The introduction of engineered organ-on-a-chip platforms has brought a revolutionary dimension to the current fibrosis studies and discovery of anti-fibrotic therapeutics. Advances in human induced pluripotent stem cells and tissue engineering technologies are enabling significant progress in this field. Some of the most recent breakthroughs and emerging challenges are discussed, with an emphasis on engineering strategies for platform design, development, and application of machine learning on these models for anti-fibrotic drug discovery. In this review, we discuss engineered designs to model fibrosis and how biosensor and machine learning technologies combine to facilitate mechanistic studies of fibrosis and pre-clinical drug testing.Human fibrotic diseases constitute a major health problem worldwide. Fibrosis involves significant etiological heterogeneity and encompasses a wide spectrum of diseases affecting various organs. To date, many fibrosis targeted therapeutic agents failed due to inadequate efficacy and poor prognosis. In order to dissect disease mechanisms and develop therapeutic solutions for fibrosis patients,  Human fibrotic diseases constitute a major health problem worldwide owing to their high incidence rate in most chronic inflammatory diseases . The datFibrosis is generally initiated by a tissue repair process, followed by a scarring phase over the regeneration limit . The repin vitro models that can faithfully recapitulate human pathophysiology.Organ transplantation is the only therapeutic option for many end-stage diseases given the inefficiency and poor prognosis capabilities of fibrosis-modifying therapeutics . Drug diin vitro monolayer-based assays. Animal models, despite being the gold standards in current drug testing, present considerable discrepancies in both physiology and pathology compared to humans. Moreover, monolayer cell cultures are cost-effective alternatives for pre-clinical studies and have been widely used as established systems for pre-clinical drug testing. However, many of these models poorly replicate pathological factors responsible for disease onset and progression, because they lack of the structural and compositional complexity of native human tissues.Conventional disease models mainly rely on animal experiments and in vivo environment such as functional and structural properties, and molecular and metabolic signatures over the course of drug treatment. Ideally, it should also allow real-time and non-invasive tracking of multiplexed functional outputs  in response to drug candidates under investigation.An ideal model for fibrosis studies and drug development should  produce in vitro models that are not only capable of modelling of fibrotic disease, but also test feasible therapeutic strategies. Organ-on-a-chip (OOC) technology, as a multi-disciplinary field that combines bioengineering, regenerative medicine, and platform manufacturing, has led to the establishment of authentic bio-systems to emulate human biology and diseases. This field has grown appreciably over the past decades with versatile approaches to generate disease- and patient-specific models for preclinical drug testing. Some of the most recent advances in modeling of fibrotic disease will be discussed, with an emphasis on the development of engineering techniques in the aspects of platform design, tissue construction, biosensor integration, and machine learning aided testing , and plasminogen activator inhibitor-1 (PAI-1), which directly recruit and activate fibroblasts to produce ECM proteins . In addiAs a key process of wound healing, various immune cells engage in the manifestation of fibrosis after tissue injury. Macrophages are an important source of matrix metalloproteinases (MMPs) and inflammatory mediators that drive the initial tissue response after injury. Specifically, pro-inflammatory macrophages are predominant during the initial phase of wound healing, secretes pro-inflammatory cytokines, and produces MMPs for ECM degradation, which have potential antifibrotic effects. In later phases of wound healing, reparative macrophages secrete anti-inflammatory and profibrotic cytokines, such as Transforming Growth Factor β (TGF-β), Platelet Derived Growth Factor (PDGF), Insulin-Like Growth Factor 1 (IGF-1), and Vascular Endothelial Growth Factor α (VEGF-α), in response to inflammatory factors, such as interleukin 10 (IL-10) . With thECM cues refer to undergoing dynamic, structural and biomechanical changes in the extracellular matrix during fibrotic diseases. Because of the continuous activation of myofibroblasts, extracellular matrix proteins are excessively deposited in the injury sites. A hallmark of all fibrotic tissues is an increase in ECM stiffness. Major drivers of ECM stiffening include phenotypically converted myofibroblasts, activation of TGFβ pathway, and matrix cross-linking. For example, fibrotic myocardium is reported to have up to a 10-fold increase in Young’s modulus compared to normal myocardium .in vitro fibrosis model.The increased stiffness is sensed by the resident cells through mechanotransduction by the surface integrin receptor and triggers downstream pathways such as Hippo followed by YAP/TAZ, which contributes to the upregulation of the profibrotic gene expressions . The proDecellularized ECM may represent an ideal substrate for fibrosis modelling as it preserves the intrinsic biochemical and topographical micro-environments. ECM derived from hypertrophic myocardium in swine model was decellularized and reseeded with human cardiomyocytes . The resin vitro and in vivo  complex in ECMs. Macrophages can also produce TGF-β during the inflammation process. After binding with receptors on the cell surfaces, Smad 2, 3, and 4 will form complex and translocate to the nucleus to upregulate transcription of profibrotic molecules, such as α-SMA, collagen I, and tissue inhibitor of matrix metalloproteinases (TIMP) hence induce myofibroblast transformation of the cells , Rho GTPase 1, and T-type calcium channels, as well as increased proarrhythmic early after depolarization events, along with significantly reduced peak systolic stresses. In a recent study from our group, a Biowire II disease model was constructed to recapitulate myocardial response to Ang II in a temporal manner and evaluate compounds that target Ang II-induced cardiac remodeling (Angiotensin II (Ang II), the main peptide of Renin-Angiotensin System (RAS), is believed to induce progressive deterioration of organ function. Its upregulation is implicated in various pathological conditions such as cardiac hypertrophy and fibrosis, offering another important mediator for the disease model development. Ang II can induce fibrosis indirectly by activating proinflammatory mediators such as cytokines, chemokines, adhesion molecules, and directly by regulating extracellular matrix synthesis and degradation, increasing tissue stiffness and leading to the impaired function. modeling .Many other pro-fibrotic mediators recently came into focus, such as IL-10 cytokine family members. These pro-inflammatory cytokines exert host defense mechanisms and facilitate tissue healing . Recent in vitro fibrosis disease modeling have been created to recapitulate chemical, mechanical, and biological conditions in fibrosis of various organs  citrate] (POMaC) wires to recapitulate contractile, biomechanical, and electrophysiological complexities of fibrotic myocardium . The strin vitro model of human cardiac fibrosis was developed using human fetal cardiac fibroblasts (hfCF) encapsulated in a gelatin methacryloyl (GelMA) hydrogel. Subsequent TFG-β1 stimulation in hfCF-laden GelMA resulted in a fibrotic phenotype characterized by myofibroblast activation and ECM accumulation. This fibrotic model was utilized to investigate the effect of cardiac progenitor cells (CPC) on the disease process, supporting previous in vivo findings of the anti-fibrotic potential of CPC in conjunction with hfCF (Another engineered ith hfCF .in vitro model of human liver fibrosis was developed in a hepatic organoid system. Multilineage 3D spheroids composed of hepatocytes (HepG2) and hepatic stellate cells (LX-2) were constructed and exposed to free fatty acids to induce steatosis and fibrosis. This fibrotic organoid model was used to study molecular mechanisms underlying lipid accumulation induced fibrogenesis and screen compounds that are in clinical trials for the treatment of NASH -derived lung organoids were generated and showed to recapitulate important phenotypes of Hermansky-Pudlak syndrome-associated interstitial pneumonia (HPSIP). They further validated the hiPSC-derived lung organoids as a useful tool to identify potential drug targets. Genome-wide expression analysis from the HPSIP mutant fibrotic organoids deciphered the crucial role of interleukin 11 (IL-11) in the fibrotic process .in vitro models of other organs and tissues. in vitro  based biosensors were developed to measure molecular events that occur relatively fast in single cells or engineered tissues with high signal to noise ratio and spatio-temporal resolution .via a process dependent upon an intact cytoskeletal network. In a follow-up study, the authors further developed two membrane-anchored FRET biosensors of different spectra and used the system to concurrently visualize the activities of Src and MT1-MMP in a single live cell. The result delineated the temporal and spatial differences between MT1-MMP and Src activation upon EGF stimulation  activity in living cell culture. In this report, Electrophysiology is an important feature of electroactive tissues including cardiac and neuronal tissues. The fibrotic process is particularly detrimental as the heart is characterized by limited regenerative and repair capacity. In the case of cardiac fibrosis, overpopulated cardiac fibroblasts result in ECM deposition and hence altered cardiomyocyte electrophysiology and disrupted electrical impulse conduction of the myocardium . The actMicroelectrode array (MEA) is a widely used approach for recording electrical signal as it can be easily integrated in microphysiological systems. Traditional MEAs are confined to 2D substrates that render spatiotemporal electrical recordings of a single plane. More recently, 3D bioelectrical interfaces were reported, enabling a more complex electrophysiological readout. in situ recording of conduction velocity is of increasing research interest. Cardiac conduction is another important functional readout of the heart and is often impaired by fibrotic remodeling . OpticalBeyond the cardiac system, integrated electrodes are also applied in other organ models. These applications include the assessment of barrier integrity of cultured cells using trans-endothelial electrical resistance (TEER) measurements. Overall, chip embedded electrochemical sensors could be used as a promising substitute for conventional end-point assays for studying fibrosis in microphysiological systems. In future, this fast-developing field has great value in illuminating mechanisms of diverse fibrotic diseases and accelerating discovery of novel anti-fibrotic drugs.Fibrosis represents a major health problem worldwide, yet there are still limitations in current knowledge of the disease mechanisms. Development of antifibrotic therapies relies on comprehensive understanding of profibrogenic mechanisms in organ-specific systems. Due to heterogeneity in disease etiology and phenotypes, and lack of clinically validated biomarkers, there is a no effective disease-modifying therapeutic agents specific to fibrosis. Conventional treatments rely on pharmaceutical compounds targeting inhibition of fibrosis-related molecular pathways. Various potential novel strategies have been investigated to combat fibrotic tissue damage in different organs .In the past few decades, research has highlighted potentially targetable pathways and related individual factors involved in the fibrogenesis process. These include transforming growth factor-beta (TGF-β), which has long been recognized as a master activator in fibrosis . TGF-β aThere is a limited therapeutic feasibility and unpredictable off-target effects in direct targeting of TGF-β, due to the functional pleiotropy of this growth factor in a wide variety of complex biological processes. Recent discoveries in fibrotic pathways and factors regulated by TGF-β have facilitated the identification of alternative therapeutic targets and showed promise in achieving an attainable inhibition of fibrotic response .Fibroblast growth factor 2 (FGF2), one of the members of the mammalian fibroblast growth factor family, is a cytokine with well-documented cardioprotective effects to attenuate fibroblast activation through antagonizing TGF- β signaling. in vitro and in vivo investigations, and to develop small-molecule agonists for systemic administration.Bone morphogenetic protein-7 (BMP7) belongs to the TGF-β super family and is found to have an anti-fibrotic role that is correlated with the inhibition of TGF-β-mediated profibrotic signaling . VariousPeroxisome Proliferation Activator Receptors (PPARs) are nuclear transcription factors that form obligate heterodimers with retinoid-X receptors to modulate the transcription of target genes. Preclinical studies have implicated all three PPARs as potential targets for antifibrotic therapy .