A molecular brain atlas reveals cellular shifts during the repair phase of stroke.

Ischemic stroke triggers a cascade of pathological events that affect multiple cell types and often lead to incomplete functional recovery. Despite advances in single-cell technologies, the molecular and cellular responses that contribute to long-term post-stroke impairment remain poorly understood. To gain better insight into the underlying mechanisms, we generated a single-cell transcriptomic atlas from distinct brain regions using a mouse model of permanent focal ischemia at one month post-injury. Our findings reveal cell- and region-specific changes within the stroke-injured and peri-infarct brain tissue. For instance, GABAergic and glutamatergic neurons exhibited upregulated genes in signaling pathways involved in axon guidance and synaptic plasticity, and downregulated pathways associated with aerobic metabolism. Using cell-cell communication analysis, we identified increased strength in predicted interactions within stroke tissue among both neural and non-neural cells via signaling pathways such as those involving collagen, protein tyrosine phosphatase receptor, neuronal growth regulator, laminin, and several cell adhesion molecules. Furthermore, we found a strong correlation between mouse transcriptome responses after stroke and those observed in human nonfatal brain stroke lesions. Common molecular features were linked to inflammatory responses, extracellular matrix organization, and angiogenesis. Our findings provide a detailed resource for advancing our molecular understanding of stroke pathology and for discovering therapeutic targets in the repair phase of stroke recovery.

APOE4 Increases Energy Metabolism in APOE-Isogenic iPSC-Derived Neurons.

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

Dataset on stroke infarct volume in rodents: A comparison of MRI and histological methods.

This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024). Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 µm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace. Infarct areas and volumes are assessed through MRI at two distinct time frames-three days (acute) and 28 days (chronic) following photothrombotic stroke induction. Subsequently, the brains are sectioned into 40 µm thick coronal slices, stained with NeuroTrace, and imaged as whole sections. The dataset holds considerable value for reuse, particularly for researchers focused on stroke volume estimation methods as well as those interested in comparing the efficacy of MRI and histological techniques.

A toolkit for stroke infarct volume estimation in rodents.

Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p < 0.001). The Dice similarity coefficient (DC) showed a high degree of coherence (DC > 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri­infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Beneath the radar: immune-evasive cell sources for stroke therapy.

Stem cell therapy is an emerging treatment paradigm for stroke patients with remaining neurological deficits. While allogeneic cell transplants overcome the manufacturing constraints of autologous grafts, they can be rejected by the recipient's immune system, which identifies foreign cells through the human leukocyte antigen (HLA) system. The heterogeneity of HLA molecules in the human population would require a very high number of cell lines, which may still be inadequate for patients with rare genetic HLAs. Here, we outline key progress in genetic HLA engineering in pluripotent stem and derived cells to evade the host's immune system, reducing the number of allogeneic cell lines required, and examine safety measures explored in both preclinical studies and upcoming clinical trials.

From attention-deficit hyperactivity disorder to sporadic Alzheimer's disease-Wnt/mTOR pathways hypothesis.

