The nearshore area of the Toronto and Region is used as a case study to illustrate the eco-accounting framework and how habitat banking could be incorporated along with planned restoration to remediate this degraded but continually developed area. The framework represents significant advances in managing cumulative habitat effects in an integrated way, moving away from a focus on only project- or site-level assessments. We feel this approach could be adapted to other ecosystem types in addition to the lake, nearshore area example provided here.A key aspect of contemporary fish habitat management is the need to account for losses and gains associated with development and offsetting measures while protecting high quality features. We propose an ecological accounting framework for aquatic ecosystems using habitat equivalents scaled to aquatic productivity, and using fish-to-habitat associations by life stage, based on local fish community needs. The framework uses both landscape-scale and site-level evaluations of pre- and post-project habitat changes to assign and track habitat parcels, using ecological baselines and fish-habitat target setting. Concepts of natural capital reserves and productivity-based ecotypes are used for trading losses and gains between impacts from development projects and offsets, including restoration actions, while maintaining ecologically important areas intact. Traditional accounting terms such as deposits, withdrawals, and transfers are defined using scaled habitat-equivalents as the currency. Other key features of the framework include setting a service area that is ecologically meaningful, and conducting habitat transactions guided by habitat conservation, protection, and restoration ( CPR plans), and to calculate habitat equivalency trades within a landscape-scale, service area. This approach would incorporate habitat offsetting into the overall integrated spatial planning and management of aquatic ecosystems. Elements of an aquatic, ecological accounting framework have been used individually, though have rarely been unified or integrated as we suggest.In Canada, evaluations of projects in or near water have typically focused on site-level, one-time fish habitat evaluation and assessment when offsetting (i.e. trading unavoidable adverse effects for offsetting measures) on a project-by-project basis. What is needed is an environmental planning process to set habitat trades within an integrated aquatic landscape. An ecological accounting framework could be used to weigh decisions about unavoidable impacts from new or proposed development within integrated conservation, protection, and restoration planning , that may be valuated through standard ecological measures , and could include deposits from natural capital generation, such as gains from habitat restoration projects. An integrated management plan may identify particular conservation projects (banked or true deposits) that are needed within a service area where future development projects or other adverse activities  are anticipated, but also can exceed equivalency if the current situation is degraded.Good accounting practices are central to the successful operation of financial institutions. The same is true for implementing integrated planning for aquatic habitat CPR. Practically, there are many useful analogies between commercial and ecological banking systems, and terms such as capital reserves; areas with significant ecological importance or productivity that are set aside to be protected and are not available for trading. Recent investigations for bird communities recommend that where possible, up to 50 to 85% of managed areas should remain natural to preserve their ecological functionality , but at minimum, current conditions. Subsequently, valuation and tracking of all habitat transactions happens within an integrated planning or service area. Typical currencies for valuation of aquatic habitat centre around fish diversity or fisheries productivity surrogates. Moving from habitat features to productivity measures involves more uncertainty in the equivalency calculations .Quality-based equivalencies rely on habitat associations or niche information for single species, guilds, or whole fish communities, and can be based on expert opinion or quantitative approaches. Examples include habitat assessment tools like the Habitat Ecosystem Assessment Tool  is to be used;defining the time lags needed for an banked offset or restoration project to attain the full ecological value or expected productivity, and how this relates to trade timing, and;determining important existing natural features (remaining or previously restored) that should be set aside and conserved.identifying Aside from a common currency, basic conservation planning tenets need to be followed when establishing and operating a landscape-scale habitat accounting system  for an area into actionable context. We illustrate—with examples from an ongoing case study along the Toronto region’s waterfront—how the various components of a fish habitat accounting scheme may work , within which the banking or trading will be conducted, need to be defined. This area should be ecologically meaningful to the majority of the species or habitats that local management and plans are meant to conserve, protect, or restore . Fixed planning boundaries and standardized regulations for banking ensure that both the benefits and costs  are shared within the same spatial context. Some exceptions  may be necessary if offset or restoration options within a geographic area are limited. However, those out-of-area trades should be guided by consistently applied rules and transparency in decision-making.Next, and related to the ecological strategy, the ecotypes. These ecotypes, which often differ in their productivity and support different ecological communities, need to be well-defined. Measurable differences in ecotypes are needed to scale or calculate the equivalence of habitat parcels within and between ecotypes for accounting trades of habitat deposits and withdrawals. Therefore, a methodology for calculating equivalencies of habitat parcels within ecotypes is also required.Within the established boundaries, there will typically be recognizably discrete habitat areas belonging to distinct natural capital reserves, ideally based on quantitative evidence or expert local opinion. Also, it is well-documented that restoring lost ecological features does not return their equal original value  across the ecotypes present. If there is agreement, those areas should be designated as protected, Setting goals for biological or fish production, productivity, diversity, or functional use of new or restored habitats, whether for planned restoration or offsetting purposes, is essential. Of equal importance is establishing targets related to the goals in order to measure success. Ecosystem goals and targets from local management plans are a good place to begin to synthesize. For the Toronto nearshore planning area, the TWAHRS includes fishery, habitat, and ecosystem goals (TRCA Toronto and Region Conservation Authority ) as well2) of wetland, mainly in the Ashbridges Marsh . These The geographic boundaries for an integrated planning or habitat bank service area may be based on several considerations, including ecological. The area should be sufficiently large to account for human development and habitat management activities at an ecologically meaningful scale, but not be so large that costs and benefits are inequitably shared spatially . Setting the area’s spatial extent should consider existing jurisdictional and governance boundaries, but focus on conservation boundaries, natural landscape features , and other ecological factors affecting the fisheries productivity of the systems . For example, one ecological criterion to help define spatial extent could be the range within which an average, non-migratory, fish subpopulation may function, but its placement should be guided by natural largescale, eco-zonation  limits were chosen as practical and informal eastern and western boundaries for the integrated planning area; this area includes rivermouth and other nearshore ecotype habitat features, like wetlands;The southern boundary of the proposed integrated planning area was defined as the lake-based, elevation contour of 44-m ASL, roughly the 30-m depth contour . The upland boundary could be expanded if a larger riparian zone was considered an important natural feature to include, in an aquatic sense, beyond the long-term flood zone, which can vary depending on local slope .As such, the boundaries of the proposed Toronto region nearshore, integrated plan area in the northwest quadrant of Lake Ontario were based on existing jurisdictional, resource management, and ecological zones Fig. :The ToroThese boundaries make the habitat integrated planning area readily manageable within the broader landscape because they fall within existing jurisdictional watershed boundaries and are also within a similar hydrodynamic zone of Lake Ontario. Also, the TWAHRS applies within the TRCA limits, so the extent is also compatible with these local watershed and municipal boundaries, providing goal and target consistency.The 44-m ASL elevation contour or 30-m depth corresponds to a liberal limnological definition of the nearshore or littoral zone, still accounting for variability in maximum thermocline depth . This bIn some instances, higher or lower elevation boundaries may be chosen for habitat banking purposes. For example, if riparian, stream, or wetland areas immediately above the high-water elevation, or features below the profundal zone, are shown to be essential for local fishery productivity, then these boundaries could be revised on a case-by-case project basis. However, once the total area and limits of the integrated habitat planning area have been agreed upon they should be considered permanent, or at least longer-term, otherwise ecological accounting may become complex and unwieldy with constant revisions and updates. This is good rationale for being generous in setting the boundaries and spatial extent at the outset.wetland: shallow areas dominated by emergent and submerged vegetation, often with constricted connections to adjacent open coast aquatic areas;embayment: a bay or bay-like conformation defined by the Free Dictionary as “an indentation in a shoreline or coast forming a bay with intermittent or permanent connection to a larger body of water; bigger than a cove but smaller than a gulf.”  that is exposed to, and driven by, whole lake processes, and not sheltered, as with most wetlands and embayments; and,offshore: open lake habitat where water depths are greater than the littoral or maximum epilimnetic zone depth (in this case >30-m depth). Offshore waters have also been defined here as those >5 km from the shoreline along open coasts  if the maximum depth of 30 m has not yet been achieved at this distance.We define ecotypes as distinct, major habitat types within the integrated planning area, to be used for mapping, accounting, and productivity calculation purposes. However, we recognize the whole system is interconnected and gradients exist between classes, therefore we advise liberal boundaries for productive ecotypes so they are not undervalued. Nonetheless, freshwater lake ecosystems generally have easily recognizable ecotypes  are taken into account when determining the scaled habitat area required to offset the loss of open coast habitat. Other considerations in the transaction should include the relative availability of ecotypes within the integrated planning area and the underlying trade-offs made between specific fish guilds that may prefer one ecotype over another, or select habitat variables