β-catenin, the central signaling component of the canonical Wnt pathway, is believed to serve a modified target for fibrosis through activation of TGFβ pathway. Recently, several novel components of the TGF β have been discovered, including proprotein convertases (PCSKs), which is in a family of serine proteases that cleave secreted peptides. PCSKs have been reported to be involved in the activation of the TGF-β/Smad signaling pathway. Furin is one of the most ubiquitously expressed PCSK and has been implicated as a potential therapeutic target in fibrosis .The Rennin–angiotensin–aldosterone system (RAAS) plays a critical role in mediating systemic blood pressure, fluid volume, and sodium balance. Overactivity of RAAS is believed to have a pivotal role in the onset of pathological fibrosis. Angiotensin II, the main peptide of the RAS system, has been demonstrated to induce fibrosis in various organs. Many studies reported the critical role of Ang II in the cardiovascular, renal, and pulmonary systems  under pain vitro and in vivo models and shown effects on reducing fibroblast proliferation, and suppressing collagen deposition and TGF-β1 expression. Similar with ACEIs, ARBs are also frequently prescribed to treat hypertension and have comparable clinical profiles regarding anti-inflammatory and anti-fibrotic outcomes. ARBs compete with the binding of angiotensin II to AT1 receptors, thereby cause angiotensin II receptor blockade. Clinical trials looking at the potential utility of an angiotensin II type 1 receptor blocker (ARB) have demonstrated significant benefit in the prevention of myocardial fibrosis  inhibitors, Angiotensin Receptor Blockers (ARBs), and mineralocorticoid receptor antagonists has been documented to prevent fibrosis and slow the decline of organ function effectively in the process of kidney disease and cardiac hypertrophy . ACE Inhfibrosis .Direct Renin Inhibitors (DRIs), Mineralocorticoid Receptor Antagonists (MRAs) as other means of RAAS blockade also have been studied . HoweverDeveloping novel and effective anti-fibrotic therapies through targeting Ang II pathway appears an attractive strategy. Over the past decades, many novel compounds such as kinases inhibitors and hormone peptides showed promising results in alleviating Ang II signaling and compensatory fibrotic remodeling. For example, relaxin, an endogenous peptide hormone in the insulin family, has received much research attention due to the increasing evidence on this peptide hormone’s beneficial effects in fibrotic conditions. Relaxin binds with its endogenous receptor, relaxin family peptide receptor 1 (RXFP1), inhibiting the actions of major pro-fibrotic factors including Ang II .Current fibrosis therapeutic development technologies are aiming at reducing off-target effects and promoting localized action to affected or fibrotic regions. The development of biomaterial-based delivery methods offers opportunities to improve target specificity and reduce adverse effects. Nano- and micro-particles as carriers for the delivery of can improve transport efficiency and allow more circulation time for the cargo molecules to reach the disease site .via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 in a mouse model of peritoneal fibrosis.Different polymeric microspheres and microparticles have been extensively studied as drug-laden depots to achieve localized delivery of cargos including anti-fibrotic molecules. For example, poly (lactic-co-glycolic acid) (PLGA) microspheres were used to encapsulated resveratrol (RSV) and embedded in a gelatin methacryloyl (GelMA) hydrogel matrix for the treatment of epidural fibrosis . SustainIn the nanoscale range, there are successful applications of the nanoparticle-based delivery for the treatment of chronic human diseases including cancer, diabetes, and fibrosis. The advances in target-oriented delivery facilitate the therapeutic agents to anchor to their target location for maximized therapeutic function and reduced off-target effects. Controlled systems for delivering therapeutic agents to myofibroblasts have been explored for the treatment of fibrosis . Cell suCell therapy aims to introduce new, healthy cells into a diseased or injured locus, to replace the pathological or missing native cells and to restore organ function. Mesenchymal stromal cells (MSCs) have shown great potential in the treatment of inflammatory diseases and mediating profibrotic factors through their immunoregulatory properties . Averyan+ T cells. However, establishing the Car-T cell therapy in humans could take years of development. The therapy is also very expensive and accompanied by severe off-target and side effects. Continued efforts in the identification of antigens that are expressed by activated cardiac fibroblasts may contribute to the development of T cell therapy of fibrosis.Chimeric antigen Receptor (CAR) T-cell therapy is a form of immunotherapy that uses engineered T cells to target specific antigens on cancer cells. Patient-derived T cells are collected and modified to produce a modified T cell receptor or CARs on their surface . Recent Gene therapy has shown promising results in the treatment of a wide spectrum of human diseases. Gene therapy is normally generated by transferring specific genetic information into target cells to correct mutated genes that cause diseases. Many recent technologies, including genetic editing, recombinant DNA technique, mRNA, and miRNA technology, have been utilized in improving the performance of gene therapy in disease context.MicroRNAs (miRNAs) are a class of small non-coding RNAs that play a role in regulating the expression of messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including fibrosis . MediatiMachine learning (ML) is a rapidly growing field of artificial intelligence research influencing drug screening approaches and enabling new methods of drug discovery . GeneralA variety of ML methods were applied to uncover drug candidates or putative targets for drug discovery, for instance the use of support vector machines, optical flow, and tree bagger algorithms, among others. The targets of ML in the drug screening field have focused on analyzing the effects of drugs on tissue contraction, cardiotoxicity, and electrophysiology. Although current drug-centric ML approaches have not specifically focused on anti-fibrosis drugs, we will briefly review several machine learning-based methods for drug discovery and screening applications. We believe these current implementations will help establish the groundwork for future ML studies related to fibrosis as they involve similar assays and datasets.In the early stages of drug discovery there are frequently thousands, or tens of thousands of potential candidate molecules. Manual analysis of such tremendous volumes of candidates is extremely labour intensive, thus, recently this process is being enhanced using novel ML methods.Drug repositioning is a common technique in industry where after research and testing, a molecule is found to be effective on treating a different condition. Due to tremendous volume of approved compounds, ML approaches lend themselves well to determining potential repurposing targets. A novel ML approach for this problem was developed by To reduce the large volume of drug candidates, a compound’s molecular structure can be investigated to predict how it may interact with desired biological bind sites. This can be accomplished through computing the standard binding free energy between particular compound and a binding site. Using the technique of optical flow to measure cardiomyocyte monolayer contractions, The effects caused by a compound on the human ether-à-go-go-related gene (hERG) pathway is an important factor when testing for cardiotoxicity as inhibition can cause fatal cardiac arrhythmias . To predA similar study on identifying hERG pathway cardiotoxicity using ML was done by A method developed by The use of OOC disease models, as a viable alternative to traditional pre-clinical testing methods, is improving in verifications with regulatory environment and facing increasing industrial acceptance in the past decade. The upward trend in optimization, regulation, ratification, and adoption of these engineered models promises to lay the foundation of next-generation precise medicine. However, stumbling blocks remain for the widespread adoption of OOC disease models.One of the biggest challenges of hiPSC-based disease modeling is the experimental heterogeneity due to lineage variability and instability. Heterogeneity can result from a multitude of factors such as donor-specific genetic and epigenetic status, as well as clonal variations including incomplete reprogramming and changes during culture. This can hinder reproducibility and confound biological phenotypical differences. In the context of fibrotic disorders, the synergistic interaction of multiple cell types is often involved to manifest specific disease phenotypes. As a result, it is of great importance to construct the model using subpopulations of the same genetic background. The use of standardized differentiation and maturation protocols and cell sorting-based phenotypic selection can potentially minimize variability and thereby increase the reliability of phenotype comparisons in disease modeling. In addition, recent studies describe elegant protocols for the derivation of cardiac fibroblasts from genetically defined hiPSCs . Alternain vitro study of metabolic cardiomyopathies. Whilst maturity level in these engineered models is still not on par with the human level, these methods provide a significantly more adult-like hiPSC-derived cardiomyocytes. Continuous effort in improving tissue maturation is essential for developing more physiological relevant disease models.The structural and functional immaturity of hiPSC-derived cells poses another challenge for the direct application of OOC disease models. To this end, enhanced tissue maturation can be achieved using external signals, including electrical, mechanical, and hormonal stimuli. in vivo anastomosis  or hot-embossing (136), can significantly scale up the production of OOC platforms. On the tissue construction side, self-assembly is a widely used process by which cellular components adopt a defined structure without external guidance to achieve complex tissue and organ-level organization and function . Additionally, recent advances in 3D bioprinting facilitated multi-scale cell micropatterning and construction of tissues with complex topography . These techniques collectively allow rapid and reproducible automated construction of biomimetic organ-like structures. Once the manufacturing process becomes more streamlined and reproducible, widespread adoption of OOC disease models will be accelerated.in vitro models to understand the processes regulating complex human physiology and pathology. Although the application of these platforms is still confined within the research laboratories, awareness