Alzheimer's disease (AD) is the most common neurodegenerative disorder with the majority of patients classified as sporadic AD (sAD), in which etiopathogenesis remains unresolved. Though sAD is argued to be a polygenic disorder, apolipoprotein E (APOE) ε4, was found three decades ago to pose the strongest genetic risk for sAD. Currently, the only clinically approved disease-modifying drugs for AD are aducanumab (Aduhelm) and lecanemab (Leqembi). All other AD treatment options are purely symptomatic with modest benefits. Similarly, attention-deficit hyperactivity disorder (ADHD), is one of the most common neurodevelopmental mental disorders in children and adolescents, acknowledged to persist in adulthood in over 60% of the patients. Moreover, for ADHD whose etiopathogenesis is not completely understood, a large proportion of patients respond well to treatment (first-line psychostimulants, e.g., methylphenidate/MPH), however, no disease-modifying therapy exists. Interestingly, cognitive impairments, executive, and memory deficits seem to be common in ADHD, but also in early stages of mild cognitive impairment (MCI), and dementia, including sAD. Therefore, one of many hypotheses is that ADHD and sAD might have similar origins or that they intercalate with one another, as shown recently that ADHD may be considered a risk factor for sAD. Intriguingly, several overlaps have been shown between the two disorders, e.g., inflammatory activation, oxidative stress, glucose and insulin pathways, wingless-INT/mammalian target of rapamycin (Wnt/mTOR) signaling, and altered lipid metabolism. Indeed, Wnt/mTOR activities were found to be modified by MPH in several ADHD studies. Wnt/mTOR was also found to play a role in sAD and in animal models of the disorder. Moreover, MPH treatment in the MCI phase was shown to be successful for apathy including some improvement in cognition, according to a recent meta-analysis. In several AD animal models, ADHD-like behavioral phenotypes have been observed indicating a possible interconnection between ADHD and AD. In this concept paper, we will discuss the various evidence in human and animal models supporting the hypothesis in which ADHD might increase the risk for sAD, with common involvement of the Wnt/mTOR-pathway leading to lifespan alteration at the neuronal levels.

Editing a gateway for cell therapy across the blood-brain barrier.

Stem cell therapy has been shown to improve stroke outcomes in animal models and is currently advancing towards clinical practice. However, uncertainty remains regarding the optimal route for cell delivery to the injured brain. Local intracerebral injections are effective in precisely delivering cells into the stroke cavity but carry the risk of damaging adjacent healthy tissue. Systemic endovascular injections, meanwhile, are minimally invasive, but most injected cells do not cross CNS barriers and become mechanically trapped in peripheral organs. Although the blood-brain barrier and the blood-CSF barrier tightly limit the entrance of cells and molecules into the brain parenchyma, immune cells can cross these barriers especially under pathological conditions, such as stroke. Deciphering the cell surface signature and the molecular mechanisms underlying this pathophysiological process holds promise for improving the targeted delivery of systemic injected cells to the injured brain. In this review, we describe experimental approaches that have already been developed in which (i) cells are either engineered to express cell surface proteins mimicking infiltrating immune cells; or (ii) cell grafts are preconditioned with hypoxia or incubated with pharmacological agents or cytokines. Modified cell grafts can be complemented with strategies to temporarily increase the permeability of the blood-brain barrier. Although these approaches could significantly enhance homing of stem cells into the injured brain, cell entrapment in off-target organs remains a non-negligible risk. Recent developments in safety-switch systems, which enable the precise elimination of transplanted cells on the administration of a drug, represent a promising strategy for selectively removing stem cells stuck in untargeted organs. In sum, the techniques described in this review hold great potential to substantially improve efficacy and safety of future cell therapies in stroke and may be relevant to other brain diseases.

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice.

Background: Stroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent immune deficiency influences overall stroke outcome. Methods: Here, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice three weeks after injury. We performed histology, gene expression, blood serum and behavioural analysis to identify the impact of immunosuppression on stroke progression. Results: We detected changes in microglia activation/macrophage infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Transcriptomic analysis of stroked tissue revealed the strongest deviation from WT was observed in NSG mice affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. These anatomical and genetic changes did not affect functional recovery in a time course of three weeks. To determine whether timing of immunosuppression is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with delayed immunosuppression (7d) showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mice led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. Conclusions: We detected distinct anatomical and molecular changes in the stroke pathology between individual immunosuppressed mouse models that should be considered when selecting an appropriate mouse model for stroke research.

Deep learning-based behavioral profiling of rodent stroke recovery.

BACKGROUND: Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible and often unsuitable for unraveling the complex behavior after injury. RESULTS: Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of 3 weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use, and costs. CONCLUSIONS: We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Stem Cell Therapy for Repair of the Injured Brain: Five Principles.

Cell therapy holds great promise for regenerative treatment of disease. Despite recent breakthroughs in clinical research, applications of cell therapies to the injured brain have not yielded the desired results. We pinpoint current limitations and suggest five principles to advance stem cell therapies for brain regeneration. While we focus on cell therapy for stroke, all principles also apply for other brain diseases.