across ecotypes. This habitat selectivity and availability at the larger landscape and fisheries objective level were considered in developing desired targets for the integrated planning area.potential productivity index (PPI) to reflect differences in maximum potential productivity among different ecotypes. This relative index reflects expected ecosystem-level productivity that supports different fish communities or fish productivity over differing environmental conditions. Based on relative production estimates from the literature, the current PPI assignments are notional and will need to be refined once local assessment data are analyzed appropriately . Particular features within an ecotype may have higher value than others . There is also variation within each ecotype due to differing spatial configurations, parcel sizes, depth ranges, substrate types, vegetation types, and thermal characteristics. This variation must be considered as part of the habitat valuation. The potential combinations of habitat features within any ecotype, coupled with any additional environmental stresses , means that maximum productivity can only be realized under ideal circumstances within any ecotype and therefore the PPI is scaled by local habitat parcel conditions using weighted suitable area (WSA).Habitat valuations typically involve one of two approaches  were assigned for each ecotype Table . Coldwatproductivity-weighted suitable area (PWSA = PPI × WSA) to obtain a relative productivity equivalent across the whole region  and then modified using the ecotype-specific guild weightings. At this point the measure can be considered a suitable-habitat equivalency that is weighted by fisheries goals or guild preferences. The resulting WSA values are then multiplied by their ecotype PPI values to calculate the Once a full survey and accounting of the habitat supply or natural capital  that (a) have restoration or enhancement potential , or (b) might be used to create habitat  and realize a gain in quantity are identified.Habitat parcel values are assessed using their relative quality within an ecotype . Habitat supply by parcel is then scaled by the PPI of each ecotype to bring all habitat parcels to an equivalent base unit across ecotypes . For terrestrial parcels identified for potential aquatic habitat creation projects, their initial PWSA is assumed to be zero in a fish habitat sense. However, a more comprehensive planning approach might include valuation of aquatic and terrestrial ecotype areas to address those trade-offs more explicitly.The improvement or creation of the habitat parcels is planned and financial costs are estimated along with PPI. Measurable assessment benchmarks will be needed to ensure that both functional habitat and productivity targets have been achieved. This necessitates some level of short- and long-term monitoring and evidence-based criteria for use in planning and costing.Once the deposit construction is complete and the ecological performance targets have been met — time lags from habitat creation or enhancement to ecological functionality are accounted for here—the net PWSA can be deposited into the integrated planning area’s balance sheet, and become available for trade with a future offset withdrawal, if needed.The full financial costs of the habitat deposit, including costs for planning, construction, and long-term monitoring and assessment (needed to demonstrate the target productivity is achieved and sustained) are accounted for in a financial counterpart to the ecological habitat supply accounts. The financial methods are not presented here, but we feel it is important to mention. Those costs should also reflect anticipated inflation and other economic factors associated with construction, assessment, and monitoring, and possibly other valuations of the habitats . However, only one value is traded even though the deposit may have multiple benefits. This avoids stacking, or ecological double dipping  are undertaken to evaluate net change at different levels of a project using projected scenarios. The aim is to maintain a positive balance in the service area. If the evaluation indicates losses cannot be offset onsite, or located elsewhere in the integrated planning area in a timely fashion, additional withdrawals from the habitat bank could balance residual effects but would ultimately reduce gains.Once agreed upon, offset units and their habitat types and ecotypes are used in calculations of pre- and post-development comparisons to show equivalency balances. Calculations include any other ratios that need to be applied . One or more habitat parcels or ecotype areas within the habitat bank (near or removed from the development construction site) may be considered as offsets for development withdrawals—depending on CPR plans, such as the TWAHRS—and brings context to actions based on local ecological needs and knowledge over the landscape scale . The traded deposits are ‘moved’ into the integrated planning area reserves and are no longer available for offsetting. Additional habitat projects not yet constructed can be designed and then deposited for later withdrawal depending on the number and magnitude of future development projects anticipated and the negotiated timing of deposits to collective habitat banks. This landscape-based approach may require third-party banking be allowed, but with proper planning has been effective under current, single-party regulatory requirements in Canada.The timing of impacts  can be used to modify the CPR plan and update valuations accordingly as more experience and knowledge of different types of habitat loss and modifications are gained over time from the analysis of pre- and post-monitoring data.There are several steps to complete a withdrawal from the integrated planning area’s habitat supply:We have already discussed intra-service area trading—deposits and withdrawals within the same integrated planning area, a foundation of habitat banking—but there may be situations where an inter-service area transfer could be considered. Such a transfer may be needed when there is an unavoidable, anticipated loss with no, or not enough, restoration or creation potential locally. In standard approaches, efforts are made to achieve no net loss locally, but some areas may lack CPR plans, may not have a habitat bank option, or may lack the opportunities to enhance native biodiversity or fisheries production Minns . In thesFor the Toronto nearshore service area, a larger scale might include lake-wide fish community objectives . This amount is paid directly for creating future habitat bank deposits or to support required for ongoing monitoring and assessment activities. Basic monitoring and assessment at the development site will still be the responsibility of the proponent. In the Toronto example, AHT is well established with expertise in habitat creation, restoration, and monitoring within the integrated planning area and has ongoing science support to guide decision-making . Currently, the accounting framework as outlined has only partially been implemented.The ability to expand the scope of habitat supply accounting from the project/site level to an integrated plan landscape level using an ecological framework for habitat banking has been demonstrated here. The Toronto region waterfront is a relatively well-studied shoreline with a substantial recent history of efforts to restore and create aquatic habitats to offset the long-term accumulation of losses incurred over the last two centuries, and in anticipation of development needs for the 25-year plan for revitalization of the waterfront by the City of Toronto and Waterfront Toronto. Past and ongoing efforts to assess fish communities and their habitats have produced a wealth of understanding and experience in Toronto  and the analysis of its reflectance  may help. Finally, the development community and public support for restoration and enhancements are already embodied in the TWAHRS document TRCA , laying In the proposed ecological accounting framework, we adapted existing tools and incorporated general scientific knowledge about productivity differences among ecotypes, allowing a common habitat equivalency/currency, beyond weighted suitable area, to be modified. This common currency can be applied to habitat parcels across the complete range of aquatic ecotypes present in the Toronto regional nearshore. The methodology, framework, and habitat valuations can be updated as the knowledge base and tools are enhanced over time; this should be an adaptive iterative process, both locally and more generally. Habitat supply accounts can be updated on a regular basis, perhaps every 3 to 5 years as part of routine reviews of the habitat bank’s operations. Also, the natural variability in the baseline habitat features should be captured so that the ecological value of transitional and ecotone habitats between ecotypes are also captured in the valuation.Ideally, the nominal estimates for the PPIs shown here will be replaced with actual fish production or primary productivity units, but still account for differing community composition and ecosystem dynamics across ecotypes  as possible. Ironically, to achieve these goals, it is necessary to include a loss in the accounting from infilling to recreate the lost embayments and wetlands in an area where reclaiming lake from historical land infilling  is near impossible. Overall, the goal is for a targeted positive balance in habitat supply, reaching ecotype goals, and also reserves of natural capital to not duplicate historical cumulative losses.To become fully operational, the financial accounting  and governance system (how decisions are made) for any integrated spatial aquatic planning will have to be fully developed in parallel with this proposed ecological accounting system. TRCA and other similar groups already have a good appreciation of the immediate costs of creating, enhancing, and restoring habitats based on their involvement in the intensive and extensive activities along the Toronto waterfront. It will be important to make sure the full financial costs—including long-term monitoring and maintenance costs for the accumulated deposits and any longer-term science support required for adaptive science and management—are estimated accurately for different habitat modification and creation activities. These costs, including estimates for different offsetting options, could be outlined in a separate financial framework. Governance models that separate or lump the different activities within the habitat banking system and their regulatory needs will also be required, and there are good examples —to fully appraise and value riparian terrestrial trades with aquatic ones. Under the current scheme those trades are undervalued, especially for semi-aquatic species . There is also scope for the accounting scheme to be expanded beyond fish productivity units to consider habitat biodiversity (Morrison et al. For now, AHT plans to use the ecological accounting system outlined here for the Toronto waterfront, but iteratively make improvements as the methods and habitat equivalents are refined. This approach has potential for broader scale application across the Great Lakes for habitat management where landscape-scale service areas are appropriate for CPR plans to benefit fish populations, communities, and ecosystems, while allowing development to continue and implementing broader restoration goals beyond site-by-site and project-by-project considerations.Supplementary Information