of this technology is spreading fast. The potential for wide adoption across the broader scientific community, industry, and regulatory agencies is encouraging.While the field is still in the early stage of development, OOC has indeed delivered a breakthrough in disease modeling, allowing for the creation of sophisticated in vivo or in vitro approaches which are limited by the absence of physiologically relevant hallmarks of human fibrosis.Faithfully modelling disease phenotypes and providing clinically meaningful readouts are the important features of an ideal experimental system for drug development. Engineered microtissues, organoids, spheroids, and microfluidic cultures are enjoying rapid growth in the past decade. In terms of fibrosis remodeling, various engineered tools with the capacity to identify pathological factors and potential therapeutics have been devised. Such clinically relevant findings cannot be easily achieved from other commonly used in vitro. A further boost to these platforms could be achieved using multiple techniques in conjunction, such as hiPSC-derived 3D microtissues on a chip where the real-time properties can be monitored using a buit-in sensor. In addition, automation capacity is strongly desired for chronological tissue assessment and for retrieving useful data from the system. Machine learning applications through analysis of tissue contractions, electrophysiology, calcium flux, and overall toxicity are applicable to fibrosis-related drug screening. In the future, these algorithms could be integrated into anti-fibrosis drug screening pipelines to enhance screening throughput and detection of novel compounds. With highly controlled and formulated micro-environments, these avatars of the human organs can faithfully replicate disease phenotypes. These biosystems present a promising tool with unique features that can propel them to transform the present disease study and drug discovery with superior biofidelity, outperforming the conventional animal and monolayer disease models. Although tumbling blocks still exist in the widespread application of OOC models, we are optimistic that the coherent efforts from the engineers, biologists, and clinicians will entail this complex issue.OOC models are broadly applicable as novel tools for better understating the underlying mechanisms of pathological development of fibrotic diseases, and the inhibition or prevention of fibrotic phenotypes through therapeutic interventions. Each model system exhibits its unique limitations. It may be challenging to find an appropriate model to meet all kinds of experimental requirements and the efficacy of each model is mostly based on the desired assay or translational applications. Current OOC are used to identify the problem and design the system based on the elements needed by this problem, therefore, we do not need everything in the system, but only elements that are critical to reproduce the desired function. The value that OOCs bring is the ability to recapitulate these essential factors


The increasing prevalence of traumatic events requires our public health workforce to be knowledgeable about ways trauma influences population and individual health. There is a gap in student training about the various ways that traumatic events affect their capacity to perform public health work and the communities they serve. While other human services disciplines explicitly use trauma-informed terminology and concepts in student training, references to trauma-informed approaches are more implicit in public health curricula. This study examined trauma-informed principles and related terminology for use in public health coursework in the context of a community-wide water contamination public health crisis in Flint, Michigan, USA. We addressed the principles of trauma-informed approaches across key competency areas common to USA public health accredited programs, including discussion to support student understanding of the principle in action. Using trauma-informed language (1) enhances our capacity to name and respond empathetically in traumatized communities, (2) provides guiding principles for less community-engaged efforts, and (3) fosters stronger relationships for more community-engaged initiatives by providing areas of accountability for unintended consequences throughout the program’s development and implementation processes. Rising public health professionals equipped with knowledge of trauma-informed approaches can more intentionally minimize unintended negative consequences of public health initiatives. Public health students need preparation to perform public health work without causing harm in communities affected by high rates of trauma. Public health practice includes the delivery of health interventions in diverse communities, and university-based public health programs lead the training process. The COVID-19 pandemic has been a global reminder of the collaborative and cross-disciplinary efforts required for effective public health practice through traumatic events. COVID-19 has negatively affected morbidity and mortality rates , and socAccording to the US-based Association of Schools and Programs of Public Health (ASPPH) academic program finder index of the 2580 CEPH accredited programs, 938 (36%) included “community trauma” as a search keyword, 872 (34%) included “traumatized populations” as a keyword, just 148 (6%) programs included “collective trauma” as a keyword, and 53 (2%) included “traumatic stress” as a keyword . These sThe Substance Abuse and Mental Health Services Administration (SAMHSA), the leading federal agency addressing mental health services in the USA, define trauma as “an event, series of events, or set of circumstances that is experienced by an individual as physically or emotionally harmful or threatening and that has lasting adverse effects on the individual’s functioning and physical, social, emotional, or spiritual well-being”  (p. 7). As public health faculty in Flint, MI, USA, we have been challenged to deliver training experiences that prepare our students to meet public health needs in Flint. The Flint community has navigated multiple collective traumas that still affect the community, including long-term economic instability , the morLearning from our professional colleagues in human services and clinical care settings, we adopted SAMHSA’s concept of trauma-informed approaches  and guidTrauma-informed approaches (TIA) are evidence-driven practices to support the health and wellbeing of patients and clients managing traumatic experiences by avoiding re-traumatization. “A program, organization, or system that is trauma-informed realizes the widespread impact of trauma and understands potential paths for healing; recognizes the signs and symptoms of trauma in staff, clients, and others involved with the system; and responds by fully integrating knowledge about trauma into policies, procedures, practices, and settings”  (p. 9). SAMHSA provides six principles to inform TIA development: (1) safety—minimizing risk and building one’s sense of control; (2) trustworthiness and transparency—ensuring strategies and decisions are visible, described clearly, and do not violate trust in relationships; (3) peer support—voluntarily building mutual and respectful relationships; (4) collaboration and mutuality—rebalancing power differentials in decision-making; (5) empowerment, voice, and choice—acknowledging strengths and having space to use them; and (6) recognizing cultural, historical, and gender issues—avoiding stereotypes, promoting nurturing cultural practices, and addressing historical trauma  (p. 11).Several human services and public-health adjacent disciplines have adopted trauma-informed principles to offer better (and cost-saving) services to their clients, including health care , social This manuscript explores how trauma-informed principles and terminology can be incorporated into public health student coursework using FWC events as examples. We discuss how trauma-informed principles align with community-engaged public health practices to drive equitable and inclusive processes and health outcomes. This work is to ensure that the lessons we learned through Flint-based work for minimizing unintended consequences are accessible to public health educators seeking strategies to reduce harm in public health.We first provide an overview of the connections between trauma-informed principles and existing public health training content related to community engagement and equity/inclusion with regard to both engagement processes and health outcomes . The secOur experiences working in the Flint, MI community were vividly shaped by historical and pressing community-wide experiences that were traumatic for many residents. Informed by the history of community health inequities, we adopted community-engaged approaches to partner with local residents toward developing beneficial, effective, and sustainable public health programs. The engagement continuum ,25 can bNumerous decisions of the past have been made with limited attention to the public health impact across diverse communities and, subsequently, require significant investment to correct. To reduce unintended, avoidable, and negative consequences, decision-making should include an array of stakeholders from the communities that are impacted by the decisions being made. In practice, this means engaging potential users in the development process of public health programs/interventions to avoid barriers to program use. Engagement and participatory practices have been recognized as critical to improving health and health equity , includiThese processes are necessary in nearly all forms of public health practice. Every organization and program uses processes for decision-making in the course of public health work. Such processes commonly include defining public health goals and needs, conceptualizing service offerings, selecting implementation strategies, and evaluation. Even without malicious intent, decision makers rely on information they deem relevant. Using community engagement approaches, decision makers can build (their) awareness about the potential effects (unintended consequences) of their program decisions. Engaged stakeholders can raise their concerns, inform solutions, or be partners for program implementation. While some processes can yield similar outcomes across different communities, other processes may need modification to achieve the desired health outcomes. Importantly, the final decisions should reflect attention to the needs of the communities being served. In vulnerable communities, additional consideration is necessary to ensure the request for engagement does not generate greater vulnerability. Given process work requires ongoing engagement and proper compensation for community members’ time, and expertise is warranted.There is a broad range of primary, secondary, and tertiary outcomes associated with public health practice. Furthermore, communities can have notably different expectations about what they determine as the benefits and important outcomes of public health programs and interventions. For example, communities with high disease rates may benefit from medication adherence interventions, but they may also benefit from transportation improvement  or eliminating disease-related community exposures. Engaged community stakeholders can support identification of key impactful outcomes based on their experiences. They can also highlight process outcomes that may need to be addressed in order to reach the public health goal, and likely have greater community participation along the way.Accredited public health training programs use competency-based models to ensure students can demonstrate their capacity as practitioners. The Council on Education for Public Health agency is the nationally recognized accrediting body for schools of public health and for public health programs outside schools of public health in the USA . UndergrThese competencies provide spaces within public health curricula to incorporate trauma-informed principles, terminology, and practice. As students develop competencies to perform