, PsycINFO and Cochrane. Seven hundred and ninety-nine articles were screened by two independent reviewers using PRISMA reporting guidelines of which 30 were included for qualitative analysis. Only five studies explicitly mentioned strategies used during visual processing, namely gaze strategies, reading strategies and search strategies. We define a viewing strategy as a conscious and systematic way of viewing during task performance. The results of this review are integrated with different attention network systems, which provide direction on how to design future interventions targeting the use of viewing strategies to improve different aspects of visual processing. The etiology of visual impairment in childhood is very heterogeneous. Visual impairment in children is caused by ocular diseases and/or genetic factors affecting the eye or by cerebral visual impairment (CVI). CVI comprises visual malfunction due to retro-chiasmal and visual association pathway pathology  the benefits of compensatory strategies in subjects with brain damage, (2) in current visual rehabilitation a variety of strategies is used without consideration of the different etiologies of visual impairment, (3) viewing strategies hold potential benefits for visual attention, and (4) oculomotor behavior provides insights into viewing strategies.The diversity of visual deficits in CVI is considerable, and the impact of CVI on education and rehabilitation strategies is less well-understood than for ocular visual impairments. A recent review states that educational strategies employed for children with ocular impairments are largely ineffective for children with CVI  visual impairment. Based on the variety of causes of  visual impairment and clinical practice it would be conceivable that children with CVI use different viewing strategies compared to children with ocular visual impairment and children with normal vision. Knowledge about these strategies is needed to adapt rehabilitation techniques and training to the different problems in visual functioning.CVI is most often accompanied by impairments in higher order visuospatial processing, visual selective attention, and object recognition  to provide a clear concept of viewing strategies, (2) to map differences in viewing strategies between children with ocular visual impairment, children with CVI and children with normal vision and (3) to identify interventions that can improve visual processing by targeting viewing strategies.Studies were identified through electronic databases searching in PubMed, EMBASE, PsycINFO and Cochrane Library. The final search was run on 20 December 2020. In addition to the articles found with our search query, reference lists of included articles were scanned and experts were consulted. No limitation regarding year of publication was applied. The search was developed by an experienced clinical librarian. The selection was made by the first two authors of the article. The following search strategy was used to search for all databases using MeSH terms and keywords: visual perception (MeSH), viewing strategy or viewing skills, reading strategy or reading skills, visual processing or perceptual exploration or perceptual processing or fixation strategy or perceptual learning or visual development. An example of the search strategy used in PubMed is shown in Two authors (AF and BH) independently reviewed the list of results and identified relevant articles, based on predefined in- and exclusion criteria regarding viewing strategies of school-aged children. The inclusion criteria are presented in Included articles focused on the concept of viewing strategies or viewing behavior during near school-based tasks in a variety of children. Since limited research about children with  visual impairment is available, we choose to include articles describing viewing behavior of children with normal vision . For example we included articles concerning reading strategies by disabled or dyslexic children and controls. Control data provide information about viewing strategies used in children with normal vision which makes their inclusion relevant for this scoping review. On consensus five articles regarding the use of Irlen filters were included, since the use of aids may count as a strategy used by the visually impaired child. Since visual training programs commonly are assigned to children in primary school, the age was limited to 4–12 years old.The included studies appeared very heterogeneous in subjects, paradigm and outcome measures. There was too little information provided on quantitative outcome measures regarding viewings strategies to conduct a meta-analysis. Therefore, the results of the studies will be described in a narrative manner. To assess the quality of the articles included in this scoping review, we modified the QUADAS tool  and 724 articles were screened by abstract. In addition one article was identified by an expert  studies regarding viewing strategies, (2) studies describing viewing behavior in children with normal vision, (3) studies describing viewing behavior in children with  visual impairment, and (4) intervention studies focused at improving viewing behavior. We found five studies regarding viewing strategies VI. Finally, thirteen studies were found which evaluated the effectiveness of behavioral treatment on visual processing proficiency in children with normal vision and/or children with (C)VI. The characteristics and outcomes of these studies are presented in A variety of viewing strategies were mentioned in five studies. None of these five studies involved children with  visual impairment. The tasks for which viewing strategies were described varied across these studies: three studies mentioned strategies during reading , reading disabled children (n = 26), and backward reading children (n = 12). The control group performed better on the temporal dot counting task than reading disabled children, while there were no group differences on the spatial dot counting task. This study emphasizes the relationship between visual temporal processing skills and reading performance. Spatial abilities might however, play a role in predicting reading ability in young children. Fisher et al.  in the study by Eden et al. . Three gr et al.  found evr et al. .The relation between visual learning and reading skills for children with and without reading problems was evaluated by Tong et al.  and GarcLutzer  measuredIn sum, oculomotor studies demonstrate that: (1) average readers use less fixations and regressions during reading, and (2) a shorter fixation duration found in average readers is linked with greater amplitudes of saccades during reading. Eye movements take a developmental leap when children learn to read more fluently, but are not essential to learn to read. Visual attentional processes seem to influence reading performance as well: average readers more often used global before local processing and performed better on visual temporal processing tasks (like dot counting and coherent motion) compared to children with reading problems.Three studies were included with viewing behavior as an outcome measure in children with (C)VI see . These sOverall, children with (C)VI showed longer reaction times to visual stimuli than controls. In addition, children with low visual acuity and children with nystagmus showed increased gaze fixation areas on cartoons.Twelve non-randomized controlled trials (non-RCT) were included in this review, and one cohort study see . Seven oOne training program was focused on improving visual processing skills in children with poor visual processing , effects on academic outcome measures are often not reported or absent  visual impairment. The main goals of this scoping review were to define a concept of viewing strategies and to compare viewing strategies between children with normal vision and children with  visual impairment. In this scoping review we found no published research regarding viewing strategies for  visually impaired children which makes a comparison between groups impossible. The lack of published research illustrates a giant gap between daily child vision rehabilitation practice, in which viewing strategies are a key component, and scientific evidence. Even for school-aged children with normal vision, literature about viewing strategies is scarce.To create a concept of viewing strategies, information could be extracted from a total number of five studies in which the use of viewing strategies is mentioned VI. Since improving viewing strategies is a common training goal in visual rehabilitation, more research concerning viewing strategies used by children with (C)VI is needed. Till date, it remains unknown whether there are differences in viewing strategies between children with normal vision and children with (C) VI. Also, even if school-aged children use the same kind of viewings strategies, it remains unclear if the quality of the viewing strategies differs between these groups, for example in speed of processing or accuracy of visual identification. Three studies showed lower visual processing speed in children with (C)VI VI. The influence of attentional processes on viewing behavior is shown in a variety of studies and stresses the importance of investigating the potential benefits of targeting task-relevant attention networks in visual training. VAS dysfunction, or more specific disorders in visual selective attention processes, can be one of the visual dysfunctions in children with CVI  visual impairment differ from viewing strategies in children with normal vision. Clear definitions of viewing strategies were not found in the included studies. Gaze strategies, search strategies and reading strategies were described as elements contributing to visual processing. The use of viewing strategies might influence different visual attention networks . We propose that for the development of an effective viewing strategy, the role of these three networks should be considered. Although the scoping review revealed attentional processes that are involved in viewing behavior, we did not find any interventions that targeted visual attentional processing in children with  visual impairment. The relation between strategic viewing processes and academic performance therefore remains unclear.The original contributions presented in the study are included in the article/Literature screening and selection, data extraction, and synthesis was performed by AF-V and BH. Preparation of the first draft of the manuscript was done by AF-V. Review and approval of the manuscript was performed by BH and FB. All authors read and approved the final manuscript.This research was supported by Novum. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.