Obsessive compulsive disorder (OCD) and chronic tic disorders (CTD) including Tourette Syndrome (TS) are often comorbid conditions. While some patients present with distinct symptoms of CTD and/or OCD, a subset of patients demonstrate a unique overlap of symptoms, known as Tourettic OCD (TOCD), in which tics, compulsions, and their preceding premonitory urges are overlapping and tightly intertwined. The specific behaviors seen in TOCD are typically complex tic-like behaviors although with a compulsive and partially anxious nature reminiscent of OCD. TOCD is not classified within the Diagnostic and Statistical Manual of Mental Disorders fifth edition (DSM-5) as an independent diagnostic entity, but mounting evidence suggests that TOCD is an intermediate neuropsychiatric disorder distinct from either TS or OCD alone and as such represents a unique phenomenology. In this review of TOCD we discuss clinical, genetic, environmental, neurodevelopmental, and neurocircuit-based research to better characterize our current understanding of this disorder. TOCD is characterized by earlier age of onset, male predominance, and specific symptom clusters such as lower tendency toward compulsions related to checking, cleaning, and reassurance seeking and higher tendency toward compulsions such as rubbing, tapping, or touching associated with symmetry concerns or thoughts of exactness. Functional magnetic resonance imaging (fMRI) imaging suggests that TOCD symptoms may arise from involvement of an intermediate neurocircuitry distinct from classic OCD or classic CTD. Small cumulative contributions from multiple genetic loci have been implicated, as have environmental factors such as infection and perinatal trauma. In addition, this review addresses the treatment of TOCD which is especially complex and often treatment resistant and requires pharmacology and behavioral therapy in multiple modalities. Given the distressing impact of TOCD on patients’ functioning, the goal of this review is to raise awareness of this distinct entity toward the goal of improving standards of care. Obsessive compulsive disorder (OCD) and tic disorders such as Tourette Syndrome (TS) are well established entities within the pediatric population that can occur independently, but are often comorbid within the same individual. Over the last 10–15 years, there has been increasing awareness of the overlapping neurocircuitry of tics and OCD and the existence of an intermediate phenotype, known as Tourettic OCD (TOCD), in which symptoms are influenced by features of both OCD and TS and differ from either disorder alone  and a second wave of new cases with mean onset in the early 20’s , 7. In cDRD2, DAT1 (SLC6A3), HTR2A, and EAAT1 but later switched to GWAS and CNV studies (HDC enzyme that converts L-histidine to histamine) (Tourette syndrome is inherited in 70–80% of cases  making i studies . TSAICG  studies . A similstamine) –20, agaiGRID2, DLGAP1). Genome-wide association studies (GWAS) have estimated the genetic heritability for OCD at approximately 0.37 in adults with OCD and 0.43 in childhood onset OCD  and the OCD Collaborative Genetics Association Study (OCGAS). The meta-analysis from the two consortia of 2,688 individuals with OCD and 7,037 matched controls found that no SNPs reached genome-wide significance  but sevenset OCD . The agenset OCD .Previous data suggests that OCD and TS share some genetic blueprint. Among pediatric patients with OCD, greater than 50% exhibit tics. Likewise, an estimated 30–60% of patients with TS manifest symptoms of OCD . PatientThe genetic architecture for TOCD is still unknown. Given that TOCD manifests with components of both TS and OCD, it is postulated to share genetic similarities to both disorders. Coffey et al. suggest that TOCD is more genetically similar to TS than to OCD because patients with TS and TOCD have higher rates of comorbid ADHD compared to patients with OCD . IronicaIt is widely known in the literature that environmental factors exacerbate tic/OCD symptoms. In addition to the known tendency of environmental stressors to exacerbate existing symptoms within an individual, prospective studies are elucidating a potential link between environmental factors and the onset of tics or OCD in individuals with no personal prior diagnosis but with a first degree relative with a CTD . While mA variety of neuropsychiatric disorders that include pre-existing tic, OCD, and TOCD symptoms are known to increase during infectious or inflammatory processes, suggesting immune-mediated mechanisms for these symptoms . Not onlThe possibility of a specific connection between streptococcal infection and tic/OCD symptoms has been widely debated in the literature. While some studies have shown a link, others have failed to corroborate these results. A very recent European study did not confirm this specific association between tics and streptococcal infection . The proThe hotly debated connection between GAS infection and tic/OCD symptoms is disputed not only as it relates to symptom flares in diagnosed patients, but also the potential for GAS to trigger abrupt onset CTD or OCD in previously healthy children. The diagnostic term PANDAS – Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus – was introduced in 1998 to describe a presumed subset of acute onset OCD and tic disorders that occur spontaneously in response to GAS infections, primarily in children , 36. ThiIn a timely example of the effects of infections, both tic and OCD symptoms have been shown to sometimes increase in relation to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen behind COVID-19. This increase is thought to be related to both the infectious agent itself as well as to increased environmental stress of quarantining and the effect of social protocols that may encourage compulsions such as cleaning, checking, and hand washing .A 2016 study examining 1,113 patients from the Tourette International Collaborative Genetics Study evaluated 586 patients with CTD and 527 healthy family controls and found that pre- and perinatal complications result in increased incidence of CTD and OCD . These cPsychosocial stress has been linked to the development of CTD and OCD as well as to symptom severity, suggesting that the same is true in TOCD patients –44. Lin either OCD or CTD, confirms that these disorders do not follow simple inheritance patterns, nor are they likely to be exclusively determined by genetic factors. Clinically, we have observed that CTD are prevalent among patients with a parent with CTD or OCD and vice versa, as compared to the general population. Likewise, it is common for one sibling within the family to develop a CTD, while another sibling may develop OCD suggesting epigenetic or environmental contributions to an underlying genetic predisposition. Unfortunately, no definitive data has yet elucidated why one family member may develop a CTD while another may develop OCD or TOCD. This remains an important and outstanding question that requires further study.Taken together, genetic, epigenetic, and environmental factors play a role in driving both tic and OCD symptoms as described above. The strong familial tendency for first degree relatives with a shared family history to develop The overlapping symptomatology of OCD and TS suggests similar mechanisms for reduced cognitive control over motor and behavioral inhibition. Therefore, it is not surprising that ADHD and ASD run comorbid with TOCD because ADHD and ASD both arguably manifest with difficulties of inhibition indicated by impulsivity (ADHD) and repetitive, stereotyped behaviors (ASD).The neurocircuitry of tic disorders and OCD are among the best characterized within neuropsychiatric disorders. In recent years, studies have implicated up to five circuits that play critical roles in the neurocircuitry of OCD and may explain some of the heterogeneity of this disorder , 48. MulThe CSTC circuit is comprised of three primary sub-loops as described below. A full description of these circuits can be found in the medical literature , 48.1.Motor Circuit connects the sensorimotor and premotor cortices to the basal ganglia via the posterior-lateral putamen which in turn projects to the globus pallidus externus (GPe), the globus pallidus internus (GPi), and the subthalamic nuclei (STN). The GPi outputs to the ventrolateral thalamus en route back to the cortex. This loop is involved in habit formation and top-down motor control.The 2.Associative Circuit  involves circuits from the dorsolateral prefrontal and lateral orbitofrontal cortices, which project through the caudate nucleus to the other basal ganglia structures described above. This circuit is thought to play a role in goal-directed behavior.The 3.Limbic Circuit involves the orbitofrontal and anterior cingulate cortices, which project to the caudate nucleus (CN), putamen, GPe, GPi, and STN and thalamus before returning to the cortex. This circuit is involved in motivation and reward.The balance of these loops has been postulated to differ between TS and OCD. While both symptom clusters involve the associative circuit, OCD is thought to more heavily involve the limbic-associative circuits (associated with anxious distress), while TS involves the motor-associative circuits (associated with a somatic premonitory urge)  or somatic (premonitory urge) triggers. However, the relative ry urge) , 55. As ry urge) . Such inThese findings have been supported by fMRI and Positron Emission Tomography (PET) in separate studies of patients with tics or OCD. In a study of four patients with OCD exposed to progressively more distressing triggers, McGuire et al.  correlatAlthough further studies are required, preliminary data suggest that TOCD symptoms may arise from involvement of all three sub-loops as part of an “intermediate” neurocircuitry that lies along the “impulsive-compulsive spectrum” . Indeed,In an attempt to modulate the neurocircuitry for purposes of treatment, deep brain stimulation (DBS) has been used for adults with treatment-refractory Tourette syndrome (TS) since the late 1990s. Several different nuclei within the CSTC network have been explored as potential targets for DBS –62. FourUnfortunately, as described above, TOCD is not independently classified within the DSM-5. It is best but inadequately captured under the diagnostic specifier of “tic-related OCD,” defined as an OCD “diagnostic subtype based on whether the individual has a past or current tic disorder.” As a result, pharmacological treatment for these patients may mistakenly concentrate on medication specific solely to OCD, which is often inadequate for remission of TOCD symptoms. To better account for TOCD holistically when considering the best treatment, clinicians should consider their condition as a case of comorbid OCD and tics disorder rather than a tic-related subtype of OCD .First line agents differ between CTD and OCD . OCD andLike with OCD, tic disorders are felt to respond well to antipsychotic medications but have high risk of side effects. Two in particular, haloperidol and pimozide, are FDA approved for this purpose but are typically deferred until first line agents, the alpha agonists, have been tried. Alpha agonists, such as clonidine and guanfacine, are considered first line agents for tic disorders including Tourette syndrome and act by stimulating the post-synaptic alpha-2A receptors in prefrontal cortical pyramidal cells which may stimulates the frontal cortex to regulate attention and thus suppress tics. They may also decrease arousal. Although generally recognized as less effective than antipsychotic medications, they are favored as first line agents due to their lower side effect profile which can include hypotension and sedation. Children with TOCD often require combined treatment due to their overlapping symptomatology. They are more likely to require polypharmacy and higher dosing regimens .A previous meta-analysis reviewing 15 clinical studies noted that tic