public health work, it is critically important that they also recognize potentially harmful and unintended consequences of their decisions and actions on vulnerable and traumatized communities. Trauma has been addressed across core knowledge areas of public health, including biostatistics ,29, epidIn nearly any case, the goal of public health practices is to improve public health conditions. No public health disciplines are removed from community engagement processes and information gathering. In vulnerable communities, resident expectations may include inclusive problem solving and solution-building practices to achieve, not just better, but equitable improvements in health outcomes.Trauma is an unavoidable component of public health work, especially in addressing health disparities, inequities, and social determinants of health. Having and using a framework of trauma-informed principles provides lasting guidelines for community engaged work that is less likely to cause unintentionally harder circumstances for people affected by traumatic events.This background information is our reference example for applying trauma-informed principles with public health competency areas. The FWC grabbed national attention in 2015 as evidence emerged of lead-poisoning resulting from a 2014 water source shift . In 2014Negative psychological effects ,39 emergArea activists and scientists pushed for government action to improve municipal water quality; the quality was improved to meet state and federal standards by July 2016 . Local wcollaboration and mutuality when power differentials are leveled. When stakeholder input is meaningfully incorporated, community members may feel more empowered to voice their perspectives. Ongoing engagement that addresses intent and motivation builds trustworthiness and promotes transparency for external stakeholders, which is particularly important in communities with high levels of institutional distrust. Maintaining space for community engagement in public health practice fosters safety and a sense of control about the program components, and over the longer term, encourages peer support and voluntary relationship building with stakeholders from various backgrounds. Tailoring the program approaches across diverse community settings can be a form of recognition of issues related to culture, gender, and historical trauma. When engagement is an iterative process, decision makers can grow to be more mindful about the community impact of decisions being made when external stakeholders and community members are not engaged. With empowered stakeholders at the table, we can ensure the evaluation and data interpretation processes are transparent and properly framed and represent inclusive and realistic outcomes across community subgroups.Engaging community members as stakeholders in program management where their expertise is respected represents With the FWC as an example, While public health students attending any institution need to have a strong grasp on course content, students at institutions in vulnerable communities such as Flint must also understand the strengths and challenges of the social landscape where the institution is located when they live, work, and play there. Courses designed to address current events are opportunities to connect our public health competencies with trauma-informed principles, particularly if the participating students are entering the workforce during an ongoing traumatic event (such as the COVID-19 pandemic or FWC). At the University of Michigan—Flint, a free community-centered course with a panel format and small group discussions was developed to facilitate bidirectional learning between policy makers, public health practitioners, community members, and students. With this approach, we advanced the reach of traditional public health course materials outside of the classroom and into community spaces to intentionally support participatory engagement, equitable, and inclusive information processing and discussing outcomes important to diverse communities impacted by the FWC. This is trauma-informed thinking in public health practice. There are certainly opportunities to integrate trauma-informed thinking into public health curricula in other forms, and these will be examined in future studies.The increasing prevalence of traumatic events requires our public health workforce to be knowledgeable about ways trauma influences health at population and individual levels. Collective traumatic events have an impact across public health sub-disciplines, and students who are equipped with knowledge of trauma-informed principles may be better able to navigate complex public health actions. The main implication of this work is that the language of trauma-informed approaches belongs in public health content and aligns with existing public health competencies. Trauma-informed content is becoming more common in professional degree programs, including in medical education  and sociThe second implication references the benefits of using the language and terminology of trauma-informed approaches. Using trauma-informed language to characterize major events as traumatic stressors signals public health workers to recognize the potential for major impact in their professional capacities and to take action, including decision-making , with emThe third implication of using trauma-informed language is centered on community-engaged efforts. Using trauma-informed language also reflects an intentionality in the process of community partnering as in existing public health work . For iniFuture work can address training around trauma-informed practice in preparations for experiential learning experiences such as internships, capstones, culminating experiences, service learning, and even volunteerism settings where complexity and trauma are common, as in social work . Rising From our work through the FWC, and as evidenced by workforce wellness concerns during COVID-19 , the neeFuture research is warranted to evaluate the practice and implications of using trauma-informed approaches in public health. This includes evaluation of our disciplinary integration of trauma-informed approaches, language, and community engagement practices into the student training process that is currently guided by competency-based education. This research should also address public health use of trauma-informed approaches with adjacent human services disciplines, including the use of appropriate metrics for health, process, community, and population outcomes.Rising public health professionals will be leading solutions to respond to widespread collective experiences that negatively impact population health. They will be expected to understand how and why diverse communities deserve tailored, inclusive, and trauma-informed programs. This preparation can begin or be enhanced by intentional introduction of trauma-informed terminology, principles, and situational examples throughout the public health curriculum. This provides anchoring concepts to inform their thinking about the approach to serving individual clients and communities. It exposes them to the many existing resources, such as those from SAMHSA, designed to support people working in helping professions to provide effective services and support their professional capacity.Other service disciplines have integrated trauma-informed approaches into their curricula and public health could benefit from the frameworks that have been developed for use in those disciplines. It is imperative that we are educating future practitioners to address public health issues from a trauma-informed lens to improve community engagement and health outcomes while not adding to or re-traumatizing the communities in which they serve.


Tissue engineering has paved the way for the development of artificial human cardiac muscle patches (hCMPs) and cardiac tissue analogs, especially for treating Myocardial infarction (MI), often by increasing its regenerative abilities. Low engraftment rates, insufficient clinical application scalability, and the creation of a functional vascular system remain obstacles to hCMP implementation in clinical settings. This paper will address some of these challenges, present a broad variety of heart cell types and sources that can be applied to hCMP biomanufacturing, and describe some new innovative methods for engineering such treatments. It is also important to note the injection/transplantation of cells in cardiac tissue engineering. Heart failure is one of the leading causes of death worldwide, especially due to the increasing number of patients with end-stage heart failure, despite the many treatments available MI, in particular, is a disease with high global mortality and morbidity caused by the permanent loss of myocardial cells (MC) leading to progressive pathological left ventricular (LV) remodeling and heart failure. Heart muscle can not regenerate; instead, the regenerative capacity of the adult heart declines rapidly after birth, with less than 1% of the heart’s MCs being replaced each year . HoweverA particular study developed cell myocardoplasty; a cell administration method where one injects pluripotent cells directly into the heart muscle instead of transplanting a solution directly to the coronary artery by using a syringe . InjectiOriginally, many investigations in myocardial regenerative medicine have focused on CMs, cells difficult to proliferate and recover once damaged. CMs are responsible for generating a contractile force in the myocardium . In the CMs, endothelial cells (ECs), smooth muscle cells (SMCs), and cardiac fibroblasts (CFs) have been previously used for hCMP production, in order to comprehensively reproduce the physical structures and signaling pathways present in native heart tissue. ESCs and iPSCs are the most readily available sources of CM for human strains because they can multiply indefinitely and differentiate into cells of different lineages. Both ESC-derived and iPSC-derived CM are also more structurally similar to neonatal cells than adult CM, hindering applications of hCMPs composed of immature iPSC-derived CM. However, clusters of other cell types  have also been incorporated into the heart patch and evaluated in preclinical models of myocardial injury . It is aSMs is the first discovered cell source for cell sheets and is now attracting many researchers for the treatment of acute myocardial infarction (AMI). In basic research, SM has been applied following MI in various animal models such as rats, hamsters, dogs, pigs, etc. . SMs havMSCs’ immunomodulatory abilities and growth factor secretion capabilities show promise in the field of tissue engineering, leading to many clinical trials for their use in the extensive treatment of ischemic heart disease . A few sThe myocardium is similar to a skeletal muscle but is instead made up of many CMs, where each CM is electrically connected to adjacent CMs by gap junctions allowing for synchronization of contraction and relaxation. CMs, like skeletal muscle, are difficult to differentiate and mass-culture because CMs in healthy adults are rarely available. Several research teams have reported that cell sheets derived from fetal or neonatal CMs can be used to treat MI in animals. Pioneering studies, using fetal CMs from 15-day-old mouse embryos have demonstrated that donor cells survive after transplantation and administration to the heart . While tIn 1981, the discovery of pluripotent stem cells (PSCs) using mouse ESCs led to the creation of myocardium using PSCs with the characteristics of infinite proliferation and highly efficient CM differentiation . ESCs an2 increase in force production, suggesting maturation [It is also worth noting how electrical stimulation or excitation-contraction coupling can affect the amount and duration of action potentials in CMs, thus increasing the number of synchronous contractions. Ruan-Circ et al. conducted a study with collagen-based, bioengineered tissue created with CMs derived from hiPSCs; electrical pacing combined with static stress conditioning led to a ~1.34 mN/mmturation . Electrituration . Howeverturation . Moreoveturation ,28,29.Blood vessels have a major role in the heart, transporting nutrients and oxygen using the flow of blood. Blood flow is essential in regenerative medicine due to the large size of myocardial tissue, which requires a functional vascular network within. Stevens et al. and others have shown that a cohesive heart patch composed of CMs, ECs, and fibroblasts derived from human ESCs develops vascular structures that are essential for successful transplantation in vivo. Arai et al. also used a combination of iCell, ECs, and fibroblasts to form a cardiac spheroid of interest. Based on this idea, Arai et al. hope that blood flow can be ensured by mixing human umbilical vascular ECs when creating structures based on iPSC-derived CMs . RecentlAdvances in stem cell biology have made it possible to use various cells such as adult stem cells and iPSCs, and advances in bio-3D printing have made it possible to create large tissue structures for transplantation. An approach utilizing scaffolds, a method of seeding a vesicle into a three-dimensional and porous scaffold using a biodegradable polymer material involves mixing and pouring cells and gel-like scaffolds  into a mold, then arranging the cells using a 3D printer and a small amount of gel. Another method, bioprinting, has been pursued as well where fiber-like cell-containing gels are bundled together. Spheroids and organoids-clumps of cells have also attracted attention. By seeding these cell masses in metal needles or metal devices, organizing then extracting them, one can create a three-dimensional structure that does not contain scaffolds a.Most clinical studies use sheets for their approach; for example, the cell sheet method developed by Professor Okano of Tokyo Women’s Medical University consists of synthesizing myocardial tissues with a pulsating ability, without utilizing cell supports . The celBecause the cell sheet lacks exogenous/synthetic scaffolding, there are no concerns about the potential immunogenicity of the scaffold material. A major advantage of creating tissues this way is that they maintain adhesion proteins on the surface of the cell tissue body, so when transplanted into living organs, they have an admirable integration function with the transplanted organ. Furthermore, cells in adjacent layers can form connections between the layers that facilitate communication, including gap connections necessary for electrical bonding.Early research used tissue printing techniques, where the printed constructs worked well with the cardiac system and demonstrated high survival rates after 7 days of culture . HoweverBreckwoldt et al. developed hCMPs by differentiating human ESCs into hiPSC-CMs, a design that allows one to analyze human heart diseases by checking the EHT force strength .A system for evaluating the 3D contractile force of the heart’s structure is important for new drug development. Although some research groups have already reported scaffold-based heart constructs, these scaffolding-based heart structures cannot accurately predict the heart’s drug response in vivo due to the interaction between the drug and scaffolding material . Heart sAnother study without the use of a scaffold consists of a tubular EHT (T-EHT), created using bio-3D printers with hiPSC-CO and needle arrays, which produced beating conduits for patients suffering from monoventricular disease. In this study, we created a hiPSC-derived cardiac organoid composed of hiPSC-derived CMs, human cord vein endothelial cells, and human fibroblasts . A bio-32) are still made relatively thin (1.25 mm), jeopardizing clinical application because they cannot mimic native myocardial characteristics such as action potentials [Few studies have been conducted using appropriately sized constructs, despite significant improvements in protocols used in hCMP manufacturing. After transplantation, the diffusion of oxygen and nutrients from the vascular system limits the thickness of the hCMP. This diffusion can be controlled by mixing molecular crystals  in the matrix solution and leaching them after the matrix has solidified . Howevertentials .In theory, layer-by-layer assembly technology would allow one to produce hCMP of any thickness by simply stacking the required number of individual CM sheets. Incorporating graphene oxide(GO)-based thin films would not only enhance the formation of physical and electromechanical connections between layers, but can also improve electrical bonding, maturation, and cell tissue growth. Moreover, by culturing heart cells on a 2D cell mesh that incorporates a micromachined hook-and-loop system, the hooks and loops in adjacent layers would have the ability to work together for the hCMP to contract in response to electrical stimuli .Rather than relying solely on infiltration from the original circulatory system, thicker hCMP may require some artificial blood vessels before transplantation. Angiogenesis can be induced by encapsulating a sacrificial gelatin mesh in a scaffolding material and then melting the gelatin mesh to leave a network of interconnected microfluidic channels. When seeded with human microvascular EC, the sacrificial scaffolding generates a rudimentary endothelial network . AnotherCell and tissue delivery methods can be classified into invasive and non-invasive approaches, both of which are often characterized by low engraftment rates . ImplantAlternatively, non-invasive intravascular  delivery shows recirculation and redistribution of injected cells into other organs other than the target site, which, as previously discussed, leads to improved cardiac function due to paracrine signaling mechanisms. This method has the advantage of being non-invasive and has the potential for repeated administration, therefore being considered the appropriate selection from a clinical perspective.Preclinical studies have utilized a variety of animal models, including mice, rats, guinea pigs, pigs, and non-human primates . The selSubsequent preclinical studies have utilized large animal models such as non-human primates and pigs to demonstrate remuscularization and therapeutic effects of concern for ventricular arrhythmias. Large animal models are more reliable than compared to rodent models, although the high costs associated with large animal research will limit their use ,64.The ultimate goal of a heart patch transplant is to replace the damaged heart muscle with an extrinsically functioning myocardium. Preclinical studies from mice, rats, guinea pigs, pigs, and non-human primates showed some myocardial remuscularization of fibrous scar tissue. However, the studies faced several challenges, with one being the large amounts of exogenous CM (and/or other heart cells) required to replenish lost tissue. Another challenge is ensuring long-term graft retention for maximum therapeutic effect. Genetic engineering can be utilized to increase cell retention, where overexpression of CCND2 (cyclin D2), a cell cycle activator, increases the cell cycle activity and growth rate of hiPSC-CM, thus improving engraftment rates from an average of 10% to 25%. The lack of conductivity between the hCMP and the host tissue proves to be another challenge. One study utilized super-aligned carbon nanotubes during the manufacture of cardiac tissue, enhancing electrophysiological uniformity due to the anisotropic conductivity of their aligned structures. Another study demonstrated that electrospinning nanofiber scaffolds with enhanced conductivity promoted the electrical bonding of patches, indicating the potential for the manufacture of clinically relevant hCMP products. As discussed in the previous section, one major aspect that needs to be carefully studied when addressing the electromechanical integration of exogenous CM and host myocardium is the high probability of causing arrhythmias . The occurrence of arrhythmias is a serious safety concern for the clinical applicability of heart patch therapy. Large animal models are being used to study arrhythmias in cardiac patch studies, due to their sinusoidal waves providing a more accurate representation of the physiology, function, and anatomical structure of the human heart. Although preclinical studies of non-human primates showed transient non-fatal arrhythmias 2 weeks after transplantation, after which these arrhythmias decreased, ventricular arrhythmias in pig models were more frequent and fatal (2 out of 7 pigs) . It is cThe potential benefits of hCMP for the treatment of MI are readily observable in preclinical studies, and at least one small trial in patients is currently underway . Traditi


Postmenopausal women represent an important target population in need of preventative cardiometabolic approaches. The loss of estrogen following the menopause eliminates protections against metabolic dysfunction, largely due to its role in the health and function of adipose tissue. In addition, some studies associate the menopause with reduced physical activity, which could potentially exacerbate the deleterious cardiometabolic risk profile accompanying the menopause. Meanwhile, exercise has adipocyte-specific effects that may alleviate the adverse impact of estrogen loss through the menopausal transition period and beyond. Exercise thus remains the best therapeutic agent available to mitigate menopause-associated metabolic dysfunction and represents a vital behavioral strategy to prevent and alleviate health decline in this population. The menopausal transition period is associated with increased body weight and unfavorable changes in body composition and fat deposition ,2. In adWhile there are multiple molecular mechanisms that explain these adverse metabolic fluctuations ,9, a comMetabolic health impacts every bodily system and is predictive of many chronic diseases . WhereasThe risk of obesity increases drastically during the menopausal transition period ,30, as eWhile it is difficult to determine the precise cause of menopause-associated obesity in humans, rodent studies provide a highly controlled environment in which to lend insight to potential mechanisms. Estrogen-sufficient female rodents have consistently proven to be resistant to diet-induced obesity compared to males ,37, yet Not only does the menopause facilitate weight gain, which directly increases obesity susceptibility, it modifies the pattern of fat distribution. Premenopausal females and age-matched males exhibit different patterns of adipose storage , as maleIt is not excess fat per se that is metabolically harmful; when functioning properly, fat protects other organs, such as liver tissues, from ectopic lipid deposition. In fact, estrogen associates with increased subcutaneous adipose tissue storage, which may actually confer protection against metabolic disturbances among premenopausal women . GlutealIn addition to increasing weight gain and shifting body fat distribution, the menopause may directly affect adipose tissue metabolism . Over thIn addition to the adverse effects of estrogen loss on adipocyte metabolism, obesity causes adipose tissue dysfunction, hence the combination of estrogen loss and obesity may be particularly problematic for aging women. Dysfunctional adipocyte metabolism leads to systemic metabolic dysfunction, including insulin resistance. This is because adipose tissue expansion during obesity creates a cycle of inflammation and lipotoxicity, likely due to the inability of adipose tissue to effectively store excess dietary lipids, leading to the ectopic deposition of lipids in other organs  ,75,76. SEstrogen also has anti-inflammatory effects  and is pThe protective role of estrogen extends further to insulin sensitivity , which mThe mechanisms by which estrogen is metabolically protective likely involve estrogen receptor (ER) signaling ,102. ThuWhen local estrogen production is inhibited via knocking out aromatase, animals become obese and insulin resistant. A similar phenotype is caused by selective knockout of ERα ,104, sug2 max scores, and higher exercise volumes resulted in statistically significant reductions in BMI, weight, fat mass, and fat percentage. Interestingly, weight loss among the exercise groups was not significantly