Isoform- and cell-state-specific lipidation of ApoE in astrocytes.

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP-binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10-20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes.

The apolipoprotein E4 (APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs) (APOE4, E3, E2, and APOE knockout [APOE-KO]) to functional "iAstrocytes". Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 >E3 >E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 >E3 >E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 >E3 >E2 >KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced ß-amyloid uptake, while APOE2 iAstrocytes show opposing effects.

Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture.

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming. NEW METHOD: We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. COMPARISON WITH EXISTING METHODS: In contrast to other hydrogel-based 3D culture techniques, which require full encapsulation of cells, our hydrogel allows the seeded iPSCs and iPSC neurons to simply infiltrate the gel. RESULTS: IPSC-neurons grew to a depth of 500 µm into the hydrogel. Cell viability was comparable to 2D cultures over the course of three weeks, with even better neuronal survival in 3D cultures at the one-week time point. Levels of neuronal and synaptic maturation markers, namely, neural cell adhesion molecule 1 (NCAM1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2, were strongly increased in 3D cultures. Furthermore, we identified 4-repeat (4R) tau in 3D cultures, which was not detectable in 2D cultures. CONCLUSIONS: We describe a simple, hydrogel-based method for 3D iPSC culture that can serve as a fast and drug-screening-compatible platform to identify new mechanisms and therapeutic targets for brain diseases. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment.

Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model.

Blood brain barrier (BBB) damage is an important pathophysiological feature of ischemic stroke which significantly contributes to development of severe brain injury and therefore is an interesting target for therapeutic intervention. A popular permanent occlusion model to study long term recovery following stroke is the photothrombotic model, which so far has not been anatomically characterized for BBB leakage beyond the acute phase. Here, we observed enhanced BBB permeability over a time course of 3 weeks in peri-infarct and core regions of the ischemic cortex. Slight increases in BBB permeability could also be seen in the contralesional cortex, hippocampus and the cerebellum at different time points, regions where lesion-induced degeneration of pathways is prominent. Severe damage of tight and adherens junctions and loss of pericytes was observed within the peri-infarct region. Overall, the photothrombotic stroke model reproduces a variety of features observed in human stroke and thus, represents a suitable model to study BBB damage and therapeutic approaches interfering with this process.

Familial Alzheimer's disease mutations at position 22 of the amyloid ß-peptide sequence differentially affect synaptic loss, tau phosphorylation and neuronal cell death in an ex vivo system.

Familial forms of Alzheimer's disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the ß-amyloid peptide (Aß), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aß sequence, so-called intra-Aß mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aß sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aß mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aß mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aß. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aß40 or Aß42 containing the respective intra-Aß mutations. Our analyses revealed that wt Aß and E22G Aß, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aß showed stronger effects than the respective Aß40 isoforms. In contrast, E22Δ Aß neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aß are likely reflected in different disease pathomechanisms.

A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain.

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models.

Alzheimer's in a dish - induced pluripotent stem cell-based disease modeling.

BACKGROUND: Since the discovery of the induced pluripotent stem cell (iPSC) technique more than a decade ago, extensive progress has been made to develop clinically relevant cell culture systems. Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for approximately two thirds of all cases of dementia. The massively increasing number of affected individuals explains the major interest of research in this disease as well as the strong need for better understanding of disease mechanisms. MAIN BODY: IPSC-derived neural cells have been widely used to recapitulating key aspects of AD. In this Review we highlight the progress made in studying AD pathophysiology and address the currently available techniques, such as specific differentiation techniques for AD-relevant cell types as well as 2D and 3D cultures. Finally, we critically discuss the key challenges and future directions of this field and how some of the major limitations of the iPSC technique may be overcome. CONCLUSION: Stem cell-based disease models have the potential to induce a paradigm shift in biomedical research. In particular, the combination of the iPSC technology with recent advances in gene editing or 3D cell cultures represents a breakthrough for in vitro disease modeling and provides a platform for a better understanding of disease mechanisms in human cells and the discovery of novel therapeutics.