symptoms in OCD patients are a substantial indicator of treatment-refractory response following SSRI monotherapy and that augmentation of SSRIs with antipsychotics allows for more sustained improvement in patients with comorbid TS and OCD , 73. SimIn addition, pharmacologic treatment of comorbid ADHD with alpha-agonists, atomoxetine or stimulants can help control the disinhibition and impulsivity that is common with TOCD patients. Historically, stimulants have been avoided in patients with tics; however, a recent Cochrane review analyzing eight studies with 510 participants with comorbid tics and ADHD discredited this cause of concern. It showed that symptoms of ADHD and tics, including impulsivity, actually improved when several stimulant and non-stimulant medications were used, including methylphenidate, guanfacine, clonidine, and a combination of methylphenidate and clonidine. The single exception to this was one study of 3 weeks duration that found exacerbation of tics when using high dose dextroamphetamine . As alwaFirst line agents differ between these disorders. OCD is treated with SSRIs, SSNRIs, and less commonly with TCAs as first line agents. CTD are treated initially with alpha agonists. In both disorders, antipsychotics can be used for augmentation or as primary agents for children who do not respond or cannot tolerate first line agents. TOCD often warrants combined treatment as well as higher dosing regimens due to overlapping symptomatology. Management with antipsychotics is required more often as compared to CTD or OCD alone.Various psychological and behavioral therapies are used as monotherapy or in conjunction with pharmacologic therapy for OCD or TS depending on the patient’s underlying symptoms . CognitiThe focus of these therapies differs based on the identified disorder and the symptoms being treated. CBT and ERPT start by identifying obsessional thinking patterns, and slowly train a patient to tolerate increasing levels of anxious distress while suppressing the desired compulsions. CBIT is arguably more behaviorally focused on learning to recognize premonitory urges and the occurrence of tics which are often out of a patient’s conscious awareness. It considers the circumstances that trigger tics and seeks ways of diminishing these triggers. The emphasis is on finding the best competing response for each individual tic, focusing on one discrete tic at a time. Unfortunately, because tic disorders and OCD are often conceived of as distinct diagnoses, patients are often referred to only one therapeutic modality.and traditional Cognitive Behavioral Therapy (CBT) had significantly greater reduction of OCD symptoms compared to medication alone (ES = 0.85). Because TOCD is even more nuanced than tic-related OCD, not only can its pharmacologic management be challenging but also the type of behavioral therapy employed. Similar to the need for combined medication management of both tic and OCD symptoms, we postulate and have found in our own clinical practice that TOCD responds best to adjunctive behavioral interventions targeting both tic and OCD symptoms. In light of the complex neurobehavioral presentation of TOCD, outcomes are significantly improved by focusing on the anxious-somatic distress of OCD through either CBT or ERPT in addition to targeted behavioral interventions for tics using CBIT. The emphasis on unhelpful thinking styles in CBT or ERPT can decrease the anxiety that triggers tics and repetitive behaviors and help develop strategies for coping with the debilitating nature of TOCD . The risk of bullying, social anxiety and fear of being different from peers can be very real. Children with TOCD often work hard to suppress tics at school to the extent possible, although this may cause them to avoid social activities, become temperamentally more withdrawn, and in some cases worsen anxiety, which has the paradoxical effect of exacerbating their behaviors. Friendships and age-appropriate social development are impeded. Shame and embarrassment may cause children to become withdrawn and decline to share their struggles with parents and providers, exacerbating academic decline and delaying treatment. Children with tics/OCD/TOCD are at increased risk of developing additional disorders such as ADHD, anxiety or depression, which further impede social, emotional, and academic development .It is important for the medical professionals taking care of children with tics, OCD, or TOCD to provide them not only with the right pharmacotherapy and psychotherapy but to provide support for the school and family settings. Family guidance, individual therapy for the child, family therapy (taking into account the needs of siblings as well), adequate contact with the school nurse, psychologist, teachers, and coaches can be very beneficial .For patients with TOCD, it is important to encourage physical activity rather than the use of electronics given the known worsening effect of screen time on the prevalence of tics . VigorouIn addition, there is evidence that sufficient sleep and a good sleep-wake cycle can decrease anxiety and other mood-related comorbidities . We therTourettic OCD is less commonly recognized as a diagnostic entity compared to OCD or tics alone and often necessitates a broad treatment approach and psychoeducation for all involved. Even some mental health clinicians may not be familiar with the unique treatment needs of TOCD. Medication management typically requires polypharmacy, while therapy may require more than one modality such as Exposure Response Prevention Therapy (ERPT) for OCD with concurrent Cognitive Behavioral Intervention for Tics (CBIT). Close communication between parents, clinicians, and schools is beneficial. Furthermore, educating peers, neighbors, coaches, and even non-mental health clinicians who may be caring for the child can be illuminating and may decrease ostracization, bullying, and can increase a child’s functioning by providing a more supportive environment.Since the initial characterization of TOCD  there haTourettic OCD has a later age of onset than classic tic disorders although tics may be the first presentation of TOCD prior to the onset of OCD features. Part of the challenge with TOCD is that symptoms not only wax and wane over time, similar to OCD and TS flares, but that the central nature of the behaviors also evolves over time. Specifically, patients may first present with classic simple tics which morph into complex tics, and later take on the impulsive-obsessional quality of complex repetitive movements that need to be done “just right.” For example, vocal tics may begin as a single vocalization that over time becomes a word, then a phrase, then eventually the need to repeat the phrase multiple times in a particular way. The later tendency to repeat complex behaviors in a specific way, although seemingly akin to compulsions, is distinguished from classic OCD in that the behaviors of TOCD are driven by a somatic urge or feeling of somatic distress rather than anxious, intrusive, obsessional thoughts. Behaviors characteristic of TOCD involve repetitive complex motor movements often with several steps, including tapping, arranging, adhering to certain numbers of repetitions. Patients report that their mixed behaviors are more difficult to suppress than OCD or TS alone.This moving target of symptoms poses diagnostic challenges even to providers familiar with OCD and Tourette patients. The established way of conceptualizing mental health disorders is to put them into specific taxonomic systems , categorThere is a newer trend, however, to think about these disorders as overlapping or spreading across the continuum of clinical presentations. The modern transdiagnostic approaches to neuropsychiatric disorders challenge researchers and providers to think about mental health disorders more broadly than categorizing them into specific symptoms and cause driven nosologies. The interplay between biological, behavioral, psychosocial and cultural processes are not limited to established diagnostic boundaries . Given tTransdiagnostic approaches help us describe and understand cases that share characteristics of both disorders and do not care if they fall into the specific realm of psychiatry or neurology. We are trying to describe processes within one brain comprised of millions of synaptic connections. These neuropsychiatric symptoms do not exist in discrete silos but rather may arise from overlapping neuropsychiatric circuits. Genetics plays an important role in defining the predisposition to OCD or tic behaviors, but epigenetic and environmental factors might determine the varying presentation in different individuals. Environmental factors potentially include dietary habits, the status of gut microbiome composition, physical exercise, or the type of behavioral conditioning that is present in various families. The individual’s socioeconomic status, demographics, family dynamics, access and initiation of pharmacotherapy and behavioral therapy might also contribute to the specific presentations.This review suggests that TOCD exists on a continuum with TS and OCD that crosses the boundaries of each and presents with specific characteristics of both disorders. We propose that TOCD should be included in the next version of DSM as a specific, separate diagnosis that requires a multifaceted therapeutic approach. Our understanding of why some individuals present with isolated TS, others with tic-related OCD, while others develop TOCD, is still very limited. More research should be directed at understanding if any factors can be modulated in the developing brain, given that the multigenic background cannot be changed. Most patients will first present with waxing and waning motor and vocal tics, then subsequently develop OCD tendencies, and a subset of patients will develop TOCD. Perhaps earlier treatment with multiple pharmacologic agents to encourage synergistic effects might help the diagnostic spectrum not to progress too far. Behavioral therapy should extend beyond the boundaries of current techniques, including “customized” CBT, ERPT, and CBIT techniques. We hope that new research avenues will include detailed genetic analysis combined with imaging studies such as functional MRI or PET to elucidate the pathogenesis of these disorders. Collaborations between clinicians and researchers from diverse fields of expertise including psychiatry, neurology, psychology, genetics, and molecular biology would maximize recruitment of patients for large prospective observational studies and randomized controlled studies for interventions specifically addressing TOCD patients. Our hope is that increased awareness of this clinical entity will yield downstream interventions and quality of life improvements for those suffering from TOCD.Tourettic OCD is a unique endophenotype that shares features of both Tourette Syndrome (on the left side of the diagram) and OCD (on the right side). The need to perform tics in a complex and precise way, known as the “just right phenomenon,” is the hallmark of TOCD and lies at the interface of the premonitory somatic urges of tics and the anxious obsessions of OCD. This diagram highlights the unique symptomatology of TOCD as it manifests symptoms of both CTD and OCD.TK and KT contributed equally to this research and writing of the manuscript, including research and new content, and share equal intellectual contribution to this document. JW and TB provided assistance with formatting, literature review, proofreading, creation of figures, and wrote selected sections of the document. GB assisted with research and formatting. All authors contributed to the article and approved the submitted version.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.