different, although there was a trend for a dose–response effect. The effects of exercise dose on body fat percentage were greater among women randomized to the low-volume group who already had a low BMI. However, among women with a high BMI, there was a trend for a greater reduction in total fat mass with increasing exercise volume, and the weight loss experienced among those women was due to a loss of fat while lean mass was maintained. Most importantly, the authors observed a greater reduction in intra-abdominal and subcutaneous abdominal fat in conjunction with a greater exercise volume, the first study demonstrating this effect in postmenopausal women [The benefits of exercise are broad and affect virtually all body systems. Across populations, higher levels of physical activity are associated with greater attenuation of body fat gain and preservation of lean mass across the lifespan . Indeed,al women . Anotheral women , thus suIn studies using rodents, rats bred to have higher life-long levels of physical activity, and consequently a higher fitness capacity (measured via running capacity), are protected from the metabolic dysfunction that accompanies ovarian hormone loss via ovariectomy . ConversWhile the numerous benefits of increased physical activity are clear across populations, the mechanisms through which the menopause affects responsiveness to exercise must be considered. Ample evidence indicates that exercise improves fat loss among postmenopausal women when compared to diet-only strategies, yet some studies have suggested that, compared to premenopausal women, postmenopausal women may be partially resistant to the adipose tissue-targeted effects of exercise. Other research has revealed that postmenopausal women may have impaired exercise-induced fat oxidation, making exercise-mediated weight loss more difficult. Abildgaard et al. examined the effect of fat oxidation in a cross-sectional study on pre-, peri-, and postmenopausal status and its influence on the oxidative capacity of skeletal muscle via skeletal muscle biopsies taken before and immediately after an exercise bout, in addition to measurements of indirect calorimetry during exercise. Postmenopausal women had significantly lower whole body fat oxidation and energy expenditure during exercise than the premenopausal women. Furthermore, postmenopausal women demonstrated a blunted exercise-induced increase in phosphorylation of AMPK, which coincides with the suppression of fat oxidation during exercise. The authors ultimately concluded that the reduction in whole body fat oxidation after the menopause is likely attributed to a reduction in lean body mass . FurtherBoth sex and age have been shown to affect adipose tissue ERs and adipocyte metabolism. Porter et al. compared omental  and abdominal subcutaneous adipose tissue genes and proteins in a total of 28 premenopausal women, 16 postmenopausal women, and 27 age-matched men undergoing bariatric surgery . With the exception of fasting non-esterified fatty acids, which were higher in women, no other differences were found in other indicators of systemic glucose and lipid metabolism. Upon examining the gene and protein levels of the ERs, omental adipose tissue ERβ levels were higher in older women than in younger women and older men, and aromatase expression was higher in older men than in older women. Overall, among the patients studied, the subcutaneous adipose tissue immunometabolic profile was heavily influenced by both age and menopause status, more so than omental adipose tissue . SubsequIt is unclear how changes in the ERα and ERβ density of adipose tissue affect glucose in lipid metabolism or how such changes may affect exercise-mediated fat oxidation. However, in the study by Porter et al. described previously, the protein content of both ERα and ERβ was highly correlated with the mitochondrial protein uncoupling protein 1 (UCP-1) across sexes and ages . Those sDue to the strong influence of aging and the menopause on body composition, including the loss of lean mass, exercise designed to improve and maintain muscle mass is critical. In this regard, strength training has been the focus of some studies examining how exercise may mitigate metabolic dysfunction following the menopause. A study investigating the association of resistance training exercise frequency and volume with changes in body composition among postmenopausal women found that resistance training was successful in preventing weight gain and deleterious changes in body composition. The authors determined that training frequency was significantly and inversely associated with changes in body weight, adiposity, and trunk fat . This was true even after adjusting for age, years on hormone therapy, changes in lean soft tissue, baseline body composition, and baseline habitual exercise .Since loss of estrogen is at least partially responsible for the accompanying metabolic dysfunction observed during the menopause, it is possible that combining hormone replacement therapy with resistance training may be even more beneficial than exercise alone. Exercise and estrogen therapy may synergize to improve metabolism in this population. One study determined the effects of resistance training with or without transdermal estrogen therapy (ET) on adipose tissue mass and the metabolic risk profile in early postmenopausal women. Here, untrained postmenopausal women were allocated to supervised resistance training with a placebo or transdermal ET for 12 weeks. Body composition, metabolic-health-related blood markers, body fat percentage, adipocyte cell size, and lipogenic markers in subcutaneous adipose tissue from the abdominal and femoral regions were assessed. While resistance training improved those outcomes, the ET group unexpectedly experienced a lesser reduction in total and visceral adiposity compared to the placebo group. However, the ET group improved their metabolic blood profile as indicated by reduced low-density lipoprotein, glucose, and hemoglobin A1c .The metabolic benefits of high-intensity interval training (HIIT) are increasingly well established. A recent meta-analysis concluded that HIIT is a time-efficient, feasible, and effective intervention to modify body composition by reducing abdominal and visceral fat mass . AccordiSome studies have suggested that the effects of HIIT may be diminished following the menopause. In fact, the Dupuit study described above determined that HIIT was significantly more impactful in reducing weight through total and abdominal adiposity prior to the menopause . NonetheIn a randomized controlled trial assessing the effects of a 16-week functional fitness protocol utilizing elastic resistance bands in conjunction with balance and agility exercises, significant improvements in all body composition variables related to fat were observed among the postmenopausal women studied. Significant improvements in total cholesterol and HDL were also observed . Thus, fOne study looked at tai chi as a means to improve the adverse metabolic symptoms of the menopause. The authors investigated the effects of a 12-week training regimen and saw improved body composition, muscle strength, functional capacities, blood pressure, and general health perception in postmenopausal women . FurtherSimilar studies have looked at the effects of yoga on menopausal symptoms and sleep quality. Some studies observed significant decreases in menopausal symptoms in both peri- and postmenopausal women following 20 weeks of participation in yoga classes, with greater effects in postmenopausal women. Additionally, sleep quality improved among all of the participants, indicating that yoga can improve health and wellbeing during the menopausal transition period through multiple mechanisms . A diffeWhile there is ample evidence in both human and animal models that weight gain occurs following the menopause and the loss of ovarian hormone production, whether this metabolic disturbance is due to increased energy intake through dietary changes or reduced energy expenditure is still unclear. The MONET study, which followed a group of women through the menopausal transition period while annually tracking dietary patterns and energy expenditure, found that observed weight gain was attributed to reduced physical activity and total energy expenditure and not increased energy intake . StudiesWhether or not dietary patterns change during the menopause may be a difficult question to address, but new research may shed some answers. Decades ago, during the Women’s Health Initiative (WHI), a dietary intervention study was implemented to determine the effectiveness of diet in mitigating postmenopausal health impairments. Postmenopausal women were instructed to adopt a low-fat, high-fiber diet for a 9-month period and through a moderate reduction in energy intake, they weighed ~2.2 kg less on average than those in the control group by the study’s end. Notably, in addition to consuming less fat, the intervention group consumed more fiber, vitamins and minerals, dairy products, and fish ,149. AloResearch has also shown the benefits of exercise in modifying gut microbial populations . A recenWomen spend up to three decades in the postmenopausal phase of life, which is often accompanied by a wide variety of disruptive manifestations, including vasomotor symptoms, sleep disturbances, cognitive decline, mood disorders, and metabolic dysfunction . While a


Respiratory infectious disease outbreaks such as those caused by coronaviruses and influenza, necessitate the use of specialized aerosol test chambers to study aspects of these causative agents including detection, efficacy of countermeasures, and aerosol survivability. The anthrax attacks from 2001 and earlier biowarfare and biodefense also influenced the study of biological aerosols to learn about how certain pathogens transmit either naturally or through artificial means. Some high containment biological laboratories, which work with Risk Group 3 and 4 agents in biosafety level -3, biosafety level-4 containment, are equipped with aerosol test chambers to enable the study of high-risk organisms in aerosolized form. Consequently, the biomedical, military and environmental sectors have specific applications when studying bioaerosols which may overlap while being different. There are countless aerosol test chambers worldwide and this number along with numerous high containment biological laboratories underscores the need for technical standards, regulatory and dual-use compliance. Here we survey common aerosol test chambers and their history, current use, and practice. Our findings reinforce the importance and need for continued collaboration among the multi-disciplinary fields studying aerobiology and biological aerosols. Aerosol test chambers (ATC) are specialized facilities used to house studies for detection, animal models, and related efforts involving dispersal of biological aerosols (bioaerosols). ATCs are common among academic, commercial, government, and non-profit research organizations where they often employ researchers who perform studies involving these facilities and maintaining such equipment. For example, ATCs are used to study aerosols containing biological material and minimize the physical loss of particles to isolate key variables and study specific aerosol parameters over time or to infect animal subjects to a relevant dose . These fAlthough the World Health Organization designates SARS-CoV-2 as a Risk Group 3 biological agent, it can be handled at the BSL-2 level for routine diagnostic analysis ABSA, . HoweverNational state programs became active in biodefense in the first half of the 20th century, as consequence of World War II (WWII), and during the Cold War. The Geneva and Biological Weapons Conventions banned offensive use of biowarfare agents (BWA) thus reinforcing biodefense measures for studying detection, survivability and dispersal. Biodefense studies involving BWA may include actual pathogens or simulants and dispersals occurring indoors such as aerosol chamber and outdoors such as field test . The ForIn general, aerosol work would be difficult to detect, monitor, and