Pulsatile tinnitus synchronous with heartbeat is rare and normally has vascular origin: arterial  or venous . Early etiology identification is essential for appropriate treatment to be established. Magnetic angioresonance makes the vascular identification possible and precise. We report a case of arterial anatomical variation in which the treatment was propranolol and clonazepam, showing tinnitus improvement. Tinnitus is a frequent complaint in the office of otorhinolaryngologists and neurologists, but pulsatile tinnitus synchronous with heartbeat is less frequent and may be caused by different arterial or venous vascular etiologies. Identification of the etiology of this type of tinnitus is essential to define the appropriate therapy and to prevent sequelae that may result from late or incorrect diagnosis. Arterial pulsatile tinnitus may result from malformations of arterial-venous fistulas, atherosclerosis, vascular stenosis, ectopic intratympanic carotid artery, persistence of stapedial artery, stria vascularis aberrant artery, high cardiac output . Venous pulsatile tinnitus may be primary  or secondary to intracranial hypertension.The purpose of the present study was to report a case of tinnitus of arterial vascular etiology that was difficult to solve and the approach followed to reach relief of tinnitus.Male 51-year-old patient without systemic or otological disease, complained of tinnitus of pulsatile nature on the left, synchronous with heartbeat, which had started 9 months before and hindered his professional performance (administrative assistant) and resulted in marked anxiety. The patient had taken antidepressants, anxiolitic and vasoactive drugs prescribed by the neurology, without any response. ENT physical examination presented normal otoscopy and the patient had past history of septoplasty two years before the onset of tinnitus. Neurological examination had no significant findings.–Pure tone audiometry – mild decrease in high frequencies  bilaterally.–Brainstem evoked potential did not evidence any abnormalities.–Nuclear Magnetic Resonance of the head, with gadolinium– isolated focus of reduced signal in T1 and elevated in T2, not impregnated by the contrast medium in the left frontal radial crown, suggestive of cerebral lacuna. Basilar artery was elongated and tortuous, describing anterior-lateral loop on the left under the pons.–Head angioresonance - brain images in T2 showed hyperintense focus on left radial crown, which may correspond to small area of gliosis or lacuna infarction. Tortuous vertebral-basilar transition towards the pontine-cerebellum angle cistern on the left .Figure 2We conducted the following complementary tests:In both neuroimaging studies the other head and vascular structures were normal.After excluding the hypothesis of surgical treatment, we prescribed clonazepam 2mg/day associated with propranolol 20mg TID, which made tinnitus more tolerable to the patient.Propranolol is an example of a typical beta-blocker. Its main effects are reduction in heart rate and myocardial contractility, reduction of systolic volume, reduction of cardiac output and reduction of arterial blood pressure. Pulsatile tinnitus may be generated by blood flow turbulence caused by vascular malformations that affect vessel lumen, as observed by comparing the normal anatomy of vertebral-basilar system  with theUnilateral pulsatile tinnitus is not normally followed by specific audiometric alterations and should be investigated by neuroimaging exams to be properly diagnosed. In this case there was no indication for surgical intervention and we decided to prescribe benzodiazepine agent associated with beta-blocker to reduce the perception of tinnitus and heart rate allowing the patient to carry on with his daily activities.


Over the past decade, cancer diagnosis has expanded to include liquid biopsies in addition to tissue biopsies. Liquid biopsies can result in earlier and more accurate diagnosis and more effective monitoring of disease progression than tissue biopsies as samples can be collected frequently. Because of these advantages, liquid biopsies are now used extensively in clinical care. Liquid biopsy samples are analysed for circulating tumour cells (CTCs), cell-free DNA, RNA, proteins and exosomes. CTCs originate from the tumour, play crucial roles in metastasis and carry information on tumour heterogeneity. Multiple single-cell omics approaches allow the characterisation of the molecular makeup of CTCs. It has become evident that CTCs are robust biomarkers for predicting therapy response, clinical development of metastasis and disease progression. This review describes CTC biology, molecular heterogeneity within CTCs and the involvement of EMT in CTC dynamics. In addition, we describe the single-cell multi-omics technologies that have provided insights into the molecular features within therapy-resistant and metastasis-prone CTC populations. Functional studies coupled with integrated multi-omics analyses have the potential to identify therapies that can intervene the functions of CTCs. Liquid biopsies are minimally invasive and have the potential to provide diagnostic and prognostic information that can aid in the treatment of patients with various solid tumours –3. CircuResearch over the past decade has resulted in immense improvements in methods for CTC detection, separation, microfluidic isolation and genomic analysis. Whereas ctDNA can only be analysed at a genomic level, CTCs can be dissected at transcriptomic, genomic and proteomic levels either in bulk or as single cells. The precision medicine era has led to an in-depth investigation of CTCs. In this review, we discuss what is known about CTC biology and explore how single-cell multi-omics has contributed to our current understanding of these rare cancer cells and their potential in personalised medicine.CTCs are shed from the primary tumour and are directly involved in the metastatic cascade based on Paget’s “seed and soil” hypothesis. CTCs are extraordinary cancer cells disseminated from the primary tumour that survive in the systemic circulation and initiate tumour formation at a site distant from the primary tumour. During the metastatic process, cancer cells undergo the epithelial-mesenchymal transition (EMT), through which they acquire the migratory and invasive ability and invade the surrounding stroma. They then intravasate and survive in the vasculature as CTCs, eventually extravasating at distant organs resulting in metastatic tumours , 11. AltThe mesenchymal-epithelial transition (MET), which is the reverse of EMT, plays a crucial role in the colonisation and formation of macro-metastases. CTCs can exist in different states across the EMT spectrum , 15. HowCTCs serve as a privileged gateway to study mechanisms of metastasis and have prognostic and predictive value in breast and other cancers –31. The The activation of EMT occurs at the early stage of metastatic progression. During EMT, carcinoma cells lose their ability to adhere to other cells and the matrix, lose apicobasal polarity and gain mesenchymal characteristics. The gain of mesenchymal-like features enables cells to migrate from the primary tumour  and promEMT is a spectrum rather than a process with binary states –56. ThreThe detection and isolation of CTCs from the blood is routinely performed using the epithelial marker EpCAM , but levCTCs with hybrid E/M properties express aldehyde dehydrogenase 1 (ALDH1), which is associated with stemness, and is expressed in hybrid E/M CTCs together with cytokeratins and nuclear TWIST1  these CTCTC detection could play an important role in the early detection of cancer and recurrence. Phenotype identification and molecular analysis of CTCs are expected to yield a significant amount of information about metastatic disease progression. However, CTCs are rare cells in the blood, are present at very low and undetectable levels. This poses a major challenge for detection, isolation and further downstream applications. Due to their rarity, a preceding CTC enrichment step is essential to separate CTCs based on the physical and biological properties of the tumour cells.+ CTCs ..,. IMC aProteomics studies are far more challenging than nucleic acid analyses mainly due to the complexity of the proteome and the need for highly specific probes and amplification tools. Moreover, highly sensitive detection methods are urgently needed to allow the study of low-abundance proteins and post-translational modifications. The use of proteomics to study rare CTCs using panels that include established therapeutic markers will help discover novel markers and mechanisms behind CTC immune escape. Targeted single-CTC proteomics has the potential to crucial insights into the process of metastatic progression .Liquid biopsies to analyse cell-free DNA, exosomes and CTCs present in the blood can provide information on the tumour in a far less invasive way than a tumour biopsy. Currently, the enumeration of CTCs in a blood sample is used to guide treatment decisions. Although many cancer patients have very low or undetectable levels of CTCs, the presence of CTCs at the time of cancer diagnosis or recurrence is correlated with prognosis, and sequential liquid biopsy collection is used to evaluate disease progression. Several studies determined that between 60 and 80% of breast early breast cancer patients have detectable CTCs , 195. Th+ CTCs [Clinically useful single-cell ‘omics approaches that can reveal prognostic phenotypic information about CTCs are needed. Epithelial markers, such as cytokeratin and EpCAM, are often used to detect CTCs –198. Cur+ CTCs . CTCs thAll single-cell technologies give molecular insight into the makeup of CTCs, which we can correlate with clinical predictors and outcomes to identify CTC features that are detrimental for patients with solid cancers. Targeting these CTCs is challenging, not only because of our limited knowledge of which CTCs cause metastasis but also due to their heterogeneity. In-depth analyses of CTCs might provide insight into the cells that drive metastatic progression since the subclones that can become CTCs are subclones within the tumour. The true drivers of cancer progression might go undetected by performing multi-omics on the tumour bulk because these are rare clones with stem cell properties. The fitness to metastasize is represented in the CTCs, which can be observed in early disease before the existence of metastases. Likewise, drug resistance predictions could be determined based on the molecular makeup of CTCs. Over 90% of patients with cancer die of their metastases . CTCs arHER2 is frequently amplified and overexpressed in breast cancer, and the detection of changes in HER2 gene expression in CTCs might contribute to the effective stratification of breast cancer patients and personalised treatment strategies targeting HER2. Mutations in the ESR1, which encodes an estrogen receptor, can have large consequences on the efficacy of hormone therapy [ESR1 in CTCs [KRAS mutations, which are a strong predictor for resistance to EGFR inhibitors [Targeting alterations in CTCs identified from the tumour with established compounds is an attractive approach to treat cancer. For instance,  therapy , 201. Th in CTCs , 203. CThibitors . These sIsolation and analysis of CTCs from liquid biopsies can offer non-invasive diagnostic and therapeutic information in a wide variety of cancers. Emerging research suggests the existence of different CTC subpopulations with differing phenotypic compositions and metastatic capabilities in individual patients. This underscores the importance of targeted studies to identify the phenotypes and genotypes of CTCs with specific emphasis on characterising their metastatic potential. Although the detection of CTCs remains a challenge due to low numbers of CTCs in the blood, recent advancements in isolation methods and analysis technologies suggest that CTC phenotyping will soon be used clinically.Characterisation of single CTCs via the integration of genomic, epigenomic, transcriptomic and proteomic approaches has shed much light on intra-tumour heterogeneity and therapeutic resistance mechanisms Table  141, 20–209. SinFurther information on research design is available in the Reporting Summary


Stem cell niches are composed of dynamic microenvironments that support stem cells over a lifetime. The emerging niche is distinct from the adult because its main role is to support the progenitors that build organ systems in development. Emerging niches mature through distinct stages to form the adult niche and enable proper stem cell support. As a model of emerging niches, this review highlights how differences in the skeletal muscle microenvironment influence emerging versus satellite cell (SC) niche formation in skeletal muscle, which is among the most regenerative tissue systems. We contrast how stem cell niches regulate intrinsic properties between progenitor and stem cells throughout development to adulthood. We describe new applications for generating emerging niches from human pluripotent stem cells (hPSCs) using developmental principles and highlight potential applications for regeneration and therapeutics. Precise control over stem cell self-renewal and differentiation is essential for proper organogenesis and tissue homeostasis. Immediately juxtaposed to stem cells are specialized microenvironments, termed ‘niches’, containing multiple cell types guiding stem cell behavior. Just as adult stem cells are dynamically regulated by their niche, embryonic niches formed in development equivalently control cell fate decisions to mature from an early precursor or progenitor and eventually to an adult quiescent stem cell. Importantly, embryonic niches also change over these developmental contexts. This review seeks to identify how changes during development in both the stem cell and its niche affect behavior and characteristics of one another. We examine the idea of the emerging niche, defined as initial niches in development that help to capture or support fetal progenitor cells and direct their proliferative nature and eventual generation of quiescent adult stem cells. We specifically focus on niche emergence in skeletal muscle, but we highlight universal themes in emerging niche biology broadly across multiple stem cell systems and compare these with adult niches.The initial niches formed in development are likely the most regenerative and pristine but may evolve over a lifespan or become altered in disease. Niches across tissue systems share common features, such as regulating progenitor to adult cell maturation, self-renewal, and activation states during regeneration.A common theme for many niche systems is supporting waves of developing progenitors and stem cells. One of the best understood emerging niche systems is the hematopoietic stem cell (HSC) niche, which supports two HSC waves that give rise to all blood cells. The first primitive hematopoiesis wave produces transient hematopoietic cells to meet the immediate needs of the embryo, including primitive erythroid progenitors required for oxygenation. Definitive hematopoiesis then generates HSCs that give rise to all mature blood cells and have long-term repopulation potential.Hematopoiesis occurs in discrete anatomical niches, including from the aorta-gonadmesonephros region, arteries, yolk sac, and placenta, that are age-matched for appropriate maturation and lineage specification ,2. FetalInterestingly, another common theme for many adult niche systems is reversion toward the developmental-like niche after an injury to support stem cell regeneration. The brain houses at least two well-studied neural stem cell (NSC) niches in the subgranular zone in the hippocampal dentate gyrus and the subventricular zone around the lateral ventricles ,7. A recThe adult skeletal muscle SC niche is a precise anatomical location between the plasma membrane of myofibers and a laminin-rich basal lamina. In contrast, emerging niches of skeletal muscle loosely consist of minimal basal lamina and immature myofibers that enable progenitor expansion of the rapidly growing muscle ,10.Skeletal muscle precursor/progenitor cells (SMPCs) in trunk and limb originate through segmentation of the paraxial mesoderm, leading to somitogenesis, which results in the formation of distinct anterior/posterior compartments important for specification of the dermomyotome and myotome structures that produce embryonic SMPCs. Key components of the dermomyotome and myotome niches, including signals from the emerging neural tube/ectoderm and mesenchyme, are reviewed elsewhere .et al., unpublished). As multiple myofibers aggregate to form myobundles, a subset of progenitors become encompassed by fusing myofibers, noted by spectrin-positive cross-bridges [Once embryonic progenitor cells emerge from the myotome, one of the first steps in the formation of the niche is cell–cell adhesion between SMPCs and the sarcolemma of newly formed myofibers through interactions with M- and N-cadherins and CD82 ,12. This-bridges   and Aurora kinase pathways were identified as key determinants of SC polarity through peroxisome proliferator-activated receptor-γ and CARM1 [Upon formation of the skeletal muscle niche, cells begin to establish an apical and a basal side, which enables polarity and asymmetric divisions of SCs both to form myoblasts and to self-renew. In contrast, fetal SMPCs are more prone to symmetrically divide . Throughnd CARM1 . In contnd CARM1 . ERBB fand CARM1 . Indeed,nd CARM1 .The calcitonin receptor  is uniquely expressed by quiescent adult SCs and inhibits SCs from escaping the SC niche . The Notin vivo and lack a strong ability to form adult myotubes in vitro [in utero, where the developmental stage would more closely match the fetal niche. In other tissue systems, on the one hand, developmental timing has been shown to be important for cell competition following transplantation in which immature cells lose stemness when transplanted into more mature skin niches [In mouse, a single adult SC transplanted into a recipient muscle can self-renew to form many myofibers and re-establish the stem cell pool  and can in vitro . It reman niches  or immatn niches . On the in vitro systems able to increase PAX7 expression by 1000-fold [in vitro niche that is not equivalent to the typical well-studied adult niche. Across many lineages, differentiation of hPSCs results in phenotypes more closely aligned with embryonic to fetal progenitor cells [in vitro niche for PAX7 SMPC generation. Many supportive cells found in emerging fetal niches . During regeneration in the adult, new myofibers are formed, and PAX7 cells associate with regenerating myofibers [Emerging niches can be generated from hPSC SMPCs that recapitulate the early stages seen in muscle regeneration yofibers . Both inIt is interesting that as niches in development support more progenitor cells than the adult, it could be possible to rejuvenate the aging or diseased niches by recapitulating the niche set points, such as those that are created in development. Recently, overexpression of the Yamanaka factors  in myofibers caused changes to the myofibers, which included upregulation of cytoskeletal reorganization pathways and induced the activation of SCs, which could be similar to the SMPC state and in turn accelerated muscle regeneration in young mice . Regulatin vitro [It has been shown that injecting niche factors that target the receptors and ECM can modulate regeneration. For example, monoclonal antibody augmentation of β1-integrin activity can restore Fgf2 sensitivity and improve regeneration after experimentally induced muscle injury . To imprin vitro . Others in vitro , and depin vitro .It is important to note that the niche is not static but changes over the lifespan to regulate stem cell function in aging and disease contexts . For exaSome of the worst muscle diseases occur from genetic defects in key components of the SC niche. Mutations in laminin-2 or collagen-6 genes result in congenital myopathies and Emery-Dreifuss disease, respectively, and lead to loss of Pax7 cells and muscle wasting . In one A key unresolved question is whether defects in emerging niche formation lead to incomplete maturation of the stem cell or a propensity for birth defects or developmental delays. Convincing evidence has demonstrated that some pediatric leukemias can originate before birth and that these cancers retain fetal niche properties postnatally ,111. UndStem cell niches range in biological diversity across development to adulthood. The niches not only promote regeneration but also regulate the maturation and functional potential of the progenitors or stem cells they support. Niches should be considered as a therapeutic target to readily influence both intrinsic and extrinsic activity of progenitors and stem cells in development and disease. hPSCs offer a powerful system to generate robust regenerative cells, and this could have implications for use across many different cell-based therapeutic applications, especially if they can be directed to generate fully functional adult SCs. Evaluation of the differences in emerging compared with adult niches using new tools, including spatial sequencing, multiomics, and niche-specific computational platforms, will accelerate our understanding of how progenitor and stem cells are controlled differently in each niche state. Harnessing the power of emerging niches could shed light on new approaches to enhance regeneration and support human health see .