verify. This is especially true for indoor aerosol studies using ATCs where it is hard to discern dual-use equipment. Aerobiology study methods are often straight-forward but descriptions for equipment set up, agent dispersion, sampling, collection, and detection are lengthy due to the inherent physical and biological nature. Frameworks such as the Australia Group were established to maintain an export control list of dual-use equipment . TrackinAlthough there is no standard biosafety oversight, WHO and CDC provide widely accepted biosafety guidelines which aid at the national level . WHO andIn addition to bioaerosol research aspects and dual-use applications, recent publications also address the need for standardized methods, harmonized regulations, and increased multi-disciplinary initiatives . AlthougGenerally, an aerosol particle per volume (liter or cubic meter of air) does not indicate that particle contains the target agent. A simple particle counter like an Aerodynamic Particle Sizer® would measure particle per liter (P/L) of air. For culturing bacteria and viruses, biologists commonly use colony forming unit (CFU) and plaque forming unit (pfu) which can also be used to measure bacteria (i.e. CFU/liter of air) or viruses  in the air from liquid or solid collection. The US Department of Defense (DOD) has historically used the term Agent Containing Particles of liter of air (ACPLA) to describe a particle that has a detected target organism within it; to differentiate it from simply a particle per liter of air which provides comparable data when evaluating detectors but little operational utility . FurtherDue to their broad and multi-disciplinary use, aerosol chambers generally exist as one of three categories, Static Chambers, Flow-Through Chambers, and Rotating Chambers . Static Flow-Through Chambers, also referred to as Dynamic or Wind-Tunnel Chambers, are more complex and incorporate air that is continuously introduced into the chamber using airflow velocity to counteract the loss of aerosol due to gravity over the course of an experiment. This enables researchers and testers to mimic a realistic airflow environment, however they require continuous aerosol dissemination to maintain aerosol concentration over time. Rotating Chambers, which are also referred to as Goldberg Drum or Rotating Drum Chambers, are typically constructed as a cylinder that rotates over time to reduce the effects of gravity and to maintain aerosol aloft for longer time when compared to a static chamber . The rot• Suspected use of chemical and biological weapons during the two Gulf Wars in 1991 and 2003.• The terrorist attacks on New York City and Washington DC on 11 September 2001.B. anthracis attacks in the United States in October 2001.• The 2001 • Outbreaks of SARS, MERS-CoV, ebolaviruses in 2000s.• Outbreak of SARS-CoV-2 in 2019 and COVID-19 pandemic.As mentioned previously, biodefense programs in the US, United Kingdom, and Canada have continued bioaerosol work domestically and jointly since WWII. In the US, academic, industry, and Government organizations with ATC capabilities have performed research and related activities in their respective niche market that have traditionally experienced low demand. Some of these markets included industrial hygiene applications such as monitoring exposure to environmental chemicals and molds. Nonprofit research organizations have also historically provided related biotech research services for translational sciences including diagnostics, therapeutics, and vaccines . The conThere are numerous aerosol test chambers worldwide represented across academia, industry and government. A few examples of each category are shown . For theAnother example of an entity repurposing their expertise to study SARS-CoV-2 is the US Army Dugway Proving Ground (DPG). In addition to biodefense, DPG provides testing and support for counter CBRE hazards , and theSimilar to the United States, foreign governments have entities that focus on infectious disease, to include BWA. An allied counterpart in the United Kingdom is their Defence Science and Technology Laboratory (Dstl) which the Ministry of Defense operates at Porton Down. Prior to the COVID 19 outbreak, Dstl conducted numerous studies using a Rotating Drum to study the stability and viability of aerosolized infectious diseases, including Lake Victoria Marburgvirus, Zaire ebolavirus, and Reston ebolavirus . FollowiB. anthracis and bioterrorism was high. However, ATCs and related bioaerosol efforts have historically been a niche and service-oriented market resulting in a lack of standardized methods for ATC use and measuring bioaerosols.Recent descriptions of high containment biological laboratories that demonstrated their utility and lessons learned during COVID-19 pandemic offer unique capability that has become essential during the recent outbreaks of respiratory infectious disease . BesidesOften the use of ATCs does not accommodate the different kinds of research being conducted. As described, the basic types of ATCs are inherently inflexible. For example, if a researcher is studying infection pathways or infectious dose, they are primarily limited to ABSL Flowthrough chambers, or if studying aerosol survivability in the environment, researchers will primarily use Rotating Chambers or Static Chambers. Another consideration is the artificiality inherent in using ATCs such as the inability to replicate the relevant real-world environment including UV radiation and pollutants. Researchers have attempted to address this artificiality over time by enhancing capabilities, but it is important that researchers consider these parameters when analyzing results.While the number of HCBLs continues to increase for many reasons as countries and states will choose to prioritize and build them, the number of ATCs will likely increase since the small, portable facilities have lower startup cost. In addition, ATCs are often not under governmental oversight, which make it difficult to estimate their exact numbers. Further work is needed to define methods according to perspectives of health-risk to humans, product-performance of detectors and efficacy of treatments. While challenging, the multi-disciplinary nature of the work offers potential opportunities for greater collaboration among respective communities, which encourages greater transparency through trust building.Responsible development of bioaerosol capabilities should also be considered in the future as related to maintaining resilient biotech industrial base. As demonstrated in the COVID-19 pandemic, the so-called “warm-base” model is essential for preparedness for supplementing manufacture of disposable consumables such as swabs and personal protective equipment. In addition, biomanufacturing of commonly used reagents such as viral transport medium and vaccines require specific materials and scale-up process which are difficult and expensive to have on-demand. These capabilities and associated markets support the bioeconomy ecosystem as a whole.


JCI, Colligan et al. demonstrate that BRQ restored terminal differentiation of MDSCs. Using in vivo models of immunotherapy-resistant breast cancer, the authors uncovered a mechanism by which BRQ promoted myeloid cell differentiation by limiting their suppressive function and enhancing the efficacy of immune checkpoint blockade therapy. The findings offer insight into the biogenesis of MDSCs, provide an alternative avenue for cancers that remain unresponsive to conventional therapies, and may be extended to future translational studies in patients.Myeloid-derived suppressor cells (MDSCs) hinder antitumor immunity in multiple cancer types. While brequinar (BRQ), an inhibitor of dihydroorotate dehydrogenase, shows cytotoxicity in hematological malignancy, it has not yet been adapted to attenuate MDSCs by augmenting bone marrow progenitors in breast cancer. In this issue of the  Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses and limit the efficacy of cancer immunotherapies through a diverse collection of mechanisms . Among tJCI, Colligan et al. addressed the concept of targeting MDSCs at the bone marrow progenitor phase in a series of preclinical studies  that led to the maturation and functional restoration of MDSCs. For example, in vivo administration of BRQ to mice with triple-negative breast cancer (TNBC) did not reduce splenic or peripherally circulating MDSC numbers, however it did affect their maturation, as evidenced by increases in CD101 (a neutrophil marker) and Ly6G and Ly6C on polymorphonuclear (PMN) MDSCs. Consequently, BRQ also reduced the ability of PMN-MDSCs to elicit a suppressive function on T cells and dampened the expression of multiple pathways and downstream functional mediators of MDSC biogenesis, including Arg1, iNOS, etc. . This fer models  4)..+ T celltrans retinoic acid (ATRA). This pharmacologic agent promotes myeloid differentiation through ERK1/2 pathway signaling and increased glutathione generation (While the data point to modulation of endogenous pyrimidine synthesis as a culprit responsible for the antitumor action of BRQ, overlapping mechanisms probably also contribute to its effects on MDSC biogenesis. For example, clues arise from other strategies that enforce MDSC differentiation into macrophages and DCs, such as the use of al-neration . The datneration . In anotneration . For myeneration , BRQ mayneration .This enticing collection of data on BRQ encourages further investigation of its mechanism of action on MDSC biogenesis. For instance, it is tempting to speculate that BRQ might also act in part by modulating other critical regulators of MDSC biology such as IRF8 or other transcription factors that may align with gene expression data . Since tThe data presented in Colligan et al.  support


Here, we review how technical advances in cell culture, robotics, computational biology, and development of reporter systems have transformed drug discovery, enabling screening approaches tailored to clinically relevant functional readouts . Illustrating with selected examples of “success stories,” we describe the process of phenotype-based high-throughput drug screening to target malignant cells or the immune system. Second, we describe computational approaches that link transcriptomic profiling of cancers with existing pharmaceutical compounds to accelerate drug repurposing. Finally, we review how CRISPR-based screening can be applied for the discovery of mechanisms of drug resistance and sensitization. Overall, we explore how the complementary strengths of each of these approaches allow them to transform the paradigm of pre-clinical drug development.Multi-omic approaches offer an unprecedented overview of the development, plasticity, and resistance of cancer. However, the translation from anti-cancer compounds identified  The systematic analysis of cancer genomes has led to the identification of recurrently mutated drivers and, on a fundamental level, to a greater understanding of the diverse mechanisms of oncogenesis . ClinicaUnfortunately, several challenges persist in the clinical translation of cancer genetic information. Importantly, several cancer alleles lead to loss of function that cannot be directly rescued by pharmacological means. In addition, the net phenotypic result of complex genetic interactions may be difficult to predict. Further, targeted inhibition of single biological pathways in malignant cells often leads to resistance that operates at several levels: outgrowth of genetically distinct subclones , epigeneTranscending the mutational profile of cancer, screening approaches that focus on functional surrogates of biological activity, such as changes in cellular phenotype, gene, or protein expression, directly assess the link between perturbagen and desired clinical effect. Such screening strategies do not require prior understanding of the molecular target of the disease, nor of the mechanism of action of the compound. Instead, the process of phenotypic screening directly converges on biological effect. phenotypic screens can accelerate the identification of compounds targeting these adaptations, thus