As one of the core executive functions, inhibitory control plays an important role in human life. Inhibitory control refers to the ability to suppress task irrelevant information both internally and externally. Modern cognitive neuroscience has extensively investigated the neural basis of inhibitory control, less is known about the inhibitory control mechanisms in aging. Growing interests in cognitive declines of aging have given raise to the compensation-related utilization of neural circuits hypothesis (CRUNCH). In this review, we survey both behavioral, functional, and structural changes relevant to inhibitory control in aging. In line with CRUNCH, we found that older adults engage additional brain regions than younger adults when performing the same cognitive task, to compensate for declining brain structures and functions. Moreover, we propose CRUNCH could well take functional inhibitory deficits in older adults into account. Finally, we provide three sensible future research directions. Inhibitory control is the ability to suppress actions when they are unlikely to accomplish valuable results. Indeed, we are usually controlled by the older habits we have and the external stimuli in the environment more than we realize . Having Contemporary cognitive neuroscience research has also shed light upon the neural basis that supports this fundamental cognitive process. For example, several pioneering studies e.g., , 2004 usAlthough the neural basis of inhibitory control is widely investigated, recently there is growing interest in the age-related behavioral and brain alterations related to inhibition. For example, SST makes it possible to determine the stop-signal reaction time (SSRT), which is a measurement of latency of the processes that underpins the behavioral inhibition. Several earlier studies suggest that older adults have worse performance in SST compared to younger adults . MoreoveSeveral commonalities between these studies were that most studies reported decreased activities in the core inhibitory control region but increased activities in additional brain regions in aging adults. These findings could be explained by the compensation-related utilization of neural circuits hypothesis (CRUNCH), which suggests that older adults engage additional brain regions than younger adults when performing the same cognitive task, to compensate for declining brain structures and functions.a priori required the activity of only one hemisphere, thus favoring an increase in the success rate. In the end, the ELSA model (early-to-late shift in aging) places particular emphasis on the fact that neurocognitive differences found in activation patterns also lead to a differential temporal pattern with age, which seems to show that older adults resort to action control strategies that compensate for deficits  model suggests that age-related reduction in occipital activities accompanied with increased frontal activities is associated with cognitive declines due to aging e.g., . The HARdeficits .Over the several decades, predominant theory suggests that the region within the rIFG/aIns in the human brain is dedicated for inhibitory control. However, there is growing evidence suggesting that inhibitory control should be one of the broader classes that is supported by the same sets of frontoparietal networks [see The remaining part of this review is organized as follows: first we introduce the CRUNCH. After that, we shed lights upon the behavioral and neural alteration in older adults relating to inhibitory control and evaluate the evidence and counter evidence for CRUNCH in inhibitory control ability; we propose that CRUNCH could well explain the functional changes of inhibitory control abilities in aging. Morever, we also investigate on inhibitory control related neurological disease in aging. Finally, we discuss the potential limitations of this review and outline some sensible directions for future research.Before the invention of neuroimaging techniques, traditional behavioral methods and logic in cognitive aging factors view the minimal age differences in cognitive performance between older adults and younger adults are explained by the minimal alteration in neural substrates involved. However, subsequent neuroimaging studies have proven this account is erroneous [see Inspired by research in impaired working memory and the ability to restrain impulses, inhibitory control deficit is proposed to be one of the most essential causes of cognitive declines . The behA recent study recruited 49 healthy participants in a fMRI session and asked participants to perform a go/no-go, a Simon, and a SST. Moreover, these tasks are qualitatively different from each other, especially in terms of response related complexity. For instance, a simple go/no-go task only requires a unimanual response, whereas, Simon and SST could have two alternative responses thus require bimanual responses. These differences contribute quantitatively to inhibitory control demands, and therefore inhibitory control load. The logic is that preparing a single response as in the simple go/no-go task is much easier than in tasks that require choosing between two or more alternatives as in the Simon’s task and SST. Moreover, canceling an ongoing motor actions as required in the SST could further increase the inhibitory control demands comparing to suppressing motor response tendencies. Functional imaging studies indeed provide insights into the alterations in brain activation patterns during aging. Moreover, previous studies have found reduction in white matter tracts between the rIFC, pre-SMA, and subthalamic nucleus (STN) could predict declines in inhibitory control abilities . The assHowever, there is some other counter evidence that argues against the CRUNCH . For insAdditionally, There are notable pieces of evidence detectable from using CRUNCH quantitative model of compensation to characterize compensation in neurological diseases . For exaImportantly, However, pre- and symp-HD individuals demonstrated increased neural activities in the context of intact behavioral performance , which iInhibitory control is an important ability declining with aging but much less investigated. We propose there are several directions for future research: (1) It is important for future research to consider other cognitive demands in inhibitory tasks, for example, The present review focused on the neural basis of inhibitory control and compared the behavioral, structural, and functional changes during aging. Moreover, we evaluated evidence both for and against the CRUNCH and conclude that CRUNCH is useful to take inhibitory control declines in older adults into account. Older adults reach an asymptote in both behavior and brain activation at lower levels comparing to younger adults . The indHowever, experimental evidence against CRUNCH is provided by Our work provided a novel perspective on the brain activation patterns in inhibitory control tasks during aging and foundations for future neuroscientific research. Our conclusion may be biased by the neuroimaging methods and the preexisting conditions given currently available studies are cross-sectional rather than longitudinal according to our knowledge. Future studies should (1) consider other cognitive demands in inhibitory tasks, (2) investigate whether inhibitory control tasks could be utilized as biomarkers for various neurological conditions during aging, and (3) conduct longitudinal research which rules out the possibility of pre-existing conditions.WK: conceptualization, writing – original draft and review and editing, visualization, and funding acquisition. JW and AM: writing – review and editing. All authors contributed to the article and approved the submitted version.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.


Combining synthetic approaches with multi-level phenotyping (phenomics) will supply a detailed, quantitative characterization of how internal and external stimuli shape the morphology and behaviour of living organisms. We advocate integrating high-throughput experimental data with mathematical and computational techniques from a variety of disciplines in order to pursue a comprehensive theory of evolution.Identifying the general principles by which genotypes are converted into phenotypes remains a challenge in the post-genomic era. We still lack a predictive understanding of how genes shape interactions among cells and tissues in response to signalling and environmental cues, and hence how regulatory networks generate the phenotypic variation required for adaptive evolution. Here, we discuss how techniques borrowed from synthetic biology may facilitate a systematic exploration of evolvability across biological scales. Synthetic approaches permit controlled manipulation of both endogenous and fully engineered systems, providing a flexible platform for investigating causal mechanisms This article is part of the theme issue ‘Genetic basis of adaptation and speciation: from loci to causative mutations’. Life is infinitely stranger than anything which the mind of man could invent.The Adventures of Sherlock Holmes (1891)– Sherlock Holmes, ‘A Case of Identity’ in . 1cis-regulatory regions may avoid the pleiotropic effects of mutating proteins [Over a century of advances in molecular techniques have gifted evolutionary biologists greater access to the source of heritable variation than Mendel or Darwin could have imagined . We are proteins , recent proteins . Indeed,proteins  even wheproteins –14. ThesWe face a number of fundamental challenges to elucidating phenotypic evolution through regulatory mutations. On a very basic level, it is still difficult to predict gene expression levels from an arbitrary promoter by an enhancer of known sequence. Recent progress in mathematical and mechanistic modelling has introduced general thermodynamic frameworks for the interactions of regulatory factors with enhancers , and relAdditionally, we have only a limited empirical understanding of the possible paths for regulatory evolution. Most evidence derives from either observational measures of standing variation or biased perturbations of genomic regions that are already known to bind transcription factors strongly. This methodological skew reflects a general trend of relying more on genome-scale experimental data than would be typical for focused experimental studies . Even whHere, we discuss gene regulation as a motivating example of how synthetic approaches can uncover evolutionary principles across biological scales. Many of these approaches are borrowed from synthetic biology, an interdisciplinary field that emphasizes rationally designing organisms to execute predefined functions, predicated on a practical understanding of the genetic and biophysical causes behind phenotypic features. Recent advances in synthetic technologies and theories have targeted throughput and control across multiple levels of biophysical and functional organization ,26. We o. 2Studies of transcriptional regulation often focus on developmental enhancers, which specify the precise time, level and location of gene expression during embryonic growth  and therin vivo [Drosophila melanogaster used an automated robotics pipeline to survey an unbiased mutation library for a developmental enhancer, finding that almost all mutations altered gene expression and that the location and levels were highly interdependent [We start by exploring how synthetic approaches may clarify regulatory logic. Mutational scanning provides a powerful means to assay the activities of regulatory elements and to explore evolutionary potential –38. Thesin vivo . A recenependent . The resependent  and promependent , indicatependent ,48. In tMost studies such as the one just described rely on reporter constructs to evidence phenotypic responses to genotypic changes. While reporter gene-based assays can provide information on the activity of individual regulatory regions, there are several potential caveats to generalizing the conclusions. First, removing an enhancer from its native location and placing it in a novel chromatin environment may facilitate the binding of transcription factors to sites that are not used in their native context . Second,in vivo [Classical reporter-gene assays can be complemented by a number of methods and techniques from synthetic biology for interrogating native loci. Unlike screens performed in transgenes, assays that modify endogenous sequences can be used to directly test the physiological effects of different genetic variants. This capability is crucial given that changes in gene expression levels alone are not necessarily correlated with changes in evolutionary fitness . New tecin vivo –73. Actiin vivo , introdueven-skipped stripe two (eveS2) enhancer, one of the most well-characterized developmental enhancers [Coupling synthetically perturbed enhancers with live imaging holds enormous potential to shed light on how enhancers establish precise expression patterns and confer phenotypic robustness. Recent technological advances have enabled real-time imaging of transcription by tagging mRNA with multiple repeats of a stem-loop sequence that is recognized by a binding protein fused with a fluorescent protein. The resulting ‘transcription spots’ are then visualized using standard live microscopy techniques , revealinhancers , is actinhancers  and drivnhancers . Simultanhancers  will be We have presented here just a few of the available techniques for modifying regulatory regions and measuring their effects. Future efforts in technological development should focus on comprehensive approaches to interrogating regulatory elements in their native context. Analysis of the resulting data will be facilitated by our enhanced capacity to detect genetic variants with small effect sizes, as well as continued improvements to the sensitivity and throughput of live-imaging methods. With a combination of these approaches, we may begin to tease out the precise relationships between regulatory sequences, transcription factors and the expression patterns of target genes—the first step toward understanding the potential of genetic variants, realized or unrealized, to influence downstream phenotypes.. 