facilitating the bi-directional feedback between drug design and clinical observations. Complementing this approach, novel bioinformatic strategies can discover relationships between chemical compounds, molecular targets, and biological pathways, and genetic perturbation screens can identify drivers of resistance. In this review, we describe how these technological advances operate and how they have transformed cancer drug development.HTS assays offer the potential to accelerate the discovery and development of new pharmacological compounds for various types of medical indications . In factGiardia lamblia carbamate kinase, a crucial enzyme for the metabolism of this parasite. In fact, carbamate kinase converts carbamoyl phosphate into several products including ATP. The resulting ATP is then used by the luciferase luminescent enzyme to generate light. Therefore, a greater light production correlates with higher carbamate kinase’s activity. Screening almost 4,100 compounds, this assay identified enzyme inhibitors that could potentially serve as drugs against the targeted pathogen. Although biochemical screens succeed in identifying target-specific compounds, many complex biological processes induced by the drugs screened will be omitted, such as unexpected activities, toxicities or responses . A better characterization of molecular pathways in cancer  has inspFor example, in an elegant study, Similarly, knowledge of molecular pathways that are dysregulated in cancer can inform the development of reporter cell lines in which the activity of the pathway is coupled with expression of a reporter (fluorescence or bioluminescence) amenable to HTS, a concept previously termed “mechanism-informed phenotypic drug discovery” (MIPDD) . Wnt sigWe recently described a fluorescence-based lymphocyte assay designed as a tool to screen for immunomodulatory compounds . This asIn 2015, Vincent et al. formulated a “rule of 3” for phenotypic screening with three criteria to assess the relevance of phenotypic assays: 1) “System” to assess how representative of the disease is the cellular assay; 2) “Stimulus” to assess how well can the experimental conditions reproduce the cellular response to study . 3) “Readout” to assess the relationship between the assay readout and the clinical end point. From a technical standpoint, to properly identify inhibitors or activators for a given molecular target or cellular function, the quality of the assay should be first established to ensure reproducibility and the ease in quantifying its triggered signal . .Although phenotypic screens enabled the discovery of several first-in-class cancer drugs, they do not allow to elucidate the exact mode of action and/or molecular targets. Therefore, the use of other complementary strategies such as biochemical screening, RNA-seq and CRISPR-Cas9 screening can complement HTS findings, as they may easily narrow down the list of plausible targets.ex vivo, cancer stem cells derived from patients with glioma or AML, allow to correlate genotype with drug susceptibility. The Leucegene project is an example of phenopytic HTS performed on arrays of patient-derived samples in acute myeloid leukemia , genomics has enabled the first attempts to discover new druggable targets in the human genome through the analysis of genome wide association studies (GWAS), opening a new era for human genetics to inform new therapies . A populGWAS from the United Kingdom Biobank  and the Kaiser Permanente Genetic Epidemiology Research on Adult Health and Aging cohorts  also provided insights from pan cancer studies and have detected 21 genome-wide significant associations that could identify deregulated targets and carcinogenesis mechanisms across tissue-types . United One of the prominent applications of GWAS is pharmacogenomics . The latInitially, much of the pharmacogenomic (PGx) discovery was carried out in the laboratory setting through cell line resources such as the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC) . PGx finSystems biology approaches using transcriptomic data from mRNA-sequencing and microarrays have been extensively used to understand perturbations caused by drug or chemical treatments to infer novel drug-targets, mechanism of action and repurposing avenues for old and existing drugs . Public The following section will discuss the use of transcriptomics to connect or cluster similar drugs, infer novel drug-target predictions and identify novel drugs that could reverse molecular states (aka drug repositioning) as a cost-effective alternative to the classical drug discovery pipeline.in vitro and in vivo); 3) Extraction of mRNA and capturing global molecular perturbations via RNA-seq or microarray assays; 4) Applying bioinformatic methods  to extract a robust drug induced gene expression signature that discriminate treated groups at different dose levels and time points; 5) Applying statistical algorithms  to score up and down-regulated genes with respect to similar expression patterns induced by reference compounds in the connectivity map.In 2006, the connectivity map (CMap) pioneered the concept that commonalities in drug mechanism of action can be inferred from similar transcriptional responses upon treating cancer cells with 1,309 bioactive compounds . It was This “guilt by association” concept generates new hypotheses for uncharacterized compounds. This approach has been applied successfully to understand the MoA of celastrol, a natural herbal compound . CelastrGiven that structural and molecular layers are complementary, several recent studies have used integrative computational approaches to elucidate drug MoA and propose new repurposing avenues. A versatile method known as Drug Network Fusion (DNF), has fused drug-centric networks relying only on basic drug characteristics such as structural information from pubchem and NCI60, and drug-induced perturbation profiles from LINCS and CMap . DNF taxThe LINCS database contains both gene expression profiles of ∼20,000 chemical treatments and ∼13,000 shRNAs targeting 3,800 genes across 9 cell lines  . This sin vitro, in patient derived xenografts and in genetic engineered models of HCC. Interestingly, the measured reversal of expression by a compound correlates with its efficacy in the tested cell lines. This systems pharmacology approach is gaining popularity for precision medicine applications (Since the creation of the earlier version of the connectivity map (CMap), drugs that induce an anticorrelated expression profile with respect to a given disease expression signature could be considered as therapeutic agents. Using this algorithmic technique, it was hypothesized that in a given “disease state,” a set of perturbed gene sets or biological pathways can return to a baseline or “normal state” following an effective drug treatment. ications .Gene expression (microarray and RNA-sequencing) has been widely harnessed to understand drug-induced transcriptional perturbations and how it implicates drug repurposing, target and biomarker identification. Still, CRISPR/Cas9 functional genomic assays are now powerful tools to identify context dependent drug-targets; its combination with transcriptomics could offer a path forward in drug discovery and development.in vivo  alter the DNA sequence of a gene to inactivate it ; 2) alter levels of expression  repress a gene by targeting mRNA transcripts for degradation (shRNAs). The advantages of both CRISPR and shRNA screening technologies are their scalability and flexibility. Using simple rules for the design of gene targeting sequences, most protein-coding genes in the genome can be efficiently perturbed. Thousands of shRNA and CRISPR screens have been reported at the scale of the genome in various contexts, ranging from identifying genes that are essential in malignant cells, to those that mediate resistance to chemotherapy or promote immune responses in vivo .https://depmap.org/portal/) is a collaboration between the Broad Institute and the Wellcome Sanger institute  . FLT3-inDr. Daniel Durocher’s group in Toronto have similarly used genome wide CRISPR screening to study genes driving sensitivity to PARP inhibition in human cancer cell lines. In 2018, Methods for CRISPR genetic screening have rapidly improved and new tools and applications are continuously published. Using data from previous genome-wide CRISPR screens, linear regression models and deep learning algorithms have been produced to improve the design of sgRNAs and reduce the potential for off-target effects . Online From the standpoint of genetic perturbation screening, it is important to consider the different effects of CRISPR and shRNA on gene expression. CRISPR often leads to bi-allelic gene knockout through the introduction of small insertions or deletion. Consequently, if a given gene, involved in the response of a cell to a pharmaceutical compound, is also essential to survival of the cell, it will likely not be identifiable in a pharmacogenomic CRISPR screen. In contrast, shRNAs have a partial and varied effect on mRNA transcript abundance, so they might be better suited for the study of the subset of genes that are essential to survival of the cell. However, a significant drawback for shRNA screening is the greater occurrence of “off-target” effects with shRNA: the targeting sequence of shRNAs may affect other non-intended mRNA transcripts, thus affecting the interpretation of some experimental results. To circumvent this issue, the CRISPR system can be repurposed to transiently enhance or repress gene expression instead of producing a gene knockout . The CRIA variety of publicly available resources exist for the analysis of CRISPR screening data. The complete analysis of CRISPR-Cas9 screens requires multiple steps after NGS, including data normalization, quality control, and identification of positively or negatively selected genes and relevant biological pathways . Some ofThe MAGeCK  algorithm was published in 2014 and is one of the most popular comprehensive methods for analyzing CRISPR data. MAGeCK uses a negative binomial model to test whether sgRNAs differ significantly between conditions and produces a list of FDR-adjusted hits . Two updThe BAGEL  pipeline was developed to leverage data from previous CRISPR screens. BAGEL uses essential and non-essential reference gene sets to identify novel essential genes in a screen and provides a Bayes factor for each gene . AlthougMany cancer cell lines show high copy number variation (CNVs). In CRISPR-cas9 screening, this presents an issue as sgRNA targets within high CNV regions may cause multiple DNA double-stranded breaks and cell death independent of gene essentiality . CERES wThe drugZ algorithm was developed specifically for the analysis of pharmacogenomic CRISPR screens. DrugZ calculates gene-level normalized Z-scores and FDR values, and can be applied to identify genes involved in conferring drug resistance and sensitivity .Though widely used, a shortcoming of the described packages is the required programming knowledge. More recently tools such as Cas-analyzer and PinAPL-Py have been published to provide complete web-based CRISPR analysis pipelines for researchers with less computational experience .in vitro, the technology has also been adapted for in vivo use  knowledge. Besides, the identification of genes or phenotypes through pharmacogenomic or phenotypic screens do not always provide information concerning the pharmacological properties of the identified/lead compound. Thus, phenotypic screening requires almost always complementary biochemical approaches to fully characterize the molecular target binding and MoA of the compound of interest. A possible combinatory strategy or alternative would consist of designing a library of competitive antagonists or negative allosteric modulators prior to their screening in a phenotypic assay. The latter approach is particularly interesting as negative allosteric modulators are usually known for their higher target selectivity compared to competitive antagonists. This would then result in better selectivity and less side effects while exhibiting the desired biological effect . Simulta