3Synthetic approaches suitable for individual regulatory regions naturally extend to the study of gene regulatory networks (GRNs), or functional groupings of genes that produce phenotypes from genotypes by modulating expression levels in response to internal and external signals. Even small GRNs can execute involved processes such as disturbance rejection  and foldAt the level of networks, understanding causal relationships between genotypes and phenotypes is complicated in a number of ways. If a regulated gene is itself a transcription factor, then downstream genes may constrain the minimum or maximum expression of the gene . CompounSynthetic biologists have developed a toolkit sufficient for constructing entirely synthetic GRNs from engineered enhancers, promoters and transcription factors in bacteria and eukaryotes . ConsideFor example, networks are subject to hard limits on their functionality that shape, directly or indirectly, the optima of the fitness landscape. Mathematical theory has elucidated fundamental limits to such functions as noise suppression , fold-chstrength of regulatory interactions typically determines module behaviour, such that modules may vary in their robustness to enhancer mutations or noisy gene expression depending upon their quantitative biophysical properties [Similarly, both evolutionary and synthetic biologists have investigated the degree to which GRNs are modular —the formoperties ,130–132.The degree to which modules are (i) separable from the context of the full GRN or whole cell and (ii) sensitive to variation in topology or biophysical parameters begs a host of evolutionary questions. Do less parameter-sensitive modules exhibit greater standing variation? Are parameter-sensitive modules more likely to be evolutionarily co-opted? How do the relative sensitivities of overlapping modules determine evolvability –136? To a priori. For example, it has been shown that the set of regulatory interactions among three genes in a synthetic circuit biases the phenotypic variation observed after random mutation to the regulatory regions, even if the initial phenotypes are identical [A straightforward way to proceed involves using the techniques discussed in the previous section to simultaneously disturb multiple regulatory regions belonging to the same GRN. It may be difficult, however, to infer causation from experiments conducted on complex and incompletely characterized endogenous systems. A complementary approach is to explore evolutionary principles in simple synthetic networks whose constituent parts can be individually measured and manipulated, and whose functions are understood dentical . Similardentical .cis-regulatory variation to the output of GRNs will require new techniques and tools capable of manipulating and monitoring multiple genes simultaneously. Such detailed study of regulatory regions in context will be essential for understanding biophysical and evolutionary constraints, and therefore for predicting phenotypic consequences on larger organizational scales.Truly leveraging insights from simple synthetic modules will require theoretical and experimental work to predict the behaviour of full networks from constituent components under interconnection . Such a . 4the missing heritability problem [Historically, most efforts to study adaptation have involved providing lists of genetic variants that influence the phenotype of interest. Large-scale approaches such as genome-wide association studies identify regions correlated with the observed phenotypic variation, but often fall short of functional validation ,143. Thi problem  that limin situ hybridization or immunostaining methods [Phenomic characterization is becoming more feasible as high-throughput phenotyping technologies become more available, ranging from transcriptomics and epigenomics to proteomics and metabolomics. Techniques that can link single-cell data to tissue architecture will be particularly useful, as bulk datasets tend to hide effects that are restricted to specific cell types or regions but that could deeply affect organism-level, fitness-related phenotypes such as mating behaviours or survivability. Recently, single-cell RNA-seq has been performed on relatively large samples from various species –146 incl methods . Moving  methods –153. Eve methods  or free- methods  animals.phenotypic layers , thereby overriding the need for genetics to act as the preferential level of causal explanation [Though it is often assumed that causality runs from genotype to phenotype, phenomics characterization could reveal causal links between lanation . New synlanation . Thus, ilanation , alter nlanation  or induclanation  with preleverage environmental cues are still scarce [A comprehensive understanding of genotype-to-phenotype mapping will also necessitate a mechanistic description of how regulatory networks integrate the enormous range of environmental stimuli that shape phenotypic outputs. The fact that environmental inputs can modify developmental programmes and lead to altered phenotypes, a process called phenotypic plasticity, has been deeply studied by ecologists and geneticists ,161. Devl scarce .An underlying assumption of experimental biology is that the laboratory environment allows researchers to control external conditions and thus standardize the effect of the environment on the system under study. This approach, however, does not minimize the environmental dependency of the analysed phenomenon; it just provides a set of standard conditions that are used to compare different experiments from which more general rules and principles are then derived. Thus, many conclusions obtained from laboratory experiments do not hold when they are tested in natural environments (reviewed in ). If theFor these reasons, we advocate a deeper exploration of external conditions when studying genotype-to-phenotype mapping, beyond the optimal laboratory environment and closer to the actual stimuli that the studied organisms can find in their natural environments. Focusing on systems in their natural context with functional biology is gaining traction in the evolutionary community . We propEnvironmental effects on phenotype could also be systematically and selectively interrogated through synthetic interventions that trigger, disable or modify signalling pathways naturally linked to selective stimuli, such as nutrient-sensitive pathways , oxygen-Phenomics approaches will produce huge datasets that vary significantly in content as well as the method and framework of acquisition. How to integrate information and extract meaningful conclusions from diverse, high-dimensional datasets may become one of the most urgent biological challenges in the near future . AdCollectively, the technologies and methods discussed here would allow researchers to empirically test the principles of genotype-to-phenotype mapping through a wide variety of synthetic approaches coupled with phenomic characterizations and controlled exposure to a range of realistic selective stimuli. The proposed experimental pipeline could identify and modulate contributing factors across biological scales and phenotypic levels up through environmental interactions, thereby providing a framework for the causal understanding of adaptive responses. Furthermore, the ability to measure multiple phenotypic levels in individual organisms will permit correlating stochasticity or variability across organizational scales, illuminating an otherwise elusive means by which phenotypic variation may be generated –177. Sys. 5In the previous sections, we have outlined synthetic approaches to perturb individual regulatory regions and GRNs, as well as emerging technologies for manipulating and characterizing phenotypes at the level of cells, tissues or organisms. Here, we demonstrate the joint potential of these approaches using metabolism as a case study. Metabolic networks—which have been the subject of intense study by both evolutionary and synthetic biologists—are more complex than GRNs, as they are not only regulated by transcription factors, but also respond to changes in the concentrations of metabolites that are themselves part of the network. This additional regulatory layer is essential for quick responses to a constantly changing extracellular environment ,179. ThuExplaining the mechanisms that could originate such intricate networks constitutes a major question for evolutionary biologists. Although metabolic engineering has been mostly driven by industrial motivations , the quest to optimize bacteria or yeast strains to produce commercially viable compounds has also provided insight into the biophysical constraints, and thus the evolutionary potential, of metabolic networks. Metabolic engineering ranges from single genetic alterations that enhance an endogenous metabolic route ,182 to tA comprehensive understanding of metabolism may be even more relevant to studying evolution in multi-cellular than unicellular organisms, given that metabolic processes, in addition to managing the bioenergetic requirements of cells, can directly alter signalling pathways and developmental programmes . FoIn sum, as technological advances increase our throughput to measure metabolism ,195, we . 6Deep comprehension of the mechanisms behind genotype-to-phenotype mapping is essential for predicting adaptive evolution. Charting the map will require a concerted effort to blend mathematical theory and computational modelling with large-scale experiments yielding high-dimensional quantitative data. Synthetic approaches are a particularly promising avenue for empirically testing ideas inferred from genomic and morphological studies of extinct and extant lineages. The ability to manipulate individual genetic sequences has already produced insights into the origins of pleiotropy at the regulatory and network levels. Advancements in high-throughput methods for synthetic perturbation and phenomic measurement promise to elucidate macroscale morphological and behavioural changes relevant to natural selection. The sizable resource investment required by this ‘synthetic synthesis’ will be justified to produce new, qualitatively diverse data sufficient to identify not only causal factors behind specific phenotypes but also general rules for how perturbations to genotype are propagated across phenotypes. In this way, we might finally begin to develop a detailed understanding of the multi-scale interactions and constraints that shape the evolvability of biological systems.E. coli strains that found network linkages synthetically introduced into endogenous GRNs tend to enhance adaptability to selection pressures [E. coli have been observed to evolve under experimental conditions, despite the fact that the metabolic rewiring required to generate these two interactions is theoretically no less complex than to generate the other outcomes [Despite recent successes mapping targeted genomic perturbations to phenotypic outcomes, we have yet to harness the full potential of experimental evolution combined with synthetic approaches. For practical reasons, most existing efforts have focused on microbial systems, including a study of almost 600 ressures . Yet manoutcomes . The abiD. melanogaster proposed that microbiota contribute directly to speciation in host organisms [Synthetic approaches appropriate for microbial studies provide direct inroads into controlled experiments involving multi-cellular organisms. Gut microbiota are one avenue of particular interest, as they are the subject of significant medical attention as well as a growing body of evolutionary work ,199. Onerganisms , but subrganisms . The comAs technological and theoretical progress continues, it will be crucial to extend the application of synthetic approaches to experimental evolution in multi-cellular organisms. A straightforward first step involves using mutational screens in transgenes or endogenous loci to generate sexually reproducing populations with varying degrees of genetic variation, in order to systematically quantitate how heterozygosity and genetic diversity influence evolutionary fitness. Given several starting populations homozygous at the allele(s) of interest, it should be possible to quantify the effects of heterozygosity in all possible pairwise couplings before moving to mixed populations of three or more lineages combined. This sequential, ‘tiered’ approach could be especially important for teasing out combinatorial effects, such as the recent observation that larval phenotypes in annelids result from the interplay of three genomes: maternal, paternal and zygotic . Mixed pSimultaneously, synthetic approaches should be leveraged to deepen our understanding of how GRNs evolve, which will be crucial for interrogating emergent features of complex multi-cellular organisms. For example, the flexible relationship between network structure and function affords greater potential for phenotypic variation than has been described in natural systems. While individual modules of fixed topology may be constrained in their evolvability, it appears that larger or more complex networks may be less constrained, due to the presence of redundant or ‘buffering’ mechanisms as well as the combinatorial increase in possible compensatory mechanisms. Future experiments with fully synthetic networks will permit more detailed research into the evolutionary potential of GRNs that interact to varying degrees with endogenous components at different times during the life cycle of an organism. Key to such studies is the ability to construct topologies unencumbered by evolutionary lineage and to sample a larger range of parameter space than could be expected to persist in a naturally evolving population. Eventually, the general principles derived from such experiments could be used to build circuits that directly influence fitness, in order to systematically investigate the evolvability of biological networks under controlled selection regimes.Finally, multi-cellular organisms present unique challenges and opportunities for evolutionary study that should be both considered and capitalized upon. Notably, animals and plants possess a plethora of cell types and tissues that differ in behaviour almost exclusively due to differences in gene regulation . PhenomiThough their motivations differ, evolutionary and synthetic biologists are both concerned with the mechanisms that decode genetic variation into phenotypic variation. Inspired by this convergence, we have illustrated here just a few of the ways in which synthetic approaches may shed light on outstanding problems in the evolution of genetic regulation by transcription factors. The framework we propose applies equally well to synthetic interventions modulating protein function –205, gen